Chlamydia pneumoniae antigenic polypeptides, which comprise polypeptide A containing a sequence of at least 5 consecutive amino acids in the polypeptide of SEQ ID NO: 1; DNAs encoding the antigenic polypeptides, or DNAs complementary thereto; a method for production of an anti-Chlamydia pneumoniae antibody, wherein the antigenic polypeptide is used as an antigen; a method for detection and/or measurement of an anti-Chlamydia pneumoniae antibody, which comprise the antigenic polypeptide as an antigen; agents for diagnosis of Chlamydia pneumoniae infections, which comprise the antigenic polypeptide as an active ingredient; fused proteins of an antigenic polypeptide of Chlamydia pneumoniae with dihydrofolate reudctase; a method for production of an anti-Chlamydia pneumoniae antibody, wherein the fused protein is used as an antigen; reagents for detection and/or measurement of an anti-Chlamydia pneumoniae antibody, which comprise the fused protein as an antigen; agents for diagnosis of Chlamydia pneumoniae infections, which comprise the fused protein as an active ingredient.

The invention relates to Chlamydia pneumoniae antigenic polypeptides, fused proteins containing the polypeptides, DNAs coding therefor, recombinant vectors carrying the DNAS, transformants containing the recombinant vectors, a method for production of antibody, a method and reagents for detection and/or measurement of antibody, a method and agents for diagnosis of Chlamydia pneumoniae infections, probes and primers for detection and/or measurement of Chlamydia pneumoniae gene, and a method and reagents for detection and/or measurement of Chlamydia pneumoniae gene. The invention can be effectively used in the pharmaceutical industry, particularly in the preparation of agents for diagnosis of Chlamydia pneumoniae infections.
Several kinds of species are known in Chlamydia, that is, Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pecorum, Chlamydia pneumoniae and the like. Chlamydia trachomatis causes trachoma, venereal lymphogranuloma, urogenital infections, inclusion conjunctivitis, neonatal pneumonia and the like. Chlamydia psittaci causes psittocosis and the like. Chlamydia pneumoniae causes respiratory infections, atypical pneumonia and the like.
Since the symptoms of infections in the respiratory apparatus which are caused by Chlamydia pneumoniae are similar to those of infections caused by Mycoplasma pneumoniae or Influenza virus, physicians often make a wrong diagnosis. Hence, there is a need for the development of a simple method for diagnosing the infections caused by Chlamydia pneumoniae.
In general, an infection can reliably be diagnosed by detecting the causative bacterium in the infected site or by detecting an antibody against the causative bacterium in body fluids such as a sera and the like. The former method is called an antigen test and the latter is called an antibody test. Both of them are clinically important. As for Chlamydia pneumoniae, there is known an antibody test which is carried out by a method in which an antibody is detected by using an elementary body of Chlamydia pneumoniae.
However, this method has the disadvantage that the elementary body of Chlamydia pneumoniae reacts not only with an antibody against Chlamydia pneumoniae but also with antibodies against other species of Chlamydia, thus being fairly unspecific. This is because the elementary body of Chlamydia pneumoniae contains an antigen which is also present in other species of geneus Chlamydia than Chlamydia pneumoniae, that is, Chlamydia trachomatis and Chlamydia psittaciae.
As a plasmid which can be used for the expression of a large amount of a protein in E. coli, pBBK10MM is known (Japanese Unexamined Patent Publication No. Hei 4-117284). This plasmid can be used for the expression of a fused protein of an anti-allergic peptide with DHFR. The expressed fused protein also maintains the enzymatic activity of DHFR and can therefore be purified easily by utilizing the characteristic properties and activities of DHFR.
Genetic screening has been carried out to diagnose infections. In this screening, the presence of the gene of a microorganism to be detected in a sample is examined using nucleic acid probes and the like.
As for Chlamydia pneumoniae, there is known a genetic screening method which is carried out as disclosed in Japanese Unexamined Patent Publication No. Sho 64-500083, U.S. Pat. No. 5,281,518 and WO94/04549.
However, Japanese Unexamined Patent Publication No. Sho 64-500083 and U.S. Pat. No. 5,281,518 only disclose that a chromosomal DNA of Chlamydia pneumoniae or a DNA fragment which is obtained by cleaving the chromosomal DNA with a restriction enzyme or the like is used as a probe. The base sequences of these DNA molecules are not determined and the specificity of these probes are therefore unclear. In addition, it is difficult to determine the reaction conditions.
Although WO94/04549 discloses a method using a probe which is hybridized to ribosome RNA or DNA corresponding thereto, the specificity of these probes is not reliable because the homology of ribosomal RNA is relatively high in all organisms.
It is an object of the invention to provide antigenic polypeptides that do not react with antibodies against species of geneus Chlamydia other than Chlamydia pneumoniae, such as Chlamydia trachomatis, Chlamydia psittaci and the like and which react only with a Chlamydia pneumoniae-specific antibody and can thereby detect the Chlamydia pneumoniae-specific antibody.
Another object of the invention is to provide a method for synthesizing large amounts of the antigenic polypeptides by using gene recombination techniques.
A further object of the invention is to provide a method for production of an anti-Chlamydia pneumoniae-specific antibody, a method and reagents for detection and/or measurement of the anti-Chlamydia pneumoniae-specific antibody, and agents for diagnosis of Chlamydia pneumoniae infections, all by using said antigenic polypeptides.
A still further object of the invention is to provide probes and primers for detecting and/or measuring specifically Chlamydia pneumoniae gene, a method and reagents for detection and/or measurement of Chlamydia pneumoniae gene and agents for diagnosis of Chlamydia pneumoniae infections, all by using the probes or primers.
An even further object of the invention is to provide antigenic polypeptides for detection of an antibody which reacts with geneus Chlamydia including Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci and the like.
The subject matters of the invention are as follows:
(1) A Chlamydia pneumoniae antigenic polypeptide, which comprises polypeptide containing a sequence of at least 5 consecutive amino acids in the polypeptide of SEQ ID NO: 1 (hereinafter referred to as xe2x80x9cpolypeptide Axe2x80x9d).
(2) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide in which at least one amino acid is deleted from the polypeptide of SEQ ID NO: 1.
(3) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide in which at least one amino acid in the polypeptide of SEQ ID NO: 1 is replaced with other amino acid or a polypeptide in which at least one amino acid is added in the polypeptide of SEQ ID NO: 1.
(4) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide in which an amino acid or a peptide sequence is bound to a sequence of at least 5 consecutive amino acids in the polypeptide of SEQ ID NO: 1.
(5) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide containing the amino acid sequence of SEQ ID NO: 1
(6) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide containing the amino acid sequence of SEQ ID NO: 2.
(7) The antigenic polypeptide of (1), wherein said polypeptide A is a polypeptide containing the amino acid sequence of SEQ ID NO: 5.
(8) A DNA encoding the antigenic polypeptide of any one of (1)-(7), or a DNA complementary thereto.
(9) The DNA of (8), which contains the base sequence of SEQ ID NO: 3.
(10) The DNA of (8), which contains the base sequence of SEQ ID NO: 4.
(11) The DNA of (8), which contains the base sequence of SEQ ID NO: 7.
(12) A recombinant vector carrying the DNA of any one of (8)-(11).
(13) The recombinant vector of (12), which is plasmid pCPN533 xcex1 containing the base sequence of SEQ ID NO: 10.
(14) A transformant containing the recombinant vector of (12) or (13).
(15) A method for production of an anti-Chlamydia pneumoniae antibody,
wherein the antigenic polypeptide of any one of (1)-(7) is used as an antigen.
(16) A method for detection and/or measurement of an anti-Chlamydia pneumoniae antibody, wherein the antigenic polypeptide of any one of (1)-(7) is used as an antigen.
(17) A reagent for detection and/or measurement of an anti-Chlamydia pneumoniae antibody, which comprises the antigenic polypeptide of any one of (1)-(7) as an antigen.
(18) A reagent for diagnosis of a Chlamydia pneumoniae infection, which comprises the antigenic polypeptide of any one of (1)-(7) as an active ingredient.
(19) A fused protein of a Chlamydia pneumoniae antigenic polypeptide with dihydrofolate reductase, in which polypeptide containing a sequence of at least 5 consecutive amino acids in the polypeptide of SEQ ID NO: 1 is bound to the polypeptide of SEQ ID NO: 14 (hereinafter referred to as xe2x80x9cpolypeptide Bxe2x80x9d) either directly or via an intervening amino acid or amino acid sequence.
(20) The fused protein of (19), wherein said polypeptide B is a polypeptide in which at least one amino acid is deleted from the polypeptide of SEQ ID NO: 1.
(21) The fused protein of (19), wherein said polypeptide B is a polypeptide in which at least one amino acid in the polypeptide of SEQ ID NO: 1 is replaced with other amino acids or a polypeptide in which at least one amino acid is added in the polypeptide of SEQ ID NO: 1.
(22) The fused protein of (19), which is a polypeptide containing the amino acid sequence of SEQ ID NO: 15.
(23) The fused protein of (19), which is a polypeptide containing the amino acid sequence of SEQ ID NO: 16.
(24) A DNA encoding the fused protein of any one of (19)-(23), or a DNA complementary thereto.
(25) The DNA of (24), which contains the base sequence of SEQ ID NO: 17.
(26) The DNA of (24), which contains the base sequence of SEQ ID NO: 18.
(27) A recombinant vector carrying the DNA of any one of (24)-(26).
(28) The recombinant vector of (27), which is plasmid pCPN533T.
(29) A transformant containing the recombinant vector of (27) or (28).
(30) A method for production of an anti-Chlamydia pneumoniae antibody, wherein the fused protein of any one of (19)-(23) is used as an antigen.
(31) A method for detection and/or measurement of an anti-Chlamydia pneumoniae antibody, wherein the fused protein of any one of (19)-(23) is used as an antigen.
(32) A reagent for detection and/or measurement of an anti-Chlamydia pneumoniae antibody, which comprises the fused protein of any one of (19)-(23) as an antigen.
(33) A reagent for diagnosis of a Chlamydia pneumoniae infection, which comprises the fused protein of any one of (19)-(23) as an active ingredient.
(34) A probe for detection and/or measurement of Chlamydia pneumoniae gene, which comprises any one of
(a) a DNA containing a sequence of at least 10 consecutive bases in the DNA of SEQ ID NO: 3,
(b) a DNA complementary to DNA (a), or
(c) a DNA having at least 90% homology to DNA (a) or (b).
(35) The probe of (34), which contains the base sequence of SEQ ID NO: 19.
(36) The probe of (34), which contains the base sequence of SEQ ID NO: 20.
(37) A method for detection and/or measurement of Chlamydia pneumoniae gene, characterized in that the probe of any one of (34)-(36) is used.
(38) A reagent for detection and/or measurement of Chlamydia pneumoniae gene, which comprises the probe of any one of (34)-(36).
(39) An agent for diagnosis of a Chlamydia pneumoniae infection, which comprises the probe of any one of (34)-(36) as an active ingredient.
(40) A primer for detection and/or measurement of Chlamydia pneumoniae gene, which comprises any one of
(a) a DNA containing a sequence of at least 10 consecutive bases in the DNA of SEQ ID NO: 3,
(b) a DNA complementary to DNA (a), or
(c) a DNA having at least 90% homology to DNA (a) or (b).
(41) The primer of (40), which contains the base sequence of SEQ ID NO: 19.
(42) The primer of (40), which contains the base sequence of SEQ ID NO: 20.
(43) A method for detection and/or measurement of Chlamydia pneumoniae gene, wherein the primer of any one of (40)-(42) is used.
(44) A reagent for detection and/or measurement of Chlamydia pneumoniae gene, which comprises the primer of any one of (40)-(42).
(45) A reagent for diagnosis of a Chlamydia pneumoniae infection, which comprises the primer of any one of (40)-(42) as an active ingredient.
(46) A Chlamydia pneumoniae antigenic polypeptide, which is selected from the group consisting of
(a) the polypeptide of SEQ ID NO: 5,
(b) a polypeptide in which at least one amino acid is deleted from the polypeptide of SEQ ID NO: 5,
(c) a polypeptide in which at least one amino acid in the polypeptide of SEQ ID NO: 5 is replaced with another amino acid, and
(d) a fused polypeptide of any one of (a)-(c) with another amino acid or peptide.
(47) A Chlamydia pneumoniae antigenic polypeptide, which is selected from the group consisting of
(a) the polypeptide of SEQ ID NO: 6,
(b) a polypeptide in which at least one amino acid is deleted from the polypeptide of SEQ ID NO: 6,
(c) a polypeptide in which at least one amino acid in the polypeptide of SEQ ID NO: 6 is replaced with another amino acid, and
(d) a fused polypeptide of any one of (a)-(c) with another amino acid or peptide.
(48) A DNA encoding the polypeptide of (46), or a DNA complementary thereto.
(49) A DNA encoding the polypeptide of (47), or a DNA complementary thereto.
(50) The DNA of (48), wherein said DNA encoding the polypeptide of (46) is the DNA of SEQ ID NO: 7.
(51) The DNA of (49), wherein said DNA encoding the polypeptide of (47) is the DNA of SEQ ID NO: 8.
(52) A recombinant vector carrying the DNA of any one of (48)-(51).
In the specification, deoxynucleotides having only one base are referred to as xe2x80x9cmonodeoxynucleotidesxe2x80x9d and deoxynucleotides having at least two bases are referred to as xe2x80x9cDNASxe2x80x9d, unless otherwise indicated.
The invention will now be explained in detail.
Antigen Polypeptide
The antigen polypeptide of the present invention is formed of polypeptides containing at least five continued amino acid sequences in a polypeptide of SEQ ID No. 1 (hereinafter referred to as xe2x80x9cPolypeptide Axe2x80x9d) from the viewpoint of the minimum size in which a peptide is allowed to possess antigenicity.
Since the antigen-antibody reaction can be expected to gain in sensitivity in proportion as the length of amino acid sequence increases, the polypeptide A is appropriately formed of not less than 20, preferably not less than 100, and more preferably not less than 250 amino acids.
So long as the polypeptide A possesses the antigenicity inherent in Chlamydia pneumoniae, it tolerates the loss of amino acids (1-250 amino acids, for example) from the polypeptide of SEQ ID No. 1. If the number of missing amino acids is unduly large, the polypeptide A will tend to suffer the antigenicity inherent in Chlamydia pneumoniae to be impaired.
When the number of missing amino acids is large (five or more, for example), the polypeptide A prefers such missing amino acids (five or more, for example) to occur in a continued series for the sake of retaining the antigenicity of Chlamydia pneumoniae.
So long as the polypeptide A possesses the antigenicity inherent in Chlamydia pneumoniae, it tolerates the substitution of part of the amino acids (1-100 amino acids, for example) by other amino acids or the insertion of amino acids (1-100 amino acids, for example) in the polypeptide of SEQ ID No. 1. If the number of amino acids involved in the substitution or insertion is unduly large, the polypeptide A will tend to suffer the antigenicity inherent in Chlamydia pneumoniae to be impaired. When the number of amino acids involved in the substitution or insertion is large (five or more, for example), the polypeptide A prefers the amino acids (five or more, for example) to occur in a continued series for the sake of retaining the antigenicity of Chlamydia pneumoniae. The amino acids to be involved in the substitution are preferred to possess such similar qualities as are observed in the substitution between glycine and alanine, for example.
So long as the polypeptide A possesses the antigenicity inherent in Chlamydia pneumoniae, it may be a polypeptide having amino acids or peptides ligated directly or through the medium of an intervening amino acid sequence to at least five continued amino acid sequences in the polypeptide of SEQ ID No. 1.
The peptides for the ligation are appropriately formed of not more than 1000 amino acid sequences, preferably not more than 500 amino acid sequences, and more preferably not more than 200 amino acid sequences for the sake of retaining the antigenicity inherent in Chlamydia pneumoniae.
As concrete examples of such amino acids or peptides, leucine, leucine-methionine, dihydrofolic acid reductase (DHFR), and xcex2-galactosidase may be cited.
As concrete examples of the polypeptide A using DHFR or xcex2-galactosidase as a peptide, DHFR-Chlamydia pneumoniae antigen polypeptide-fused protein and xcex2-galactosidase-Chlamydia pneumoniae antigen polypeptide-fused protein may be cited. DHFR or xcex2-galactosidase may be ligated either directly or through the medium of an intervening amino acid sequence with Chlamydia pneumoniae antigen polypeptide.
As concrete examples of the polypeptide A, the polypeptides of SEQ ID No. 1, SEQ ID No. 2, and Sequence No. 5 may be cited.
Though the intervening amino acid sequence is not defined particularly, the amino acid sequences of leucine and leucine-methionine are examples.
As concrete examples of the fused protein of the present invention, the polypeptide formed of amino acid sequences of SEQ ID No. 15 and the polypeptide formed of amino acid sequences of SEQ ID No. 16 may be cited.
Among the fused proteins cited above, the polypeptide formed of the amino acid sequences of SEQ ID No. 15 including the whole antigen polypeptide of 53 kDa of Chlamydia pneumoniae proves particularly advantageous.
The method of chemical synthesis and the method of gene recombination are available for the production of the antigen polypeptide of this invention.
The polypeptide of SEQ ID No. 1 of this invention is an antigen polypeptide formed of 488 amino acid residues as shown in the table of sequences.
The polypeptide of SEQ ID No. 2 of this invention is an antigen polypeptide formed of 271 amino acid residues as shown in the table of sequences.
The polypeptide of SEQ ID No. 5 of this invention is an antigen polypeptide formed of 259 amino acid residues as shown in the table of sequences.
Among other antigen polypeptides mentioned above, the polypeptide of SEQ ID No. 1 containing the whole antigen polypeptide of 53 kDa of Chlamydia pneumoniae proves particularly advantageous.
Method for Production of Antigen Polypeptide
The method of chemical synthesis and the method of gene recombination are available for the production of the antigen polypeptide of this invention.
Among the methods of chemical synthesis is counted the MAP (multiple antigen peptide) method, for example. The MAP method befits the synthesis of a peptide formed of not more than 30 amino acid sequences. This synthesis can be implemented by the use of a commercially available peptide synthesizing device.
Among the methods of gene recombination is counted a method which comprises inserting a DNA coding for the antigen polypeptide of this invention in a vector thereby constructing a recombinant vector, inserting the recombinant vector in a host thereby producing a transformant, and isolating the peptide aimed at from the transformant.
The DNA coding for the antigen polypeptide of this invention will be described afterward.
The vector may be plasmid, phage, etc.
As concrete examples of the host, Escherichia coli, Bacillus subtilis, yeast, etc. may be cited.
Now, the method for forming the transformant and the method for refining the peptide aimed at by the use of the transformant will be described in detail below.
Preparation of Recombinant vector Carrying the DNA Encoding the Antigenic Polypeptide and Transformants Containing the Same
The xcex phage obtained by screening (see infra) is already a kind of recombinant vector carrying the DNA of the invention. Additional recombinant vectors can be prepared by inserting in a known plasmid vector or phage vector the DNA encoding the Chlamydia pneumoniae antigenic polypeptide (see infra) in a conventional procedure. In this case, a linker may be used if necessary. As the known plasmid vector, pBR322, pUC18, pUC19, pBBK10MM or the like can be used. Plasmids pBR322, pUC18 and pUC19 are commercially available and pBBK10MM is described in detail in Japanse Unexamined Patent Publication No. Hei 4-117284. As the phage vector, xcex gt11 phage, xcex gt11 phage or the like can be used. In any case, recombinant vectors corresponding to the parent vectors used can be obtained.
The recombinant vectors carrying the DNA of the invention include plasmid pCPN533 xcex1, 53-3S xcex phage and the like (see infra).
The obtained recombinant vector is introduced into a host to prepare a transformant. If an E. coli-derived plasmid or xcex phage is used, an E. coli strain such as HB 101 can be used as a host. The host is treated to become a competent cell. A competent cell obtained by treating E. coli strain HB101 is commercially available from Takara Shuzo Co., Ltd. A method of introducing the recombinant vector into a host to prepare a transformant is described in xe2x80x9cMolecular Cloningxe2x80x9d.
The obtained transformant is cultured to form colonies. Plasmid DNAs are obtained from each of the colonies and cleaved with an appropriate restriction enzyme. A transformant having a desired recombinant plasmid is selected according to the results of agarose gel electrophoretic analysis of the cleaved plasmid DNA. The plasmid vectors thus prepared include plasmid pCPN533 xcex1.
Examples of the transformant thus prepared include E. coli strain HB101 containing the recombinant vector pCPN533 xcex1.
Preparation of Recombinant Vectors Carrying the DNA Encoding Fused Protein of the Chlamydia pneumoniae Antigenic Polypeptide with DHFR and Transformants Containing the Same
The DNA molecule encoding the Chlamydia pneumoniae antigenic polypeptide (see infra) is ligated to the DNA molecule encoding DHFR (see infra) by means of a commercially available kit. In the ligation, a linker may be used if necessary. A DNA ligation kit (Takara Shuzo Co., Ltd) can be used as a commercially available kit. If the DNA obtained by the ligation does not have a replication origin and does not therefore function as a plasmid, the DNA is inserted in a separate plasmid vector, which may be pBR322, pUC18 or the like.
The ligated DNA is introduced into a host to prepare a transformant. If an E. coli-derived plasmid is used, an E. coli strain such as HB 101 can be used as a host. The host is treated to become a competent cell. A competent cell obtained by treating E. coli strain HB101 is commercially available from Takara Shuzo Co., Ltd. The method of introducing the ligated DNA into a host to prepare a transformant is described in xe2x80x9cMolecular Cloningxe2x80x9d.
The obtained transformant is cultured to form colonies. Plasmid DNAs are obtained from each of the colonies and cleaved with an appropriate restriction enzyme. A transformant having a desired recombinant plasmid is selected according to the results of agarose gel electrophoretic analysis. An example of the plasmid vector thus prepared is plasmid pCPN533T.
An example of the transformant thus prepared is E. coli strain HB101 containing the recombinant vector pCPN533T.
The transformant is cultured by shaking an incubator containing the transfomant at an appropriate temperature in a medium that allows the transformant to grow until a sufficient amount of the desired antigenic polypeptide is accumulated in the transformant. If E. coli strain HB101 containing the recombinant vectors pCPN533 xcex1 or pCPN533T are used as a transformant, the cell is cultured while shaking in ampicillin-containing LB medium at 37xc2x0 C. overnight. Subsequently, the culture is inoculated in ampicillin-containing TB medium and further cultured while shaking at 37xc2x0 C. overnight. A method for preparing the TB medium is described in xe2x80x9cMolecular Cloningxe2x80x9d.
The cultured transformant is harvested by centrifugation and suspended in a buffer. The transformant is disrupted by sonication of the suspension. If the transformant is E. coli, the cell may be lysed by successively adding lysozyme and an SDS-containing buffer to the suspension.
When the polypeptide aimed at is secretory in quality, the culture broth is centrifuged to obtain the supernatant.
After the disruption of the transformant, the cell residue is removed by centrifugation, thereby obtaining the supernatant. Streptomycin sulfate is added to the supernatant. The mixture is stirred for a certain period of time and centrifuged to precipitate nucleic acids, thereby obtaining the supernatant.
This supernatant is precipitated with ammonium sulfate and centrifuged. Generally, the precipitate is recovered as the product. Since the supernatant possibly contains the peptide aimed at, the practice of sampling and analyzing the supernatant thereby confirming the presence or absence of the peptide proves advantageous.
Either the solution of the precipitate in a small amount of buffer solution or the supernatant is fractionated by liquid chromatography. The proteins contained in the fractions are blotted by the Western blotting method using a Chlamydia pneumoniae-specific monoclonal antibody to obtain the fractions containing antigen polypeptide. When the polypeptide A is a protein fused with DHFR, a Methotrexate column can be used as the column for the liquid chromatography. Specific procedures of the removal of residues such as a cell membrane and the like, the removal of DNA by addition of streptomycin sulfate, the recovery of proteins by addition of ammonium sulfate and a Western blotting method are described in xe2x80x9cMolecular Cloningxe2x80x9d.
DNAs Encoding the Antigenic Polypeptides
In the invention, the DNA encoding the polypeptide of SEQ ID NO: 1 means DNAs selected from the group of DNAs which are obtained by translating the amino acids of the polypeptide of SEQ ID NO: 1 to triplets in accordance with the genetic code (each amino acid is assigned 1-6 sets of nucleotide sequences). This group of DNAs includes the DNA of SEQ ID NO: 3.
The DNA encoding the antigenic polypeptide A means DNAs encoding the polypeptide A. These DNAs are selected from the group of DNAs which are obtained by translating the amino acid sequence for the polypeptide A to triplets in accordance with the genetic code.
As the polypeptide A, those polypeptides which have been described under the item xe2x80x9cAntigenic Polypeptidesxe2x80x9d above may be given. As the DNA encoding the polypeptide A, nucleotides sequences which correspond to the amino acid sequences for those polypeptides may be given.
Similarly, the DNA encoding the polypeptide of SEQ ID NO: 2 means DNAs selected from the group of DNAs which are obtained by translating the amino acids of the polypeptide of SEQ ID NO: 2 to triplets in accordance with the genetic code. This group of DNAS includes the DNA of SEQ ID NO: 4.
Additionally, the DNA encoding the polypeptide of SEQ ID NO: 5 means DNAs selected from the group of DNAs which are obtained by translating the amino acids of the polypeptide of SEQ ID NO: 5 to triplets in accordance with the genetic code. This group of DNAs includes the DNA of SEQ ID NO: 7.
Moreover, the DNA encoding the polypeptide of SEQ ID NO: 6 means DNAs selected from the group of DNAs which are obtained by translating the amino acids of the polypeptide of SEQ ID NO: 6 to triplets in accordance with the genetic code. This group of DNAs includes the DNA of SEQ ID NO: 8.
DNAs encoding the fused proteins comprise codons corresponding to the amino acid sequence of the fused protein.
The DNAs include but are not limited to the DNAs of SEQ ID NOs: 17 and 18.
The base sequence of SEQ ID No. 17 is the base sequence of the DNA coding for the fused protein of DHFR and the whole antigen polypeptide of 53 kDa of Chlamydia pneumoniae and the base sequence of SEQ ID No. 18 is the base sequence of the DNA coding for the fused protein of DHFR and (part of) the antigen polypeptide of 53 kDa of Chlamydia pneumoniae.
These DNA""s can be manufactured by the method of chemical synthesis or the method of gene recombination.
Among the methods of chemical synthesis is counted the phosphoamidite method which fits the synthesis of a DNA formed in a length of not more than 100 base sequences. This chemical synthesis can be attained by a commercially available DNA synthesizing device.
Among the methods of gene recombination are counted a method for cloning the DNA from the elementary body of Chlamydia pneumoniae in the manner already described and the PCR method utilizing the already acquired DNA as a template and using a primer manufactured by adopting the base sequence at a position arbitrarily selected in that DNA. The method of gene recombination is capable of manufacturing a long DNA of more than 100 bases.
Now, the method for cloning the DNA coding for the antigen polypeptide from the elementary body of Chlamydia pneumoniae will be described in detail below.
Culture of Chlamydia pneumoniae
A suspension of cells is prepared from cultured HL cells. The supernatant of the culture is removed and the suspension of Chlamydia pneumoniae is then added to the resulting cell sheet. After incubation, Chlamydia pneuminiae-infected HL cells are obtained by centrifugation. As Chlamydia pneumoniae, strain YK41 (Y. Kanamoto et al., Micro biol. Immunol., Vol. 37, p.495-498, 1993) can be used.
Purification of Elementary Body of Chlamydia pneumoniae
The Chlamydia pneumoniae-infected HL cells are disrupted and centrifuged, thereby recovering the supernatant. The obtained supernatant is layered onto a continuous density gradient solution containing Urografin (Schering) is centrifuged.
The yellowish white band was recovered because in the preliminary experiment, it was confirmed to contain the elementary body of Chlamydia pneumoniae with the aid of an electron microscope.
Preparation of Genomic DNA of Chlamydia pneumoniae
The elementary body of Chlamydia pneumoniae is suspended in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM ethylene diaminetetra acetate (EDTA) (hereinafter referred to as xe2x80x9cTE bufferxe2x80x9d). To the resulting suspension are added a 1% aqueous solution of sodium dodecyl sulfate (SDS) and an aqueous solution of Proteinase K (1 mg/ml) and the elementary body is lysed while incubating. To the resulting solution is added phenol saturated with 0.1 M Tris-HCl buffer (pH 8.0). The mixture is stirred and centrifuged to recover an aqueous layer. The obtained aqueous layer is treated successively with RNase and phenol/chloroform/isoamyl alcohol, followed by ethanol precipitation. As a result, genomic DNA of Chlamydia pneunomiae is obtained.
Preparation of Genomic DNA Expression Library
The genomic DNA is digested with restriction enzymes AccI, HaeIII and AluI. The digest is treated with phenol/chloroform/isoamyl alcohol and subjected to ethanol precipitation to yield partially digested DNAS. To the partially digested DNAs are added a linker, adenosine 5xe2x80x2-triphosphate (hereinafter abbreviated to xe2x80x9cATPxe2x80x9d) and T4 ligase, thereby ligating the linker to the partially digested DNAs.
The linker-ligated partially digested DNAs are applied to a Chroma spin 6000 column in which the mobile phase is 10 mM Tris-HCl buffer containing 0.1 M NaCl and 1 mM EDTA. The eluate is collected and fractions containing 1-7 kbp DNA fragments are recovered. To the resulting fractions are added ATP and T4 polynucleotide kinase and a reaction is conducted to phosphorylate the 5xe2x80x2 end of the DNA fragments. The reaction solution is treated with phenol/chloroform/isoamyl alcohol and subjected to ethanol precipitation to yield 5xe2x80x2-end-phosphorylated DNA fragments.
To the resulting DNA fragments are added xcex gt11 DNA preliminarily digested with restriction enzyme EcoRI, ATP and T4 ligase and a reaction is conducted. The resulting recombinant xcex gt11 DNA is packaged with a commercially available packaging kit to prepare a gemonic DNA expression library.
Cloning of DNA Encoding Antigenic Polypeptide
Cultured cells of E. coli strain Y1090r- are infected with the gemonic DNA expression library and incubated in an agar medium. A protein produced in the cells by the expression of the inserted DNA is transferred to a nitrocellulose filter immersed in an aqueous solution of isopropylthio-xcex2-D-galactoside (IPTG). The filter is blocked with a bovine serum albumin and washed. The filter is then reacted with a Chlamydia pneumoniae-specific monoclonal antibody. As the Chlamydia pneumoniae-specific monoclonal antibody, AY6E2E8 and SCP53 can be used. A hybridoma cell line forming AY6E2E8 has been deposited with the National Institute of Bioscience and Human-Technology, the Agency of Industrial Science and Technology (1-3, Higashi 1 chome Tsukuba-shi Ibaraki-ken 305, Japan) as FERM BP-5154 under the terms of the Budapest Treaty. A hybridoma cell line forming SCP53 is disclosed in J. Clin. Microbil., Vol.132, p.583-588, 1994. After the reaction, the filter is washed and reacted with an anti-mouse IgG antibody labeled with an enzyme such as peroxidase or the like. After the reaction, the filter is washed and reacted with a color-developing substrate solution. As the color-developing substrate solution, a mixture of an aqueous solution of hydrogen peroxide and a solution of 4-chloro-1-naphthol in methanol can be used. After the reaction, the filter is washed and dried in air.
Plaques corresponding to the color-developing spots on the filter are identified and xcex phage contained in the plaques is obtained. The above procedure is repeated until all the plaques react with the aforementioned monoclonal antibody. As a result, the DNA encoding an antigenic polypeptide is cloned and A phage expressing the Chlamydia pneumoniae-specific antigenic polypeptide having reactivity with the Chlamydia pneumoniae-specific monochonal antibody is obtained.
Production of DNA Encoding the Chlamydia pneumoniae-Specific Antigenic Polypeptide
E. coli strain Y1090r- is infected with the obtained xcex phage and cultured to yield a large amount of xcex phage. DNA molecules are obtained and purified from the xcex phage using a commercially available kit. To the obtained DNA molecules are added a primer, Taq polymerase and deoxynucleotides. The steps of heating, cooling and incubating are repeated, thereby amplifying the DNA molecule inserted in xcex gt11 . xcex gt11 forward primer andxcex gt11 reverse primer (Takara Shuzo Co. Ltd.) can be used as primers and AmpliTaq DNA polymerase can be used as a Taq polymerase. A general procedure of DNA amplification is known as the PCR method, which is described in detail in J. Sambrook et al., Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press (1989) (hereinafter referred to as xe2x80x9cMolecular Cloningxe2x80x9d).
The amplified DNA is obtained and its base sequence is determined and analyzed. The amplified DNA can be obtained with a commercially available kit such as Wizard PCR Prep kit (Promega). The base sequence can be determined by fluorescence-labeled terminator cycle sequencing using Taq polymerase. This sequencing can be performed with a kit commercially available from Perkin-Elmer Japan. For analysis of the base sequence, a commercially available apparatus such as Model 373A DNA Sequencer (Applied Biosystems) can be used.
Following the determination of the base sequence, the base sequence of the DNA is analyzed using a DNA sequencing software package such as DNASIS (Hitachi Software Engineering) to estimate an editing, junctional and amino acid-translational regions.
If it is found that a full-length gene has not been obtained, DNA molecules upstream and downstream of the available DNA are obtained by genome walking. The genome walking can be performed with a kit commercially available from Takara Shuzo Co., Ltd.
Preparation of DNA Encoding DHFR
DNA encoding DHFR is obtained by digesting the DNA with a restriction enzyme from a plasmid vector containing the DNA or by amplifying the DNA by PCR using a template plasmid DNA or genomic DNA containing the DNA with an appropriate primer.
In the former method, plasmid vector pBBK10MM and recombinant vector pCPN533T of the invention can be used as the plasmid vector containing DNA encoding DHFR. E. coli containing pCPN533T and E. coli containing pBBK10MM have been deposited with the National Institute of Bioscience and Human-Technology, the Agency of Industrial Science and Technology as FERM BP-5222 and FERM BP-2374, respectively. Plasmid pCPN533T can be obtained from the deposited E. coli by a conventional method for obtaining plasmid DNA, which is described in xe2x80x9cMolecular Cloningxe2x80x9d. When plasmid pBBK10MM is used, a DNA fragment having a length of about 4.8 kbp may be excised with restriction enzymes BamHI and XhoI.
In the latter method, pBBK10MM and pCPN533T (see supra) can be used as a plasmid DNA and genomic DNA of Bacillus subtilis can be used as a genomic DNA. Genomic DNA can be obtained by a conventional method for obtaining gemonic DNA, which is described in xe2x80x9cMolecular Cloningxe2x80x9d.
The primer to be used in the latter method can be designed and synthesized in consideration of base sequences at the 5xe2x80x2 and 3xe2x80x2 ends of DNA encoding DHFR. For example, an oligonucleotide having the 1-20 sequence in the base sequence of SEQ ID NO: 17 and one having a sequence complementary to the 461-480 sequence in the base sequence of SEQ ID NO: 5 can be used. These oligonucleotides can be synthesized chemically with a commercially available DNA synthesizer.
In the antigen polypeptides mentioned above, the polypeptide of SEQ ID NO. 1 containing the whole antigen polypeptide of 53 kDa of Chlamydia pneumoniae is particularly preferred.
Method of Production of Anti-Chlamydia pneumoniae Antibody by Using the Antigenic Polypeptide as Antigen
An anti-Chlamydia pneumoniae antibody can be produced by immunizing a mouse with the antigenic polypeptide of the invention as an antigen, separating a spleen cell from the immunized mouse, fusing the spleen cell with a myeloma cell line to produce hybridomas, selecting a hybridoma recognizing the Chlamydia pneumoniae 53 kDa antigenic polypeptide from the produced hybridomas and culturing the selected hydridoma.
Exemplary myeloma cell lines include P3X63Ag8.653 (ATCC CRL-1580) and P3/NSI/1-Ag4-1 (ATCC TIB-18).
The anti-Chlamydia pneumoniae antibody is produced by a known general procedure for obtaining antibodies by immunization of mouse, except that the antigenic polypeptide of the invention is used as an antigen.
Method and Reagents for Detection and/or Measurement of Anti-Chlamydia pneumoniae Antibody Using the Antigenic Polypeptide as Antigen, and Agents for Diagnosis of Chlamydia pneumoniae Infections Comprising the Antigenic Polypeptide as Active Ingredient
A method for detection and/or measurement of an anti-Chlamydia pneumoniae antibody comprises, for example, the steps of immobilizing the antigenic polypeptide on a support, applying a sample, washing, adding a labeled secondary antibody, washing and detecting and/or measuring the label either directly or indirectly.
Examples of the support include latex particles, cellulose threads, plastic assay plates and particles and the like.
The antigenic polypeptide may be immobilized on the support through covalent bonding or physical adsorption.
Examples of the sample include human sera and the like. It is preferred to block the surface of the support with bovine serum albumin or the like before the addition of a sample so as to insure that other antibodies in the sample will not bind to the support unspecifically.
The support is washed with a surfactant-containing phosphate buffer or the like.
An example of the labeled secondary antibody is a labeled anti-human monoclonal antibody. Useful labels include various kinds of enzymes such as alkaline phosphatase, luciferase, peroxidase, xcex2-galactosidase and the like, various fluorescent compounds such as fluorescein and the like. A chemical compound such as biotin, avidin, streptoavidin, digoxigenin or the like may be inserted between the antibody and the label.
When the label is an enzyme, it may be detected and/or measured by adding a substrate and detecting and/or measuring the light emission or color development which occurs due to the catalytic action of the enzyme or by measuring the change in light absorbance. When the label is a fulorescent compound, it may be detected and/or measured by irradiating the reaction system with UV light and detecting and/or measuring the emitted fluorescence. A sensitizer may be used if necessary.
Reagents for detection and/or measurement of the anti-Chlamydia pneumoniae antibody using the antigenic polypeptide of interest as an antigen include the antigenic polypeptides which are immobilized on a support and those with which the necessary amounts of the secondary antibody and the substrate are enclosed.
The aforementioned reagents can be used as agents for diagnosis of Chlamydia pneumoniae infections. Probes and Primers for Detection and/or Measurement of Chlamydia pneumoniae Gene
DNA encoding the Chlamydia pneumoniae 53 kDa antigenic polypeptide has the base sequence of SEQ ID NO: 3.
The probes and primers of the invention comprise DNA containing any one of
(a) a DNA containing a sequence of at least 10 consecutive bases in the DNA of SEQ ID NO: 3,
(b) a DNA complementary to DNA (a), or
(c) a DNA having at least 90% homology to DNA (a) or (b).
The length of the base sequence of the probes and primers is preferably 10-50 bp, more preferably 15-20 bp.
Specific examples of the probes and primers of the invention include a DNA comprising the base sequence of SEQ ID NO: 19 and a DNA comprising the base sequence of SEQ ID NO: 20.
The probes and primers of the invention can be synthesized easily with a commercially available DNA synthesizer. DNA synthesizers are commercially available from Applied Biosystems and the like. Alternatively, the probes and primers of the invention can be prepared by chemically synthesizing a short DNA fragment and synthesizing a long DNA fragment by PCR using the short DNA as a primer.
The probes and primers of the invention include those prepared by labeling such DNAs.
Exemplary labels include chemical compounds such as biotin, avidin, streptoavidin, digoxigenin and the like; enzymes such as alkaline phosphatase, luciferase, peroxidase, xcex2-galactosidase and the like; and fluorescent compounds such as fluorescein and the like. Biotin may be bound to the probes by, for example, adding biotinated deoxyuridine 5xe2x80x2-triphosphate to the probes in the presence of a terminal transferase. A kit containing a terminal transferase and biotinated deoxyuridine 5xe2x80x2-triphosphate can be purchased from Boehringer Mannheim. In the case where a label other than biotin is to be bound, a commercially available kit can also be used. Such a kit can be purchased from Takara Shuzo Co., Ltd and TOYOBO CO., LTD. Alternatively, the label may be bound by a method described in xe2x80x9cMolecular Cloningxe2x80x9d.
If desired, radioactive isotopes can be used as labels. In this case, (xcex3xe2x88x9232P)dATP is added to the probes and primers in the presence of T4 polynucleotide kinase. A general procedure of labeling with a radioactive isotope is described in xe2x80x9cMolecular Cloningxe2x80x9d. T4 polynucleotide kinase can be purchased from TOYOBO CO., LTD. and (xcex3xe2x88x9232P)dATP from Amersham.
RNAs corresponding to the base sequences of the probes and primers of the invention, that is, nucleic acids in which thymine is replaced with uracil in the base moiety and in which deoxyriboses are replaced with riboses in the sugar chain, can be used as the probes and primers of the invention instead of the aforementioned probes and primer comprising DNAs as structural units. These probes and primers comprising RNAs as structural units can be used in the method and reagents for detection and/or measurement of the invention.
Method for Detection and/or measurement of Chlamydia pneumoniae Gene
Chlamydia pneumoniae gene is detected and/or measured by, for example, separating DNA in a sample on the basis of the difference in molecular weight by elecrophoresis, transferring the obtained DNA to a nitrocellulose filter, nylon membrane filter or the like for its identification, adding the labeled probe of the invention, and detecting and/or measuring the label. This method is called the Southern blotting technique and its general procedure is described in xe2x80x9cMolecular Cloningxe2x80x9d.
Chlamydia pneumoniae gene is detected and/or measured with the primer of the invention by, for example, the PCR method which was described above. The method for detecting and/or measuring Chlamydia pneumoniae gene by PCR using the primer of the invention comprises the following steps.
(i) A buffer containing the primer of the invention, DNA polymerase, DATP, dCTP, dGTP and dTTP is added to a sample containing DNA and the mixture is heated.
(ii) The reaction solution is cooled, held at a constant temperature and heated.
(iii) Step (ii) is repeated.
(iv) The DNA contained in the reaction solution is detected and/or measured.
The DNA-containing sample to be used in step (i) may be nucleic acids as extracted from tunica mucosa pharyngsis of a patient.
The DNA polymerase to be used in step (i) may be a Taq polymerase, which can be purchased from TOYOBO CO., LTD.
In step (i), the mixture is heated by, for example, leaving it to stand at 90-100xc2x0 C. for 0.5-10 minutes.
In step (ii), the reaction solution is cooled by, for example, leaving it to stand at 45-65xc2x0 C. for 0.5-5 minutes, held at a constant temperature by, for example, at 70-80xc2x0 C. for 1-10 minutes, heated by, for example, leaving it to stand at 90-100xc2x0 C. for 0.5-5 minutes.
The heating in step (i), and cooling, holding at a constant temperature and heating in step (ii) can be carried out by using a DNA thermal cycler(copyright) (Perkin-Elmer Cetus).
Step (iii) may be repeated any number of times, preferably about 30 times.
The DNA contained in the reaction solution is detected and/or measured in step (iv) by, for example, electrophoresing the reaction solution with an agarose gel containing ethidium bromide, and thereby separating the DNA in the reaction solution on the basis of the difference in molecular weight and irradiating the agarose gel with UV light. If the primer of the invention is a labeled one, DNA is detected and/or measured with the aid of the label.
In another embodiment of the invention, after steps (i)-(iii), the primer of the invention may be replaced with one having another base sequence and steps (i)-(iii) are repeated, followed by step (iv).
Reagents for Detection and/or Measurement of Chlamydia pneumoniae Gene
An exemplary reagent for detection and/or measurement of Chlamydia pneumoniae gene according to the invention is an aqueous solution of the probe or primer of the invention which is packed frozen in a plastic container.


Described herein are technologies related to an implementation of a time interleaved digital-to-time converter (DTC) topology to generate high frequency phase modulated local oscillator (LO) signals. A first and second DTC are connected to an oscillator where outputs of the two DTCs are combined to generate a phase modulated signal and the two DTCs have a frequency rate that is half the frequency rate of the phase modulated signal. The two DTCs can operate at a 50 percent or lower duty cycle.

A Digital Polar Transmitter (DPTX) architecture may provide a reduced size and a reduced power consumption when compared to a conventional analog radio transmitter architecture. For example, a DPTX architecture may include two main functional blocks. One main functional block is a Digital-to-Time Converter (DTC) that modulates a local oscillator (LO) carrier with phase information of a signal that is to be transmitted. While the other main functional block is a Digital Power Amplifier (DPA) that modulates the amplitude of the signal that is to be transmitted onto the phase-modulated LO signal, thereby generating a reconstruction of the original signal (e.g., the signal to be transmitted).
In modern technologies we aim to implement the DPTX in order to improve key performance indicators and to achieve better power consumption, efficiency and compatibility. For example, the DTC in the transmission chain has a very low jitter frequency offset generator and may be further used in the receive chain. However, technologies such as 5G cellular and Wi-Fi 11ac/11ax drive carrier frequency to higher frequencies in order to achieve higher signal bandwidth (BW). To this end, present phase modulator topologies are problematic when implemented to operate at high frequencies due to timing issues and power consumption which increase exponentially with frequency.

This patent is subject to a terminal disclaimer.

High frequency time interleaved digital to time converter (DTC)


The present invention is generally related to the separation, fractionation, and/or characterization of molecules and/or biomolecules in one or more mixtures. After fractionation, different phases of a partitioning system can be analyzed via an analytical technique such as spectral analysis, chromatography, or the like, to produce a spectrum or other symbolic representation of the species after fractionation, and the spectra of the various fractions/phases compared to define a comparative spectrum as a marker or otherwise providing information about the sample, including such information that is independent of the original level of abundance of the molecules in the mixture. Comparative spectra of various samples can be compared to each other and/or to controls or reference spectra and/or comparative spectra to determine a variety of information. In some embodiments, the methods can be used for discovering and/or identifying patterns in a mixture of species and/or corresponding patterns of species in a second mixture, where each mixture of species originates from biological systems with different physiological conditions as markers associated with specific diagnostics, and can be used for screening for such markers once discovered and identified during diagnostics screening.

Many diseases and/or other pathological processes or conditions are caused by dysfunction and/or disregulation of certain proteins. These disease-related proteins may have their structures altered, relative to their "normal" or "wild-type" counterparts, and/or may be expressed in larger (up-regulated expression) or smaller (down-regulated expression) quantities in a given disease state, relative to "normal" physiological conditions. In some cases, proteins having altered structure and/or function may be used as markers associated with a particular human or animal disease, for instance, as a diagnostic for earlier detection of the disease, or the like. In many cases, the particular protein(s) of relevance to a given pathological process of a disease or other condition are unknown. Identification of such protein(s) would be useful for development of new diagnostic tests, or the like.
A general approach to the identification and characterization of protein markers is based on the analysis of protein compositions of samples of biological material (biological fluids, such as blood, serum, plasma, cerebrospinal fluid, tissues, cells, etc.) using high resolution separation techniques. For instance, proteins isolated from control and experimental samples or populations can be subjected to proteolytic cleavage, and their cleavage products identified using liquid chromatography (LC) coupled with tandem mass spectrometry (LC-MS-MS). Many protein separation techniques are based on multi-dimensional separation of proteins from a sample, typically by two-dimensional gel electrophoresis (2-DE) or two-dimensional high-performance liquid chromatography (2D-HPLC). The two-dimensional (2-D) protein maps for pathological samples may be obtained and compared with those for reference samples; positions of proteins observed as "spots" on (2-DE) maps or as "peaks" on 2D-HPLC maps can be compared, and those that are present (or absent) in the maps obtained from pathological samples but absent (or present) in the maps obtained from the reference samples may be judged as being likely to correspond to pathologically relevant proteins. Additionally, quantities of proteins estimated as intensities of the spots (or peaks) may be evaluated and compared between the pathological and reference samples. Those that are significantly different may be considered as pathologically relevant in some cases.
It has also been recently established that a pattern of the presence/absence and/or the relative quantities of multiple proteins (a "signature") may also be of diagnostic relevance, where the proteins judged to be of interest are identified by peptide mapping and/or mass spectrometry. Mathematical or statistical techniques, such as pattern recognition techniques, can be used to analyze the pattern produced by these experimental techniques and produce a diagnostic classification. However, this approach is often highly inefficient, for example, due to the inherent necessity of analyzing all of the proteins in a given sample, whereas only a small portion of the proteins may have any pathological relevance.
Several different methods for reducing the analytical complexity of protein mixtures have been developed. These methods are typically based on fractionation of the original mixture prior to 2D analysis by gel electrophoresis or 2D-HPLC. One such method is the separation of proteins by the technique of free-flow electrophoresis. However, this technique, while fractionating the original protein mixture, may result in multiple 2D analysis of simplified fractions, i.e. while reducing the complexity of analysis and improving resolution, it inherently greatly increases the number of samples where further analysis is required.
Another method is fractionation based on the affinity of proteins to different natural ligands and/or pharmacological compounds; however, this approach, while allowing separation of proteins according to protein functions, may result in a large increase in the number of samples for further analysis, and often requires additional knowledge or presumption concerning the differences between the samples.
One disadvantage of most fractionation techniques is that they generally cannot preserve protein-protein or protein-ligand interactions. Differences among biological interactions are often important for elucidating and detecting changes among samples. Additionally, most of the fractionation techniques rely on separation due to a fixed physical attribute, such as molecular size or net charge. While these attributes may be very important for distinguishing among biomolecules in a complex mixture, they generally do not cover all of the potential differences between biomolecules representing, e.g., normal vs. disease states, differences in configuration etc. Another important disadvantage of present fractionation techniques is related to their inability to separate mixtures based on differences between structural changes in, e.g., glycosylation patterns and/or conformational changes. These changes are often important for identifying proteins that either participate in and/or are the result of a disease state. For example, if a protein is misfolded as a result of genetic mutation, the net charge and size of the protein may not vary significantly, and more importantly, the protein's expression level might be the same for the underlying normal vs. disease states. Finally, natural genetic variability among individuals can significantly contribute to a very large scatter in the expression levels (concentrations) of biomolecules in a biological sample. This variability generally necessitates use of statistically large number of samples to robustly detect differences innate to a particular pathological condition, rather than to genetic variability. Natural genetic variability is often a significant hindrance in implementing protein marker based diagnostics due to reduction of the sensitivity and/or specificity of the diagnostic test.
While significant advances in the field of molecular and/or sample characterization have been made, improvements are therefore needed to add specificity, versatility, convenience, and/or improve efficiency.

Systems and methods involving data patterns such as spectral biomarkers


The present invention relates to a biologically active functionalized electrospun matrix to permit immobilization and long-term delivery of biologically active agents. In particular the invention relates to a functionalized polymer matrix comprising a matrix polymer, a compatibilizing polymer and a biomolecule or other small functioning molecule. In certain aspects the electrospun polymer fibers comprise at least one biologically active molecule functionalized with low molecular weight heparin. Examples of active molecules that may be used with the multicomponent polymer of the invention include, for example, a drug, a biopolymer, for example a growth factor, a protein, a peptide, a nucleotide, a polysaccharide, a biological macromolecule or the like. The invention is further directed to the formation of functionalized crosslinked matrices, such as hydrogels, that include at least one functionalized compatibilizing polymer capable of assembly.

Over the past two decades the development of novel drug delivery systems has had a tremendous impact on medicine, allowing for the improvement of many existing therapeutics as well as enabling the use of entirely new therapies. Along with these improvements there has been a move to miniaturize drug delivery devices from the macroscale (>1 mm) to the microscale (100-0.01 μm) or nanoscale (100-1 nm). One goal of developing these devices is to provide for the in situ delivery of therapeutic compounds. Therefore, a main area of investigation concerns the development of integrated systems that combine the nanotechnology with therapeutic molecules, for example, drugs, or biopolymers.
However, these new technologies must overcome several crucial and significant obstacles. For example, new approaches are required that allow for the delivery of insoluble, or unstable compounds. In addition, these approaches must address situations in which the therapeutic molecule is rare, or difficult to purify. Furthermore, these approaches must allow for localized delivery of potent compounds, and provide for improved compliance by reducing the chances of missing or erring in a dose. Lastly, these devices cannot be so small that they are unable to deliver an adequate dose.
In addition, to date a number of peptides and proteins have been explored for pharmaceutical applications by virtue of their high biological activity and specificity. Despite potential advantages offered by these drugs, their application may suffer because of the high molecular weight, hydrophilicity and low stability, which are reflected in poor biopharmaceutical properties. In particular, peptides and proteins undergo rapid clearance from the body, which takes place by a combination of events including proteolysis, renal ultrafiltration, and liver clearance.
The development of functionalized polymers that can elicit specific biological responses, as well as the development of methods to fabricate these biologically functionalized polymers, is a viable approach for such therapeutic applications, in which delivery of molecules such as those above is desirable. For example, the generation of fibrous matrices with biological properties and fiber diameters commensurate with those of the natural extracellular matrix (ECM) may permit the development of novel materials for use not only in drug delivery, but also in wound healing or tissue engineering.
Synthetic polymers, which can be designed to mimic some functions of biopolymers, have been actively developed as drug delivery matrices due to their industrial and scientific value. They represent a primary polymeric vehicle for the delivery of drugs and biomolecules, owing to their simple synthesis, ease of processing, and range of physical properties. Polymers such as proteins, polysaccharides, and nucleic acids, which are present as basic components in living organic systems, have also found some use in drug delivery and biomaterials applications, particularly for biopolymers such as collagen and alginate, which have desired mechanical and biological properties. There remains a significant need, however, for the facile production of polymeric materials in which mechanical and biological properties can be easily modified in a modular fashion, without relying solely on the naturally occurring mechanical properties of the biological polymers or the lack of biological activity of synthetic polymers.
One approach to the use of polymers in drug delivery applications involves the response of a polymer system to certain stimuli, which is a common process for biopolymers in living organisms. Stimuli responsive polymers can provide a variety of applications for the biomedical fields. The interest in these polymers has increased dramatically due to their promising potential. Among others, temperature and pH responsive mechanisms have been investigated as they are relatively convenient and effective stimuli in a variety of applications. Such stimuli-responsive polymers have been utilized in various forms, including cross-linked (permanently) hydrogels, reversible hydrogels, micelles, modified interfaces, and conjugated solutions, among others.
There has also been developing interest by the scientific community in applying methods from engineering and the life sciences to create artificial constructs directed to tissue regeneration, and thus, providing a minimally invasive and less painful way to treat patients. The basic protocol for the tissue engineering approach includes isolating specific cells by obtaining them through a biopsy from a patient, growing them on a three-dimensional scaffold under controlled culture conditions, delivering the construct to the desired location in the patient's body and directing new tissue formation into the scaffold that will be degraded over time. Successful regeneration of damaged organs or tissues based on tissue engineering requires several critical elements, not the least of which includes biomaterial scaffolds that serve as a mechanical support for cell growth.
Materials are important in most tissue engineering strategies as they can serve as a substrate on which cell populations can attach and migrate, be implanted with a combination of specific cell types as a cell delivery vehicle, and be utilized as a drug carrier to activate specific cellular function in the localized region.
The modification of biomaterials with bioactive molecules has been found useful in designing biomimetic scaffolds that can provide biological cues to elicit specific cellular responses and direct new tissue formation. The surface and bulk modification of materials with peptide sequences or with bioactive proteins or other biomolecules, for example, can allow for the modulation of cellular functions such as adhesion, proliferation and migration through alterations of peptide concentration or its spatial distribution. A variety of cell binding peptides have been introduced into three dimensional networks through physical, chemical, photochemical, and ionic crosslinking. The molecules have been attached to a variety of polymeric substrates for varying applications, but a need still remains for methods for simple modification of materials with multiple bioactive molecules in a manner that can control their release.
Electrospinning is an attractive approach to polymer processing. The control of fiber diameter, porosity, and fiber surface morphology makes electrospun fibers useful in a range of applications including filtration, electronic, and biomedical applications. Electrospun fibers are ideal for use in biomedical applications such as tissue engineering and drug delivery due to the three-dimensional nanometer scale matrix that can be quickly produced using small quantities of starting material (<50 mg) on the laboratory scale. Larger matrices can also easily be made via the scaling up of materials that prove to have useful materials and biological properties on the laboratory scale. Collagen, fibrinogen, chitosan, poly(lactic acid), poly(L-lactide-co-β-caprolactone), and poly(D,L-lactide-co-glycolide) are just a few of the polymers being investigated for use in electrospun drug delivery and tissue engineering constructs due to their biocompatibility. Electrospun fibers mimic the size scale of fibrous proteins found in the extracellular matrix (50-150 nm) and the three-dimensional nature of the matrix allows for cells to infiltrate the matrix and proliferate. A variety of additional polymers can also be electrospun into matrices for drug delivery, tissue engineering, and other applications. For example, see Z. M. Huang, Y. Z. Zhang, M. K. Kotaki and S. Ramakrishna, Composites Sci. and Tech. 2003, 63, 2223-2253, (US Patent publication No. US20030137069).
Although there have been reports of the use of electrospun fibers as scaffolds for select drug delivery and biomaterials applications, the incorporation of bioactive molecules has not been widely exploited. In one example, tetracycline was included in a solution of poly(lactic acid) (PLLA) and poly(ethylene-co-vinyl acetate) and electrospun in a fiber membrane to study the release profile of the fiber-encapsulated drug. PLLA has also been studied as a matrix to incorporate the drugs rifampin (to treat tuberculosis) and paclitaxel (anti-cancer drug). Polymers loaded with both of these therapeutic molecules could be fabricated into nanometer to micron diameter electrospun PLLA fibers and used for delivery of the drug.
While the development of functionalized polymers that can elicit specific biological responses is of great interest in the biomedical community, as well as the development of methods to fabricate these biologically functionalized polymers, there is still a need for more facile methods to create biologically active functionalized matrices that permit immobilization and long-term delivery of biomolecules and other small functioning molecules, moieties, and/or particles.
For example, although there have been studies demonstrating the utility of functionalizing electrospun fibers, the functionalization of electrospun fibers to confer specific biological activity would be particularly advantageous for many biomaterial applications, as such modification would permit the fabrication of biomaterials that are structurally relevant and that possess properties designed to treat or address specific problems. In addition, such incorporation could serve to protect bioactive molecules, release them in relevant timeframe, and incorporate multiple such molecules. In particular, the development of simple methods to produce multifunctional electrospun fibers would advance the development of electrospun fibers for biomedical applications.
Of such bioactive molecules, glycosaminoglycans have been demonstrated to mediate a wide range of biological activities such as cell adhesion, cell mobility, cell proliferation and tissue morphogenesis via binding to various cell regulatory proteins such as the chemokines, growth factors, enzymes, enzyme inhibitors, and extracellular matrix proteins. The well studied glycosaminoglycan, heparin, is a linear, unbranched, highly sulfated polysaccharide chain, and it is well accepted that the electrostatic interactions between the sulfates of the glycosaminoglycan and basic residues of a protein play an important role in binding. In particular, the spatial orientation of the basic residues is a major determinant of heparin-binding ability, and variations in the pattern of the sulfation of the heterogeneous heparin therefore permit binding between heparin and a wide range of binding partners. Accordingly, heparin has been incorporated into covalent hydrogel delivery systems because of this ability to bind a diverse set of proteins. In addition, we and others have demonstrated that the interactions between heparin and specific HBPs can also mediate the assembly of noncovalently associated hydrogel networks (see, for example, N. Yamaguchi, K. L. Kiick, Polysaccharide-poly (ethylene glycol) star copolymers as scaffolds for the production of bioactive hydrogels, Biomacromolecules 6 (4) (2005) 1921-1930; N. Yamaguchi et al., Rheological characterization of polysaccharide-poly(ethylene glycol) star copolymer hydrogels, Biomacromolecules 6 (4) (2005) 1931-1940; B. L. Seal, A. Panitch, Physical polymer matrices based on affinity interactions between peptides and polysaccharides, Biomacromolecules 4 (6) (2003) 1572-1582).
Because hydrogels can mimic the high water content and mechanical properties of natural tissues, they are prime candidates as carriers of bioactive agents, in bioadhesive systems, or as biorecognizable materials. Given that poly (ethylene glycol), or PEG, is highly hydrophilic and generally nonadhesive to proteins or cells, it has found widespread use as a drug carrier, and many PEG hydrogels have been produced from aqueous solutions containing linear or branched PEG macromolecules via chemical crosslinking. In addition to hydrogels formed via radical crosslinking reactions, PEG hydrogels have been formed, for example, via Michael-type addition reactions upon mixing with thiol-bearing compounds or via the reaction between amino-terminated poly (ethylene glycol) and the herbal iridoid glycoside genipin. In some cases, cell adhesive peptide domains or biodegradable sequences have been introduced into PEG hydrogels to endow them more biological signaling functions, including the capacity for growth factor delivery. Accordingly, there have been a variety of covalently crosslinked hydrogel systems developed to deliver growth factors via mechanisms such as diffusion and chemical or enzymatic reaction.
Noncovalent interactions provide an alternative method for crosslinking, as introduced above, removing the need for toxic chemical crosslinking agents in gel preparation. In addition, noncovalent crosslinking strategies may offer advantages in maintaining protein integrity and bioactivity until delivery. Polymer hydrogels have been formed via specific recognition events such as reversible antibody-antigen interactions (see T. Miyata et al., A reversibly antigen-responsive hydrogel, Nature 399 (6738) (1999) 766-769) and coiled-coil interactions (see for example, C. Y. Xu et al., Reversible hydrogels from self-assembling genetically engineered protein block copolymers, Biomacromolecules 6 (3) (2005) 1739-1749; C. Wang et al., Hybrid hydrogels assembled from synthetic polymers and coiled-coil protein domains, Nature 397 (6718) (1999) 417-420; W. A. Petka, et al., Reversible hydrogels from self-assembling artificial proteins, Science 281 (5375) (1998) 389-392). There has been little attention given, however, to the interaction between peptides (particularly coiled-coils) and polysaccharides in hydrogel assembly. Further, the fabrication of biologically active fibers through such functionalization strategies is a fairly new and unexplored area in the field of electrospinning, and electrospun fibers have not been investigated for their ability to incorporate growth factor binding glycosaminoglycans, such as heparin, in a manner designed to control release.

Multifunctional and biologically active matrices from multicomponent polymeric solutions


A sound signal processing device that acquires a collected-sound signal obtained by sampling a sound collected by a microphone at a first sampling frequency, and receives a reproduced-sound signal obtained by sampling a sound for reproduction at a second sampling frequency different from the first sampling frequency, and then converts the sampling frequency of the reproduced-sound signal into the first sampling frequency so as to remove an acoustic echo from the collected-sound signal by using the reproduced-sound signal whose sampling frequency has been converted.

An electronic device including both a speaker that reproduces a sound and a microphone that collects a sound is known. In such an electronic device, an acoustic echo may occur when a microphone collects a sound reproduced by a speaker. Therefore, there are cases where echo removal processing is performed on the sound signal obtained by the microphone. The echo removal processing is processing of removing a sound signal due to an echo from a sound signal output from a microphone by using sound signal data to be input to the speaker.


The present invention provides a method of airflow transportation of methionine that can minimize the crushing of methionine, which is characterized in that when methionine is transported as airflow using carrier gas, the flow state of methionine is a low concentration floating flow type, and the mixing ratio of methionine and carrier gas is in the range of 4 to 10 kg-methionine/kg-carrier gas. In the method of airflow transportation of methionine of the present invention, if the D50 of methionine is in the range of 150 to 425 μm, the increase rate of the fine powder can be suppressed to 1.5% or less by maintaining the mixing ratio at 4 to 10 kg-methionine/kg-carrier gas, and can be suppressed to 1% or less by maintaining the mixing ratio at 5 to 10 kg-methionine/kg-carrier gas.

Methionine produced through such methionine production process as shown in Patent Document 1 is a powder or a granule, which required transport from the end unit of the manufacturing process to a storage unit such as a tank and further to a filling unit in which methionine is weighed and filled in the container to accomplish its product package.
Airflow transportation with a carrier gas in the pipe is preferable for transportation within the manufacturing apparatus because it is easy to avoid problems of scattering and contamination, but suitable conditions for airflow transportation have been unknown because there is no literature referring to airflow transportation of methionine.

Pneumatic conveyance method for methionine


A semiconductor memory device of the present invention is provided with a plurality of memory cell arrays distributed and thus arranged and having the same function, and a main control circuit and local control circuits that are structured hierarchically. Each memory cell array has its operation controlled directly by any of the local control circuits, wherein the main control circuit including a command producing circuit which responds to an externally applied signal to produce a control signal corresponding to a predetermined mode of operation, and a global control circuit which responds to a control command to generate a control signal for operating the entire semiconductor memory device consistently. The local control circuits receive the control signal from the global control circuit to cause memory cell arrays to perform a predetermined operation.

1. Field of the Invention
The present invention relates to semiconductor memory devices and more specifically to a configuration of a semiconductor memory device having a plurality of memory cell arrays distributed in arrangement and capable of operating the plurality of memory cell arrays consistently.
2. Description of the Background Art
Recently, as semiconductor memories have larger capacity, there has been the need to provide on one chip a plurality of memory circuits with the same function and operate the circuits consistently.
FIG. 26 is a block diagram showing as one example of such a memory device an entire configuration of a synchronous DRAM (SDRAM) capable of operating eight memory cell arrays having a same function consistently, shown as a conventional semiconductor memory device 100 in the figure.
Referring to FIG. 26, semiconductor memory device 100 is provided with a clock signal terminal 102, an address signal terminal 103, a control signal terminal 104, a data input/output terminal 106, a power supply terminal 108 and a ground potential terminal 109 as input/output terminals for signal communication with the external.
Semiconductor memory device 100 also includes buffer circuits 112-116 provided for signals communicated via input/output terminals, a control circuit 110, a memory cell array 150 and a sense amplifier (SA)-I/O circuit 151, a control signal bus 117 communicating a signal between control circuit 110 and SA-I/O circuit 151, and a data bus 118 for transmitting data between memory cell array 150 and input/output buffer 116.
Control circuit 110 responds to the clock, address and control signals externally applied via input/output terminals 102-104 to produce a control clock corresponding to a predetermined mode of operation and synchronizing with the signals to generally control the operation of semiconductor memory device 100. Control circuit 110 is commonly provided for the eight memory cell arrays and controls the operation of SA-I/O circuit 151 via control signal bus 117 to operate the memory cell arrays with the same function consistently.
However, since the conventional semiconductor memory device 100 has control circuit 110 provided commonly for all of the memory cell arrays, all of the signals required for controlling the operation of each memory cell array need be transmitted via control signal 131. This significantly increases the number of interconnections of control signal bus 117. Thus the load on the signal interconnections is increased and consequently the delay, distortion and the like of a signal is disadvantageously caused and chip area is disadvantageously increased.

Semiconductor memory device consistently operating a plurality of memory cell arrays distributed in arrangement


An ultrasonic sensor includes a case having a substantially cylindrical shape with a bottom. The case has a piezoelectric element provided on the bottom, and a cushion member is engaged with a side portion of an opening of the case. A substrate and pin terminals are engaged in and held by a holding portion of the cushion member. On the substrate, electrodes that are connected to the pin terminals are provided. An opening portion is provided in a top surface of the cushion member. Lead wires that are electrically connected to the piezoelectric element extend out from the opening portion and are connected to the electrodes. The inside of the case is filled with an elastic resin from the opening portion.

1. Field of the Invention
The present invention relates to ultrasonic sensors, and more particularly, to a ultrasonic sensor used, for example, for a back-up sensor on an automobile.
2. Description of the Related Art
FIG. 12 is a schematic diagram showing an example of a known ultrasonic sensor. An ultrasonic sensor 1 shown in FIG. 12 includes a case 2 having a substantially cylindrical shape with a bottom, the case 2 being formed of aluminum or other suitable material. One surface of a piezoelectric element 3 is bonded to an inner bottom surface of the case 2. The inside of the case 2 is substantially entirely filled with foamable resin 4, such as foamable silicon so as to cover the piezoelectric element 3. A substrate 6 having two terminals 5a and 5b is attached in an opening portion of the case 2 so as to cover the foamable resin 4. On opposed surfaces of the substrate 6, electrodes 7a and 7b are connected to the terminals 5a and 5b. The terminal 5a is connected to an opposite surface of the piezoelectric element 3 by the electrode 7a formed on the inner side of the substrate 6 and a wire 8. The terminal 5b is connected to the other surface of the piezoelectric element 3 by the electrode 7b formed on the outer side of the substrate 6 and solder 9 through the case 2.
Where a distance to an object to be detected is measured using the ultrasonic sensor 1 shown in FIG. 12, a driving voltage is applied to the terminals 5a and 5b to excite the piezoelectric element 3. As the piezoelectric element 3 is vibrated, the bottom surface of the case 2 is also vibrated, which causes an ultrasonic wave to be emitted in a direction perpendicular to the bottom surface as indicated by an arrow shown in FIG. 12. When the ultrasonic wave emitted from the ultrasonic sensor 1 is reflected by an object to be detected and reaches the ultrasonic sensor 1, the piezoelectric element 3 is vibrated and the vibration is converted into an electrical signal. The electrical signal is output from the terminals 5a and 5b. A period of time from the application of the driving voltage to the output of the electrical signal is measured, and thus, the distance from the ultrasonic sensor 1 to the object to be detected can be measured.
In the ultrasonic sensor 1 shown in FIG. 12, since the inside of the case 2 is filled with the foamable resin 4, the vibration of the overall case 2 is suppressed. Further, the ultrasonic wave generated in the inside of the case 2 is scattered and absorbed by a large number of foam pores in the foamable resin 4. This can efficiently suppress the vibration of the case 2 and the ultrasonic wave propagation within the case 2, thereby improving reverberation characteristics (see, for example, Japanese Unexamined Patent Application Publication No. 11-266498).
FIG. 13 is a schematic diagram showing another known ultrasonic sensor. An ultrasonic sensor 1 shown in FIG. 13 is different from the ultrasonic sensor 1 shown in FIG. 12, in that a substrate 6 having terminals 5a and 5b is not directly attached to a case 2, but instead, is covered with an elastic member 4a composed of silicon rubber or other suitable material which is filled in the inside of the case 2. A sealant 4b composed of a high-gas-barrier silicon material is provided on an exposed surface of the elastic member 4a. The terminal 5a is connected to a piezoelectric element 3 through a lead wire 8a, and the terminal 5b is connected to the piezoelectric element 3 through a lead wire 8b and the case 2.
In the ultrasonic sensor 1 shown in FIG. 13, the sealant 4b composed of a high-gas-barrier silicon material provided on the exposed surface of the elastic member 4a can effectively prevent corrosive gas from entering the case 2. Furthermore, the silicon material used as the sealant 4b can ensure a sufficient level of reverberation characteristics even in a cold temperature environment with rapid temperature changes (see, for example, Japanese Unexamined Patent Application Publication No. 2001-197592).
In the ultrasonic sensor 1 shown in FIG. 12, the substrate 6 having the terminals 5a and 5b is fixed in direct contact with a side surface of the case 2. Thus, the vibration of the piezoelectric element 3 is transmitted to the case 2 and the substrate 6 and is damped from the terminals 5a and 5b.
On the other hand, in the ultrasonic sensor 1 shown in FIG. 13, since the substrate 6 having the terminals 5a and 5b is covered with the elastic member 4a composed of silicon rubber or other suitable material which is filled in the inside of the case 2, vibration damping through the case 2 is less likely to occur.
However, in the ultrasonic sensor 1 shown in FIG. 13, the positions of the substrate 6 and the terminals 5a and 5b are often displaced by a filling process of silicon rubber or other suitable material used for the elastic member 4a, and it is difficult to position the terminals 5a and 5b at desired positions in the ultrasonic sensor. This may result in failure during mounting.
In the ultrasonic sensors 1 shown in FIGS. 12 and 13, the wire 8 and lead wires 8a and 8b inside the case 2 are bonded to the terminals 5a and 5b by soldering, welding, or other suitable method. However, due to the configuration of ultrasonic sensors 1 shown in FIGS. 12 and 13, the substrate 6, the case 2, and other components are assembled after the bonding. Thus, the length of the wire 8 and the lead wires 8a and 8b must be increased. Further, an excessive stress may be applied to the bonding portions of the wire 8 and the lead wires 8a and 8b when the case 2 and other components are assembled. Therefore, the wiring of the terminals 5a and 5b is very difficult.


The invention relates to sheet metal or a sheet metal blank provided with a lubricant coating, especially an anti-corrosion oil of the Pre-Lube or Dry Lube variety. The sheet metal or sheet metal blank comprises a layer which is formed by applying a solution containing an organic phosphoric acid ester to the metal surface of the sheet metal. Sheet metal provided with said coating has excellent tribological properties.

In the manufacture of metal sheets, such as steel sheets, it is common for these to be provided with a temporary corrosion protection for storage and transport immediately after the manufacturing or coating process and before delivery to the metal-processing industry. This is usually effected by applying a corrosion-inhibiting lubricant directly before the coiling of the sheet, such as a corrosion protection oil, a pre-lube or a dry-lube. If the metal sheet is destined for further processing by forming such as deep-drawing, the lubricants used can contain, in addition to corrosion-inhibiting substances, other ancillary substances, especially matched to the individual forming process, which improve the tribological properties of the sheet during the forming process. Metal sheets coated in this way are encountered, for example, in the automobile industry in the manufacture of bodywork made of metal sheets by deep-drawing.
With the use of lubricants for temporary corrosion protection and for improving the forming properties, it must be particularly borne in mind that these are to be easily and completely removable after the forming process.
From EP 0 489 105 B1 a method is known for the cold deep-drawing of metal sheets, in which the sheet which is to be deep-drawn and/or the deep-drawing tool are treated with an aqueous solution of an inorganic alkali phosphate and are then deep-drawn in the presence of a deep-drawing oil. The salt which is deposited on the sheet forms a soap under the pressure and temperature conditions of the deep-drawing process as a result of the reaction with the deep-drawing oil, which has the effect of reducing the friction coefficient. A disadvantage of the method is that the alkali phosphate used and the soap which is formed cannot be entirely removed from the sheet, free of any residue, after the deep-drawing.
From CH 441 594 a lubricant composition is further known for the cold-forming of metals without the removal of material, which contains a finely dispersed water-soluble inorganic salt for the improvement of the tribological properties, such as Borax. A disadvantage with this lubricant composition is that its lubrication capacity is not constant, since the salt is not distributed uniformly over the surface, which interferes with the deep-drawing process.
With the production of single-sided electrolytically galvanized fine sheet, very thin phosphating or borax passivation with a coating weight of approx. 10 mg/m2 has proved advantageous in respect of tribological properties. Both products can be applied in the existing flushing baths of the galvanizing lines, but they have the disadvantage that they may impair the phosphating process in the automobile production plant.
The use of prelubes takes account of the idea of influencing the tribological properties of steel strip directly on the surface of the steel. Prelubes contain drawing aids which are introduced into the oil coating, which is about 1 μm thick. Only the direct steel-tool interface is tribologically active, in the range of a few nanometers.
Finally, the principle is generally known of carrying out a surface treatment of the deep-drawn sheet in order to improve the tribological properties. In particular, chemical processes for the surface treatment are known, such as phosphating, by means of which the friction coefficient between the sheet and the tool is reduced, and the forming of the sheet section is thereby facilitated. Such a treatment, however, is expensive and cannot be applied to all deep-drawn sheets.

Lubricant coated sheet metal with improved deformation properties


The compact C-multiplier includes four MOSFETs operating in the subthreshold region using the translinear principle. The multiplier is controllable to meet designer requirements. A Tanner Tspice simulator is used to confirm the functionality of the design in 0.13 pm CMOS Technology. The circuit operates from a ±0.75 supply voltage. Simulation results indicate that the multiplication factor is large compared to existing designs.

1. Field of the Invention
The present invention relates to impedance scaling circuits, and particularly to a compact C-multiplier utilizing MOSFETs in a translinear loop circuit with the MOSFETs biased in weak inversion mode.
2. Description of the Related Art
Integrated circuits are playing an increasing role in implantable biomedical systems. Such circuits must be designed to meet rigorous specification especially size and power consumption. The signals of the body are low in frequency, ranging from 1 Hz or less to 1 KHz. Realization of large time constants in small area is required for integrated active filters, but unfortunately, existing circuits require large power consumption.
Thus, a compact C-multiplier solving the aforementioned problems is desired.

KING FAHD UNIVERSITY OF PETROLEUM AND MINERALS


A disposable atomizer contains a cylindrical container for a medium to be atomized, particularly a pharmaceutical medium. The container is closed by a rubber plunger plug which has a central opening into which a closure ball is pressed. For discharging the medium, the container is pressed against an actuator which pushes the ball from the opening and thereby opens the container while, at the same time, the actuator forced down from a shoulder forms the plunger of a plunger pump formed thereby.

The invention relates to a discharge device for fluid media comprising a main body for accommodating a media reservoir which forms a pump chamber, including a plunger pump, particular for single-stroke only discharge, with an actuator introducable by an actuating force into the media reservoir and thereby opening a closure of the media reservoir. One such discharge device is described in DE-A-40 21 263. In this arrangement a diaphragm forming the closure is provided in a piston plug, the diaphragm being penetratable by the actuator.
Furthermore, from DE-A-36 31 341 and 40 04 528 of Applicant ball valves are known which form the outlet valve and are actuated by the fluid pressure built up in the interior of the media reservoir.
Again in EP-0 311 863 A of Applicant there is further described that a pump barrel and a flexible stop formed as a kind of snap lock coact such that prior to discharge of a partial stroke a certain actuating pressure needs to be exterted by the operator so that discharge of the fluid at a minimum pressure does not result until this pressure point is overcome. This configuration makes sure that on atomization of a medium the pressure is sufficient for atomization right from the start. This is especially important for discharge devices affecting total discharge in only a single stroke and which are intended for the administration of medicinal drugs which as regards metering, contamination, preservation etc. thereof need to be sealed off hermetically and thus sterile until actually made use of. In the case of these normally highly expensive medicinal drugs it must be ensured that the contents are not wasted e.g. non-atomized due to actuation being too weak.

Discharge device for fluid media, particularly for single-stroke only discharge


In a metal honeycomb extrusion die which incorporates apertures for the conveyance of extrudable material through the die body toward a discharge opening in the face of the die, the aperture sidewalls are shaped by electrochemical machining or the like to develop a periodically varying aperture size, surface shape or surface finish, superimposed on the conventional randomly varying machined aperture sidewall surface, in order to reduce the adverse effects of random surface variations on extrusion process stability and extruded product quality.

The present invention relates to methods for machining steel blank materials to form steel extrusion dies, and more particularly to a modified chemical drilling process which improves the performance of such extrusion dies for the extrusion of plasticized inorganic powder batch materials.
The manufacture of inorganic honeycomb structures from plasticized powder batches comprising inorganic powders dispersed in appropriate binders is well known. U.S. Pat. Nos. 3,790,654, 3,885,977, and 3,905,743 describe dies, processes and compositions for such manufacture, while U.S. Pat. Nos. 4,992,233 and 5,011,529 describe honeycombs of similar cellular structure extruded from batches incorporating metal powders.
The manufacture of extrusion dies for the production of ceramic honeycombs by these methods requires extremely precise machining. To supply material to the slotted honeycomb discharge section of such a die, the inlet or batch supply face of the die is provided with multiple apertures or feedholes through which the plasticized material to be extruded is forced under high pressure. For softer steels, the feedhole array can be formed by mechanical drilling, but if the die is to be formed of harder materials such as stainless steels, electrochemical machining techniques offer better results.
In the electrochemical machining (ECM) process, also referred to as the STEM (Shaped Tube Electrolyte Machining) process, the apertures are formed through a controlled deplating (dissolution) of the electrically conductive steel workpiece. An electrolytic cell is formed wherein the drill comprises the negatively charged electrode (cathode), the workpiece comprises the positively charged electrode (anode), and the electrolyte is a flowing electrically conductive fluid.
In the manufacture of extrusion dies the drill (cathode) suitably comprises a metal tube of titanium having an insulated outer surface extending toward an exposed tip. This inhibits electrolytic action everywhere except at the tip of the tube. The workpiece is typically a high grade stainless steel plate or block, and the electrically conductive electrolyte fluid is nitric acid.
To drive the electrolytic dissolution of steel proximate to the tip of the shaped tube to form an aperture, an electric potential is applied between the tube and the anodic workpiece. As drilling proceeds, the electrolyte is continuously pumped through the drilling tube and into contact with the steel workpiece, thereby completing the electrolytic cell while flushing dissolved steel away from the workpiece surface.
In this "forward cycle" phase of the drilling process, the electric potential is typically controlled to a constant voltage or, more preferably, to a constant current, and the drilling rate or tube feedrate is controlled to a constant value. The purpose of these controls is to maintain as much as possible a constant aperture and an optimally smooth aperture surface finish. U.S. Pat. No. 4,687,563 to Hayes, U.S. Pat. Nos. 5,320,721 and 5,322,599 to Peters, U.S. Pat. No. 5,507,925 to Brew, and published EP Application No. EP 0 245 545 describe the ECM process as it has been applied to the fabrication of steel extrusion dies, and variations on those processes.
Although ECM remains the process of choice for forming large arrays of fine apertures in tools such as extrusion dies, problems with this process still remain. One difficulty relates to small variations in aperture surface finish, presently attributed to random variations in processing factors such as electrolyte temperature, workpiece metallurgy, electrical contact, and a myriad of other process variables or their interactions.
In the case of honeycomb extrusion dies, one effect of such variations is that surface finish can vary from aperture to aperture across the aperture array forming the inlet to the die. This can result in an uneven flow front in the material being extruded from the outlet or discharge face of the die, a condition which can produce a variety of honeycomb product defects, including but not limited to significant variations in cell dimensions, cell shapes, and cell wall thicknesses in the honeycomb structures being extruded.
In fact, feed-hole finish appears to be a fundamental factor influencing the flow characteristics of honeycomb extrusion dies which is not yet within the ability of the art to completely control. This inability to control surface finish means that the extrusion process may not be operating in a processing regime that is optimal for high quality honeycomb production. If better surface finish control were possible, engineered surface finishes providing significant improvements in extruded product quality might be attainable.