The present invention relates to polypeptides exhibiting antibiotic, and anti-viral and anti-cancer effects. In particular, the invention relates to modified T4 lysozyme, polypeptides comprising fragments of T4 lysozyme and to the production and use thereof. The fields of application for this invention are wide-ranging, and include, for example, human and veterinary uses, resistance cultivation in plants and prevention of bacterial and/or fungal-mediated food spoilage.

The present invention relates to polypeptides exhibiting antibiotic, and anti-viral and anti-cancer effects. In particular, the invention relates to modified T4 lysozyme, polypeptides comprising fragments of T4 lysozyme and to the production and use thereof. The fields of application for this invention are wide-ranging, and include, for example, human and veterinary uses, resistance cultivation in plants and prevention of bacterial and/or fungal-mediated food spoilage.
T4 lysozyme is a protein which is formed from bacteriophage T4. T4 lysozyme functions to open the bacterial host cell after it has been propagated therein, and to allow it to enter the environment. It has been assumed that the enyzme activity (muramidase) of T4 lysozyme destroys the bacteria (Tsugita, A. (1971) Phage lysozyme and other lytic enzymes; in: Boyer, P. D. (ed.) The Enzymes, Vol. V. Academic Press, New York, p. 344-411). T4 lysozyme is known to effect a specific cleavage between C-1 of the muramic acid group and C-4 of acetylglucosamine. This cleavage ruptures the bacterial muramine network ruptures and destabilizes the cell wall. However, the lysozyme must first traverse the bacterial cell membrane to access its substrate, the muramine layer. Until now it has not been possible to explain the transport route of the T4 lysozyme through the inner cell membrane to the muramine layer. It has been assumed that, as a consequence of rupture of the muramine layer the bacteria burst open through destabilization and are thus destroyed.
Further enzymatic or other biochemical functions, e.g. membrane affinities, of the T4 lysozyme have not previously been described.
DE 39 26 390 describes the introduction of lysozyme genes as exogenic or additional DNA into plants, to increase resistance to fungi and animal microbes.
Lysozyme genes are understood here to be any nucleic acids (e.g., DNA) that encode lysozymes. Lysozymes protect the transformed plants against plant-pathogenic fungi and animal microbes.
The nucleic acid used to transform plants preferably includes a promotor active in plants, a chimeric gene for T4 lysozyme, containing the coding DNA sequences for the signal peptide of alpha-amylase from barley and for the lysozyme of the bacteriophage T4, and a polyadenylation signal. The amino acid sequence of the T4 lysozyme is obtained from the phage T4.
Through a consensus sequence for the N-bound glycosylation (Asn-X-Ser/Thr) existing in the amino acids 140-142, a glycosylated form of the T4 lysozyme protein is produced in plants (Dxc3xcring, K.; Porsch, P.; Fladung, M.; Lxc3x6rz, H.; Transgenetic potato plants resistant to the phytopathogenic bacteria Erwina carotovora; The Plant Journal 3, 587-598 (1993) ). The glycosylation occurring in planta, which has not been described in patent DE 39 26 390, can be the cause of a change in the enzyme properties or functionality. It is possible, for example, that the conversion rate of the enzyme is significantly reduced.
DE 39 26 390 describes only the utilization of lysozymes for increasing the resistance of transgenic plants, containing a lysozyme-coding DNA sequence, to fungi and animal microbes. The described gene constructions are limited by the aforementioned problems. Moreover, these gene constructions do not solve the problem of potential influencing factors, which can reduce the efficiency of the described system in transgenic plants. Production of the T4 lysozyme in transgenic plants, and use of T4 lysozyme as a medical remedy in human or veterinary medicine, or as a preservative additive is not considered.
The mode of action of the T4 lysozyme (for which at the time of registration of patent DE 39 26 390 only the muramidase activity was known) on fungi and other microbes has still not been explained. The definition of lysozyme genes given in patent DE 39 26 390 relates only to translation into proteins which possess the known properties of lysozymes.
There is a need in the art for new types of polypeptides with a wide range of useful properties, such as antibiotic, anti-viral and anti-cancer properties.
The invention generally relates to antibiotic polypeptides comprising a modified T4 lysozyme, or a functional equivalent thereof, which exhibits antibiotic activity without exhibiting muramidase activity. The polypeptide preferably does not comprise full-length, native T4 lysozyme.
In a preferred aspect, the native glutamic acid residue at position 11 of the T4 lysozyme is replaced by any amino acid residue other than glutamic acid.
In another preferred aspect, the segment is selected from the group consisting of the amino acid sequence of SEQ ID NO: 1, and fragments and derivatives thereof, wherein X is any amino acid residue except glutamic acid.
A preferred set of segments includes:
amino acids 12-164 of SEQ ID NO: 1, and subsegments and functional equivalents thereof;
amino acids 126-141 of SEQ ID NO: 1, and subsegments and functional equivalents thereof;
amino acids 143-155 of SEQ ID NO: 1, and subsegments and functional equivalents thereof;
amino acids 74-164 of SEQ ID NO: 1, and subsegments and functional equivalents thereof;
amino acids 114-164 of SEQ ID NO: 1, and subsegments and functional equivalents thereof; and
amino acids 124-164 of SEQ ID NO: 1, and subsegments and functional equivalents thereof.
Another preferred group of functional equivalents includes:
amino acids 12-164 of SEQ ID NO: 1 comprising one or more mutations at any one or more of positions 140-142, which mutation(s) do not eliminate the antibiotic activity of the polypeptide.
amino acids 126-141 of SEQ ID NO: 1 comprising one or more mutations at any one or more of position s 140-141, which mutation(s) do not eliminate the antibiotic activity of the polypeptide.
amino acids 74-164 of SEQ ID NO: 1 comprising one or more mutations at any one or more of positions 140-142, which mutation(s) do not eliminate the antibiotic activity of the polypeptide.
amino acids 114-164 of SEQ ID NO: 1 comprising one or more mutations at any one or more of positions 140-142, which mutation(s) do not eliminate the antibiotic activity of the polypeptide.
The antibiotic polypeptide may also consist of the above-listed sets of segments, subsegments and functional equivalents.
The invention also relates methods for producing the segments, subsegments and functional equivalents (e.g., proteolysis of the native protein, chemical synthesis, recombinant production).
In another aspect, the invention relates to a nucleic acid encoding a polypeptide comprising a segment of T4 lysozyme, or a functional equivalent of said segment, which polypeptide exhibits antibiotic activity but which does not exhibit muramidase activity. The recombinant nucleic acid preferably does not encode full-length T4 lysozyme.
The invention also relates to a method for killing a microbe and/or controlling a population of microbes. The method generally comprises bringing the microbe and/or population of microbes into contact with a polypeptide comprising a segment of T4 lysozyme, or comprising a functional equivalent of said segment, which polypeptide exhibits antibiotic activity but which does not exhibit muramidase activity. In one aspect, the method is used to kill a bacteria or fungus, or to eliminate or control a bacterial colony or a fungal colony. In another aspect, the microbe is a pathogen of an organism and the organism is genetically modified to produce the polypeptide, thereby bringing the pathogenic organism and/or colony into contact with the polypeptide, to effect its antibiotic effect on the organism or colony. In a preferred aspect, the microbe is a plant pathogen and the polypeptide is applied to the plant and/or the plant is genetically modified to produce the polypeptide.
The invention provides microbe control compositions comprising antibiotic polypeptides formulated for application to the target microbes or their situs.
Antibiotic compositions of the invention generally contain one or more of the antibiotic polypeptides described above and a carrier acceptable for the specific use for which the composition is intended. The invention includes methods of killing or controlling microbes which involve applying such antibiotic compositions to the microbes or their environment. In one aspect, the antibiotic compositions of the present invention are administered in the form of a spray or a time release dosage unit. The antibiotic compositions can also comprise various other known antibiotic polypeptides targeting the same or different microbes.
Methods of making antibiotic compositions are also included within the scope of the present invention. These methods generally comprise bringing one or more of the antibiotic polypeptides into association with a suitable carrier, diluent or excipient therefor.
Definitions
As used herein, the term xe2x80x9cantibioticxe2x80x9d is broadly used to refer to the capacity to kill or injure cells, and specifically includes anti-bacterial, anti-fungal, anti-microbial, anti-viral and anti-cancer effects.
As used herein, the term xe2x80x9cantibiotically effectivexe2x80x9d and the like, is used to indicate an amount or concentration of an antibiotic compound which is sufficient to reduce the number of target cells in a target locus (e.g., a container of food, an infected site on an organism, or an entire organism in the case of a systemic infection), as compared to a corresponding locus in the absence of the amount or concentration of the antibiotic polypeptide.
The word xe2x80x9ctransformxe2x80x9d is broadly used herein to refer to introduction of an exogenous polynucleotide sequence into a prokaryotic or eukaryotic cell by any means known in the art (including, for example, direct transmission of a polynucleotide sequence from a cell or virus particle as well as transmission by infective virus particles and transmission by any other known means for introducing a polynucleotide into a cell), resulting in a permanent or temporary alteration of genotype and in an immortal or non-immortal cell or cell line.
The term xe2x80x9cpolypeptidexe2x80x9d is used herein to broadly refer to amino acid sequences of any length, including peptides and proteins.
As used herein, the terms xe2x80x9cfunctionally equivalent,xe2x80x9d xe2x80x9cfunctional equivalentxe2x80x9d and the like, in reference to polypeptides, refer to fragments, analogs, derivatives and the like, which retain some or all of the antibiotic activity as the exemplified peptides.
As used herein, the terms xe2x80x9cvariantsxe2x80x9d or xe2x80x9cvariationsxe2x80x9d of genes or other nucleic acid sequences refer to nucleotide sequences which encode the same peptides or which encode functionally equivalent polypeptides.
The inventors have surprisingly discovered, by structural-functional analysis of the T4 lysozyme molecule, that the antibiotic effect of the T4 lysozyme is independent of the enzymatic muramidase activity. It was found that on heating the denatured T4 lysozyme, it no longer possessed any enzyme activity, but still exhibited the full antibiotic effect.
Furthermore, an amphipathic alpha-helix was identified in the terminal-C part of the T4 lysozyme, which surprisingly is sufficient on its own to exert an antibiotic effect. However, this peptide sequence (amino acid 143-155 in the T4 lysoyzme) retains none of the native enzymatic activity. The antibiotic effect of lysozymes can be effected by a membrane-interacting function, from the T4 lysozyme, for example, by the amphipathic alpha-helix 143-155.
Contrary to the previously known properties of the T4 lysozyme, no enzymatically active protein is required to provide the antibiotic effect.
According to the invention, polypeptides have been prepared which, although derived from lysozymes, in particular the T4 lysozyme, no longer contain the lysozyme muramidase activity.
In a preferred variant according to the invention, the compounds of the invention comprise the following sequence (SEQ ID NO: 1):
in which X represents any amino acid except glutamic acid. The compounds preferably do not comprise the full-length native sequence of T4 lysozyme.
The polypeptide of SEQ ID NO: 1, segments of this polypeptide, as well as polypeptides which have been derived from SEQ ID NO: 1 or its active subsegments by mutation or fragmentation are included as aspects of the invention.
A particularly preferred variant of SEQ ID NO: 1 comprises one or more mutations of the consensus sequence NQT (amino acids 140 to 142 in the T4 lysozyme). Mutation(s) at these positions prevent N-bound glycosylation transgenic eukaryotes. N-bound glycosylation of the polypeptide can reduce the antibiotic activity.
Preferred segments of SEQ ID NO: 1 include the amino acids 12-164 of the T4 lysozyme and subsegments thereof which exhibit some or all of the antibiotic activity of the polypeptide of SEQ ID NO: 1.
The invention also relates to segments of SEQ ID NO: 1, and to polypeptides which comprise such segments. Preferred examples include:
amino acids 126-141 WDEAAVNLAKSRWYNQO, optionally comprising one or more mutations at positions 140 and/or 141;
amino acids 143-155 PNRAKRVIFTFRT;
amino acids 126-141 WDEAAVNLAKSRWYNQ, optionally comprising one or more mutations at positions 140 and/or 141; and
amino acids 143-155 PNRAKRVIFTFRT.
In a preferred aspect the invention provides recombinant proteins, comprising at least amino acids 126-141 and/or 143-155 (amphipathic helix) of the T4 lysozyme. The invention may also comprise partially homologous sequences with the same functionality, which have been formed through amino acid exchanges and/or as a component fused onto other amino acid sequences.
A further aspect of the present invention relates to addition salts, complexes, or prodrugs such as esters of the antibiotic polypeptides, especially the pharmaceutically or agriculturally acceptable acid addition salts. The acid addition salts can be prepared in standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic, ethanedisulfonic or methanesulfonic acids. Esterification to form derivatives such as the methyl or ethyl esters can be prepared by standard procedures.
The N-terminus and C-terminus of the polypeptides can be blocked to further inhibit proteolysis by metabolic enzymes. Derivation of peptides to block the N-terminus or C-terminus is known in the art. For example, the N-terminus can be acetylated by methods known to those of ordinary skill in the art; the C-terminus can be amidated as is well known in the art.
The polypeptides may also be derivatized using various polymers known in the art to stabilize, solublize and/or facilitate transport of the polypeptides across biological membranes.
In a preferred embodiment of the invention, the antibiotic polypeptides may be presented as fusion polypeptides, the amino acid sequence of which includes one or more antibiotic polypeptides of the present invention. In various specific embodiments, two or more of the antibiotic polypeptides are linked, for example, by peptide bonds between the N-terminus of one portion and the C-terminus of another portion. In other aspects, one or more of the antibiotic polypeptides can be linked to one or more heterologous peptides or proteins to form antibiotic fusion peptides. Molecules comprising such portions linked by hydrocarbon linkages are also provided. Derivatives of the foregoing fusion proteins are also provided (e.g., branched, cyclized, N- or C-terminal chemically modified, etc.).
Analogs which have one or more amino acid substitutions forming a branched polypeptide (e.g., by substitution with an amino acid or amino acid analog having a free amino- or carboxy-side chain that forms a polypeptide bond with a sequence of one or more amino acids, including but not limited to prolines) or allowing circularization of the polypeptide (e.g., by substitution with a cysteine, or insertion of a cysteine at the amino- or carboxy-terminus or internally, to provide a sulfhydryl group for disulfide bond formation), are also provided.
Nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution, added at a terminus or inserted between existing amino acid residues of the antibiotic polypeptides of the present invention. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, xcex1-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, xcex3-Abu, xcex5-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, xcex2-alanine, fluoro-amino acids, designer amino acids such as xcex2-methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogs in general.
Furthermore, the antibiotic polypeptide may comprise D (dextrorotary) or L (levorotary) amino acid residues. The presence of D-conformation amino acids can inhibit the ability of proteases to degrade the antibiotic polypeptides of the present invention.
The antibiotic polypeptide derivatives include peptides containing, as a primary amino acid sequence, all or part of the particular antibiotic polypeptide sequence including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a polypeptide which is functionally active. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Conservative substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such antibiotic polypeptide derivatives can be made either by chemical synthesis or by recombinant production from a nucleic acid encoding the antibiotic polypeptide.
Preparation of the Antibiotic Polypeptides of the Invention
The mutated polypeptides can be obtained by amino acid exchange of the lysozyme extracted from natural sources. In a preferred aspect, the compounds of the invention comprise a mutation at position 11, replacing the glutamic acid residue with another amino acid. This mutation can readily be accomplished using standard protein-technical operations. Examples include cloning of sub-fragments, polymerase chain reaction-amplification and modification and site-directed mutagenesis of certain sections of the DNA in the native state or simultaneous introduction of modifications.
In a preferred aspect, the T4 lysozyme is extracted from a natural source, and fragments are produced by cleavage with proteases of the extracted T4 lysozyme.
The polypeptides of the invention may also be chemically synthesized. A preferred technique is the Merrifield process. See Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2154 and Merrifield (1965) Science 150:178-185. This procedure, using many of the same chemical reactions and blocking groups of classical peptide synthesis, provides a growing polypeptide chain anchored by its carboxyl terminus to a solid support, usually cross-linked polystyrene or styrenedivinylbenzene copolymer. This method conveniently simplifies the number of procedural manipulations, since removal of the excess reagents at each step is effected simply by washing the polymer.
The antibiotic polypeptides can also be synthesized using a peptide synthesizer. The composition of the synthetic polypeptides may be confirmed by amino acid analysis or sequencing, e.g., see the Edman degradation procedure; see also Creighton, 1983, Proteins, Structures and Molecular Principles, W. H. Freeman and Co., N.Y., pp. 34-49.
In a particularly preferred embodiment, the polypeptides of the invention are produced by recombinant techniques, using well-known molecular biology procedures. See S. L. Beaucage and M. H. Caruthers (1981), Tetrahedran Lett. 22:1859. For example, such methods are useful for the production of partial sequences or for insertion of specific or random mutations in the coding DNA sequences. Polynucleotides encoding the antibiotic polypeptides of the invention can be synthesized readily and are a further aspect of the present invention. These polynucleootides can be used to genetically engineer, prokaryotic or eukaryotic cells for production of the antibiotic polypeptides. Examples of suitable cells include bacteria, insect cells, viruses, plant cells, fungi, algae, yeast, mammalian, etc. The antibiotic polypeptides preferably exhibit the antibiotic effects, but preferably do not exhibit the muramidase activity, of native T4 lysozyme. Additionally, organisms can be transformed to express these recombinant nucleic acids and to thereby effect a direct antibiotic effect in vivo.
If desired, the polynucleotide of the present invention can be amplified using PCR. Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki, Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn, Henry A. Erlich, Norman Arnheim [1985] xe2x80x9cEnzymatic Amplification of Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia,xe2x80x9d Science 230:1350-1354.). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3xe2x80x2 ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5xe2x80x2 ends of the PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA fragment produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as Taq polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated. Other enzymes which can be used are known to those skilled in the art.
The various methods employed in the preparation of plasmids and transformation of host organisms are well known in the art and are described, for example, in U.S. Pat. Nos. 5,011,909 and 5,130,253. These patents are incorporated herein by reference. These procedures are also described in Maniatis, T., E. F. Fritsch, J. Sambrook (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Thus, it is within the skill of those in the genetic engineering art to extract DNA from microbial cells, perform restrictions and enzyme digestions, electrophorese DNA fragments, tail and anneal plasmid and insert DNA, ligate DNA, transform cells (e.g., E. coli, or plant cells, fungal cells, algae cells, eukaryotic cells, etc.), prepare plasmid DNA, electrophorese proteins, and sequence DNA.
In one aspect the present invention is directed to a cell transformed with a polynucleotide encoding a polypeptide comprising an antibiotic polypeptide of the invention or a functional equivalent of an antibiotic polypeptide of the invention.
Hosts which may be employed according to techniques well known in the art for the production of the polypeptides of the present invention include unicellular microorganisms such as prokaryotes, i.e., bacteria; and eukaryotes such as fungi, including yeasts, algae, protozoa, molds, and the like, as well as plant cells, both in culture or in planta, and animal cells and viruses. It is preferred that the host is one which is not destroyed by the antibiotic effect of the polypeptide being produced. Examples of suitable bacteria susceptible to transformation by the polynucleotides of the invention include members of the Enterobacteriaceae, such as strains of Escherichia coli; Salmonella; Bacillaceae, such as Bacillus subtilis; Pseudomonas; Pneumococcus; Streptococcus; Haemophilus influenzae, and yeasts such as Saccharomyces, among others.
The polynucleotide sequences of the present invention can be introduced directly into the genome of the transformable host cell or can first be incorporated into a vector which is then introduced into the host. Exemplary methods of incorporation include transduction by recombinant phage or cosmids, transfection where specially treated host bacterial cells can be caused to take up naked phage chromosomes, and transformation by calcium precipitation. These methods are well known in the art. Exemplary vectors include plasmids, cosmids, and phages.
It is well known in the art that when synthesizing a gene for improved expression in a host cell it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell. For purposes of the present invention, xe2x80x9cfrequency of preferred codon usagexe2x80x9d refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. To determine the frequency of usage of a particular codon in a gene, the number of occurrences of that codon in the gene is divided by the total number of occurrences of all codons specifying the same amino acid in the gene. Similarly, the frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage in a large number of genes expressed by the host cell. It is preferable that this analysis be limited to genes that are highly expressed by the host cell.
Thus, in one embodiment of the present invention, bacteria, algae, fungi, plants, or other cells can be genetically engineered, e.g., transformed with polynucleotides encoding the subject peptides to attain desired expression levels of the subject peptides. To provide genes having enhanced expression, the DNA sequence of the gene can be modified to comprise codons preferred by highly expressed genes to attain an A+T content in nucleotide base composition which is substantially that found in the transformed host cell. It is also preferable to form an initiation sequence optimal for the host cell, and to eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA and to avoid sequences that constitute secondary structure hairpins and RNA splice sites. For example, in synthetic genes, the codons used to specify a given amino acid can be selected with regard to the distribution frequency of codon usage employed in highly expressed genes in the host cell to specify that amino acid. As is appreciated by those skilled in the art, the distribution frequency of codon usage utilized in the synthetic gene is a determinant of the level of expression.
Assembly of the polynucleotide sequences of this invention can be performed using standard technology known in the art. For example, a gene designed for enhanced expression in a host cell can be assembled within a DNA vector from chemically synthesized oligonucleotide duplex segments. Preferably, the DNA vector or construct has an operable promoter and suitable termination signals. The polynucleotide sequence can then be introduced into a host cell and expressed by means known in the art. Preferably, the polypeptide produced upon expression of the nucleotide sequence is functionally equivalent to the purified polypeptide.
With the teachings provided herein, one skilled in the art can readily produce and use the various polypeptides and polynucleotide sequences described herein.
The polynucleotide sequences and antibiotic polypeptides useful according to the subject invention include not only the exemplified sequences but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic antibiotic activity of the peptides specifically exemplified herein.
The present invention also provides chimeric polypeptides comprising two or more antibiotic polypeptides of the present invention, or one or more polypeptides of the present invention with one or more heterologous polypeptides. The polypeptides which are combined need not themselves be antibiotic so long as the combination of portions creates a chimeric protein which is antibiotic.
Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of the antibiotic polypeptides.
Polynucleotide sequences encoding the antibiotic polypeptides of the present invention can be introduced into a wide variety of plant or animal hosts. Expression of the gene results, directly or indirectly, in the production and maintenance of the antibiotic polypeptide. The result is control or eradication of the pathogenic microbe.
In one embodiment, the gene encoding the antibiotic polypeptide is only expressed or maintained by the transformed host for a relatively short period of time, such as days or weeks, so that the transformed organism does not continue indefinitely to express the antibiotic polypeptide. For example, in microbial hosts the plasmid may be constructed without plasmid maintenance systems or with insufficient plasmid maintenance function to ensure long-term survival of the antibiotic polypeptide-encoding plasmid. Alternatively, various plasmid maintenance systems known in the art can be applied, to ensure long-term survival of the antibiotic polypeptide-producing plasmid.
A wide variety of methods are available for introducing a polynucleotide sequence encoding an antibiotic polypeptide into a microorganism host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and include, for example, the methods described in U.S. Pat. No. 5,135,867, which is incorporated herein by reference.
In a preferred aspect of the invention, the polypeptide of SEQ ID NO: 1 serves as a xe2x80x9ccarrier proteinxe2x80x9d for other antibiotically active peptides, preferably amphiphatic helices, resulting in the improved stabilization of the antibiotically active peptides. For example, the polypeptide sequence 143-155 (PNRAKRVIFTFRT) can be substituted by another natural polypeptide sequence or one developed by rational design.
Methods for Using the Polypeptides of the Invention
The polypeptides of the invention exhibit some or all of the antibiotic properties of T4 lysozyme. The polypeptides of the invention are preferably at least as effective at killing and/or controlling the growth of microbes as T4 lysozyme, most preferably the polypeptides are more effective than T4 lysozyme. However, it will be appreciated by those of skill in the art that polypeptides which are less potent than T4 lysozyme are also useful. The antibiotic effectiveness of the polypeptides of the invention is illustrated by their effect on survival rates of Escherichia coli cells in Table 1.
The table shows the lack of a significant difference between the bactericidal activity of T4 lysozyme and heat denatured T4 lysozyme. Heat-denatured T4 lysozyme, which no longer dissolves 100% under the selected conditions, shows no enzyme activity (muramidase). The mutant M6K, in which the 6th amino acid (methionine) has been substituted by lysine, exhibits higher bactericidal activity, since a hydrophobic amino acid has been replaced by a polar amino acid. The peptide A4, which includes amino needs 143-155 of the T4 lysozyme (in SEQ ID NO: 1), also shows no enzymatic muramidase activity, but has a significantly higher bactericidal activity.
Table 2 illustrates the fungicidal activity of the claimed invention using germinative zoospores of Phytophthora nicotianae.
Fungus tests: Relative lengths of germ tubes
The polypeptides of the invention are useful as an additive to food (human or animal) or for other substances, to inhibit growth of microorganisms. The polypeptides may be applied to food indirectly by gerietically a food organism to express the polypeptides. The genetically modified plant is useful to prevent its own spoilage and/or as an additive to prevent the spoilage of other food components.
It will be appreciated by those of skill in the art that the antibiotic properties of the claimed polypeptides make them particularly suitable for use in therapeutic methods targeting such organisms.
A further field of application is in cancer therapy, since cancer cells have a similar cell membrane structure to bacterial cells, unlike the membranes of healthy eukaryotic cells, which have a quite different structure. In one embodiment, the polypeptides of the invention are screened for anti-cancer activity by a method comprising: (1) contacting malignant cells with the therapeutic polypeptide of the invention; (2) measuring the survival or proliferation of malignant cells; and (3) comparing the survival or proliferation of the cells contacted with the therapeutic polypeptide of the invention with the survival or proliferation of cells not so contacted (e.g., cells contacted with a control). A lower level of survival or proliferation in the contacted cells indicates that the preparation has anti-cancer activity. Examples of suitable cells are those which are derived from or display characteristics associated with a malignant disorder.
Many assays standard in the art can be used to assess whether a pre-neoplastic state, neoplastic state, or a transformed or malignant phenotype, is present. For example, characteristics associated with a transformed phenotype (a set of in vitro characteristics associated with a tumorigenic ability in vivo) include a more rounded cell morphology, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, release of proteases such as plasminogen activator, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton surface protein, etc.
Because of the ubiquitous occurrence of diseases caused by microorganisms, the polypeptides of the invention are numerous are useful in numerous fields of application. These uses range from human and veterinary medicine to resistance cultivation in plants and preventative use as bactericidal and fungicidal additives in foods. Furthermore, the properties of the polypeptides described herein are suitable for use in other fields of application, not connected with infection through microorganisms, e.g. in cancer therapy.
Another aspect of the present invention pertains to a method of controlling microbes, particularly pathogenic microbes, comprising applying to said microbes or applying to a microbe-inhabited locus an effective amount of an antibiotic polypeptide of the present invention.
The smaller partial sequences, which are preferably derived from the C-terminal part of T4 lysozyme, are particularly advantageous. For example, smaller peptides exhibit better penetration into the tissue, reduced allergenicity, higher antibiotic activity, and other advantages.
Compositions of the Invention
Amounts and locations for application of the antibiotic polypeptides and compositions of the present invention are generally determined by the target microbe and the site where the microbe is to be contacted with the antibiotic polypeptide (e.g., human or animal in vivo use, application to the surface of a plant, stored food, etc.), and the physical and functional (e.g., potency) characteristics of the specific polypeptide. Thus, it will be appreciated that a wide variety of compositions are possible within the scope of the invention.
Non-Pharmaceutical Antibiotic Compositions and Methods of Use
A wide variety of antibiotic compositions for non-pharmaceutical use are possible within the broad scope of the present invention. The antibiotic polypeptides may be encapsulated, included in a granular form, solubilized in water or other appropriate solvent, powdered, and included in any appropriate formulation for direct application to the situs of the target microbe.
The antibiotic polypeptides and their functional equivalents may be used either alone or in combination with other active or inactive substances and may be applied by any method known in the art including, for example, spraying, pouring, dipping, in the form of concentrated liquids, solutions, suspensions, sprays, powders, pellets and the like, formulated to deliver an antibiotically effective concentration of the antibiotic polypeptide. The antibiotic formulations may be applied in an antibiotically effective amount to an area of microbe infestation or an area susceptible to such infestation, e.g., a container of food, the surface of a plant, the soil in which a plant is growing or is intended to be grown, an animal, clothing, skin, and the like.
In all formulations described herein, materials which can lead to reduction in the antibiotic effectiveness of the polypeptides should be avoided but may be employed in appropriate circumstances where such materials do not entirely eliminate the antibiotic properties of the antibiotic polypeptide.
The antibiotic compositions may also include various antibiotically acceptable adjuvants known in the art. The term xe2x80x9cadjuvantxe2x80x9d is used herein to mean a substance added to a composition to aid the operation of the main ingredient. The adjuvants are antibiotically acceptable in that they do not completely diminish the antibiotic properties of the antibiotic polypeptide. Spray adjuvants are commonly employed in the application of agricultural chemicals. An effective spray adjuvant may be formulated to contain one or more surfactants, solvents or co-solvents.
Formulated antibiotic polypeptides can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other antibiotic additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may also include rheological agents, surfactants, emulsifiers, dispersants, or polymers.
The antibiotic polypeptides according to the instant invention can be utilized, in the form of the usual compositions or compositions with conventional inert (e.g., plant and/or animal compatible or herbicidally mammacidally inert) diluents or extenders, i.e. diluents, carriers or extenders of the type usable in conventional bactericidal or fungicidal compositions or compositions, e.g. conventional dispersible carrier vehicles such as gases, solutions, emulsions, suspensions, emulsifiable concentrates, spray powders, pastes, gels, soluble powders, dusting agents, granules, etc. These are prepared, for example, by extending the antibiotic polypeptides with conventional dispersible liquid diluent carriers and/or dispersible solid carriers optionally with the use of carrier vehicle assistants, e.g. conventional surface-active agents, including emulsifying agents and/or dispersing agents, whereby, for example, in the case where water is used as diluent, organic solvents may be added as auxiliary solvents. The following may be chiefly considered for use as conventional carrier vehicles for this purpose: aerosol propellants which are gaseous at normal temperatures and pressures, such as freon; inert dispersible liquid diluent carriers, including inert organic solvents, such as aromatic hydrocarbons (e.g. benzene, toluene, xylene, alkyl naphthalenes, etc.); halogenated, especially chlorinated, aromatic hydrocarbons (e.g. chlorobenzenes, etc.); cycloalkanes (e.g. cyclohexane, etc.); paraffins (e.g. petroleum or mineral oil fractions); chlorinated aliphatic hydrocarbons (e.g. methylene chloride, chloroethylenes, etc.); alcohols (e.g. methanol, ethanol, propanol, butanol, glycol, etc.), as well as ethers and esters thereof (e.g. glycol monomethyl ether, etc.), amines (e.g. ethanolamine, etc.), amides (e.g. dimethyl formamide, etc.), sulfoxides (e.g. dimethyl sulfoxide, etc.), acetonitrile, ketones (e.g. acetone, methyl ethyl ketone, methyl isobutyl ketone, cyclohexanone, etc.), and/or water; as well as inert dispersible finely divided solid carriers, such as ground natural minerals (e.g. kaolins, clays, alumina, silica, chalk, i.e. calcium carbonate, talc, attapulgite, montmorillonite, kieselguhr, etc.) and ground synthetic minerals (e.g. highly dispersed silicic acid, silicates, e.g. alkali silicates, etc.); whereas the following may be chiefly considered for use as conventional carrier vehicle assistants, e.g. surface-active agents, for this purpose: emulsifying agents, such as non-ionic and/or anionic emulsifying agents (e.g. polyethylene oxide esters of fatty acids, polyethylene oxide ethers of fatty alcohols, alkyl sulfates, alkyl sulfonates, aryl sulfonates, etc., and especially alkyl arylpolyglycol ethers, magnesium stearate, sodium oleate, etc.); and/or dispersing agents, such as lignin, sulfite waste liquors, methyl cellulose, etc. The antibiotic polypeptides may also be encapsulated in a liposomal composition (Belles et al in Pesticide Biochem. Physiol. 32, 1-10 (1988)). Esters, such as succinate ester or citrate esters, can be employed to control the buoyancy of the composition.
Another mechanism by which the antibiotic polypeptides can be made available to target microbes is through genetically engineering of organisms susceptible to infection by such microbes.
The antibiotic polypeptides of the present invention may be beneficially administered in conjunction with other active ingredients (sequentially or simultaneously), including pesticides, as well as various acaricides, algicides, antioxidants, anti-preservatives, bactericides, biocides, catalysts, chemical reactants, disinfectants, drugs, fermentation agents, fertility inhibitors, fertility promoters, fertilizers, food supplements, foods, fungicides, germicides, growth-regulating agents, herbicides, insecticides, microorganism attenuators, nematocides, plant growth inhibitors, plant growth promoters, preservatives, rodenticides, sex sterilants, and sterilization agents, and/or other agents that benefit the environment of use. For simultaneous administration, the active ingredients may be formulated into a unitary application form comprising at least one antibiotic polypeptide and one or more of such active ingredients.
Pharmaceutical Compositions and Methods of Use
The invention provides methods of treatment and prevention by administration to a subject in need of such treatment of a therapeutically or prophylactically effective amount of one or more antibiotic polypeptides of the invention. The subject is preferably an animal, including, but not limited to, animals such as monkeys, cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
Various delivery systems are known and can be used to administer antibiotic polypeptides of the invention. For example, suitable systems include: encapsulation in liposomes, microparticles and/or microcapsules; recombinant cells capable of expressing the antibiotic polypeptide; receptor-mediated endocytosis; plasmids encoding one or more antibiotic polypeptides; viral vector delivery systems, etc. The antibiotic polypeptides can be delivered in a vesicle, in particular a liposome.
Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and/oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection. An intraventricular catheter may be used to facilitate intraventricular injection, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment. For example, local administration may be achieved by topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
In yet another embodiment, the antibiotic polypeptide can be delivered in a controlled release system. A pump may be used as needed. Polymeric materials may also be employed in a controlled release system, according to methods known in the art. In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose.
In a specific embodiment a nucleic acid encoding one or more antibiotic polypeptides of the invention is administered by gene therapy methods as described herein, or as otherwise known in the art.
The pharmaceutical compositions comprise a therapeutically effective amount of one or more antibiotic polypeptides of the invention, and a pharmaceutically acceptable carrier.
The term xe2x80x9ccarrierxe2x80x9d refers to a diluent, adjuvant, excipient, or vehicle with which the antibiotic polypeptide is administered to a subject. Examples of suitable pharmaceutical carriers are described in xe2x80x9cRemington""s Pharmaceutical Sciencesxe2x80x9d by E. W. Martin. Such compositions will contain a therapeutically effective amount of one or more antibiotic polypeptides of the invention, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulation should suit the mode of administration. In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. The compositions may also be formulated for veterinary use.
Examples of suitable pharmaceutical carriers include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The antibiotic polypeptides of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
The amount of the antibiotic polypeptide(s) and/or functional equivalent(s) of the invention that will be effective in the treatment of a particular disorder or condition depends on various factors and can readily be determined by one of skill in the art using standard clinical techniques with reference to the instant disclosure. For example, dosage amounts will depend on the nature of the disorder or condition. In vivo and/or in vitro assays may optionally be employed to help predict optimal dosage ranges. Effective doses may also be extrapolated from dose-response curves derived from vitro and in vivo experiments. Ranges will vary depending on the route of administration, the seriousness of the disease or disorder, the size of the subject, and other factors known in the art.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. The kits preferably include instructions for administration of the composition. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Lysozyme-analogous polypeptides with an anti-microbial effect, their production and use

MPB Cologne GmbH Molecular Plant and Protein Biotechnology


A steering shaft double joint is provided for motor vehicles where the shaft ends are connected non-rotatably in the joint. Each of the shaft ends is movably mounted in a housing connecting two joints with the shaft ends being connected by a ball joint so that the ball is rotatable around a midpoint in a receptacle of a shaft end and is slightly movably mounted in a direction of the shaft axis of the other shaft end. The ball is spring mounted in the receptacle and the receptacle contains and surrounds a bushing with elastic material located between the receptacle and bushing.

The invention relates to a steering shaft double joint for motor vehicles with shaft ends each connected nonrotatably in a joint, each of these ends being in a joint and being movably mounted in a housing connecting the two joints; the two shaft ends being connected with one another between the two joints by a ball joint so that the ball is rotatable around its midpoint in a receptacle of the other shaft end and is slidably movably mounted in the direction of the shaft axis of the other shaft end.
A known double-jointed arrangement for example is the double articulated joint with a ball joint located between the two joint crosses. In these known arrangements of double universal joints, two joint crosses are connected movably by one articulated joint axis with one fork at each of the two shaft ends and by the other joint axis movably to a connecting housing. The middle centering is produced by an articulated connection of the two shaft ends within the connecting housing with a metallic joint ball at one shaft end and a cylindrical, likewise metallic, receptacle at the other shaft end in which the joint ball engages. The connecting housing forms a cavity in the interior that creates a space for the movement of the centering jointed connection and is adjusted in size to the maximum angular deflection of the two shaft ends relative to the stretched axis. The ends or pins of the two joint crosses are mounted suitably for easy accessibility, for example with roller bearings which are located in bores of the fork arms or in the connecting housing. The universal joint mounting with its eight bearing points as well as the central ball bearing require high precision in order to ensure ease of movement of the double universal joint. A slight displacement of the two shaft axes within the tilting plane for example can result in jamming in certain positions which can adversely affect ease of movement considerably. In addition, this also leads to undesired wear phenomena. In order to limit such disadvantages, the bearings must be made suitably precisely which causes high expense in manufacturing. Another known way to reduce the problem consists in providing rubber-elastic material as an insert for the pins of the universal joints so that the bearing can adjust itself through the spring mobility obtained in this fashion relative to manufacturing tolerances and simultaneously can have a vibration-damping effect. A rubber-elastic bearing with two to all eight universal joint pins is limiting if small sizes are required and is expensive and delicate to manufacture.
The goal of the present invention is to propose an steering shaft double joint in which the disadvantages of the prior art are eliminated. In particular, the goal consists in making a double joint which, in addition to ease of mobility, can be installed simply and economically and is insensitive to manufacturing tolerances.
This goal is achieved according to the invention as described below.
This goal is achieved according to the invention by an arrangement of the above-noted type having at least one of the following features:
(i) the ball is spring mounted in the receptacle; and
(ii) stop structure for the ball and receptacle are provided on the inside wall of the housing.
According to the invention, the receptacle for the ball which connects the two ends of the shaft movably is made spring-like or elastic. This permits a very inexpensive design since the compensation of tolerances by the sprung bearing is required only once.
The ball on one shaft end is preferably mounted, more preferably rotatably disposed, in the receptacle of the other shaft end so that the receptacle for the ball is designed as a sliding bushing and this sliding bushing surrounds the ball cylindrically. The bushing in turn is elastically mounted, with an elastic material placed between the bushing and the receptacle, advantageously a rubber-elastic material with a shore hardness in the range of 30 to 80 shore. Good elastic movements of the bushing are produced by the fact that the shore hardness is chosen in the range from 30 to 60 shore.
The elastic material between the receptacle at the end of the shaft, which is pot-shaped for example, and the sliding bushing which advantageously has a circularly round cross section can be shaped to produce favorable elastic deflection. Preferably, the outside wall of the cylindrical part is made wavy.
The sliding bushing in which the ball slides and turns is made of a sliding bearing material, with such a bushing also having a sliding coating. However, bushings with a sintered metal with a supporting sleeve are especially suitable.
The bushing itself should be made so that it together with the ball produces a zero-play bearing. This is produced by the fact that the sliding bushing abuts the ball in a spring-elastic fashion with a certain amount of pretensioning and therefore with zero play. Slitting the outside wall of the sliding bushing enables the sliding bushing to breathe in the radial direction. In this way, both radial tolerances in the diameter of the ball for example can be adjusted for and differences in tolerance of the shaft axes can be compensated by the elastic mounting of the bushing. In addition, the wear rates are compensated by the elastic adjustment.
Another advantageous embodiment consists in the fact that the plastic guide is mounted on the joint ball, so that the plastic guide slides in the sliding bushing or the cylindrical receptacle itself. In this case, it is even possible to make the bushing or the receptacle without special bearing material. The bearing bushing can even be eliminated and the plastic part that adheres to the ball can then slide directly guided in the receiving sleeve. A spring-mounted bushing can then be eliminated.
In double universal joint arrangements, especially of the type mentioned earlier, it is also important that when the joint is assembled a guide is provided which brings the ball joint together in the intended way and also that in extreme end positions of the joint which do not meet normal operating conditions, a specific stop is provided for safety reasons. By appropriately designing the connecting housing in the inner area with corresponding rotating stop surfaces, this can be defined. It is important to note that in particular this receptacle that holds the spring-elastically mounted bushing in the extreme position initially abuts the stop and then the joint ball on a second contact surface. In this way, especially in a non-installed state, assurance is provided that the stop is damped in the extreme position. This type of stop definition is especially suited for this kind of spring-elastic ball joint bearing according to the invention, but can also be used in other double universal joints without spring-elastic ball joint mounting.
For double jointed arrangements, other designs are suitable as cross joints, if for example especially high ease of movement and uniform movement are required, the double joints are preferably made with a universal joint, also called a homokinetic joint, especially of the synchro-fixed joint design. Between the two joints connected with a housing, the ball joint with the spring-elastic bearing is again located so that the shaft ends are held by the two joints so that they bend. Synchro-fixed bearings are made for example as Lxc3x6bro joints by the company Lxc3x6hr and Bromkamp GmbH, DE 6050 Offenbach.
The invention will now be described in greater detail with reference to embodiments with schematic figures.
FIG. 1 shows schematically and in section a design for a steering shaft double universal joint according to the invention;
FIG. 2 shows schematically and in section another design according to the invention of a steering shaft double articulated joint offset by 90xc2x0 and with stop means for limiting the deflection;
FIG. 3a shows a top view of a bearing bushing with shaft-shaped rubber-elastic material;
FIG. 3b is a side view of the bushing with elastic material according to FIG. 3a;
FIG. 4 shows schematically and in section a version of a design according to the invention for a steering shaft double articulated joint with a plastic sliding guide;
FIG. 5a shows schematically and in cross section a plastic sliding guide in the non-installed state;
FIG. 5b shows schematically and in lengthwise section a plastic sliding guide in an installed state;
FIG. 6a shows schematically and in cross section a plastic sliding guide in the installed state;
FIG. 6b shows schematically and in lengthwise section a plastic sliding guide in the installed state.
FIG. 7 shows the assembled ball joint of FIGS. 1 and 2 in greater detail.

Double joint for steering axles in automobiles


Snoring and obstructive sleep apnea are relieved in most instances by administering an effective dose of a muscle relaxant that acts at the spinal or supra-spinal level. A presently preferred drug is orphenadrine citrate.

This invention relates to the inhibition or prevention of snoring and the obstructive sleep apnea that often accompanies snoring.
One of the most misunderstood ailments of human beings is the noisy breathing pattern that occurs in some persons during sleep. Afflicted persons may create so much sonic disturbance that they prevent sleep in bed partners, roommates, and sometimes persons several rooms distant. Throughout recorded history, snorers have been ridiculed, harassed, and subjected to the mounting hostility of other household members. As might be expected, an almost endless variety of well-meaning or defensive attempts have been made to control snoring; see, e.g., the more than 300 devices that have received U.S. patents for the control of snoring. Most such devices function by subjecting the unfortunate subject to unpleasant mechanical or electrical stimuli as soon as snoring commences and are successful only to the degree that the snorer is kept from sleeping.
As has been pointed out in such recent papers as that of David N. F. Fairbanks, M.D. ("Snoring: Not Funny--Not Hopeless", Hospital Medicine, March, 1984), the noise of snoring comes from vibrations of soft tissues in the collapsible part of the upper airway, involving the soft palate, uvula, tonsils and tonsillar pillars, base of the tongue, and pharyngeal muscles and mucosa. The most recent medical literature expresses the opinion that, for most persons, snoring is caused by the muscle relaxation that occurs during sleep. It is postulated that, as the muscles of the mouth, nose, and throat relax, the negative pressure that occurs during inspiration encourages the tongue to fall backward into the airway and vibrate against a relaxed and floppy soft palate and uvula. At the same time, the lateral pharyngeal structures are drawn inward, further constricting the airway and increasing the speed of air flow past the vibrating structures. Obesity, which further constricts the airway, increases the work of breathing and compounds the problem. Temporary swelling caused by hay fever, inflamed sinuses, etc., also narrow the air passages and may bring on snoring in a person normally free from that affliction; however, the use of antihistamines promotes drying of the mucous membranes and further promotes vibration of the relaxed structures. Smoking, two byproducts of which are carbon monoxide and formaldehyde, tends to dry or irritate the air passages, induce hypoxemia, and even put the swallowing muscles into spasm, tending to stimulate snoring. When an afflicted person uses central nervous system depressants such as alcoholic beverages or tranquilizers before retiring, it has been found that the tendency to snore is exacerbated.
Socially unattractive as snoring may be, the frequently accompanying sleep apnea is actually dangerous. In some cases, the relaxed tissues of the airway may so effectively seal off the passage to the lungs as to completely prevent inspiration. Persons suffering from this problem may actually stop breathing 30 to 300 times per night for periods of ten seconds to two minutes (three minutes may be fatal), consequently spending as much as half their sleep time with abnormally low blood oxygen levels. Such persons resume normal breathing, albeit briefly, when they wake into a lighter sleep stage, causing the relaxed muscles to tense sufficiently to relieve the obstruction. As will be appreciated, persons with obstructive sleep apnea spend an insufficient portion of their nighttime hours in the deep sleep stages that are essential for good rest, awakening unrefreshed and feeling sleepy much of the day. Cardiac arrhythmias may occur during apneic episodes, possibly leading to death in sleep in some instances.
In recent years, the most widely heralded way of curing the related problems of snoring and obstructive sleep apnea has been the surgical removal of what has been considered excess tissue in the air passageway by means of a palatopharyngoplasty, an operation that has been characterized as a "facelift of the throat." Although effective in many instances, the operation is painful, expensive, and fraught with the dangers accompanying any operation in the blood-rich throat area. Prior to the present invention, it is believed, there has never been a simple, safe, and inexpensive way to control snoring.
The present invention provides a simple, safe, and inexpensive way of medically curing snoring in the majority of cases. No invasive surgery is required, and the snorer is not subjected to the discomfort of electrical, mechanical, or psychological treatments.
In accordance with the invention, the snorer is treated by administering a conventional amount of certain muscle relaxants. In view of the fact that snoring is almost universally believed to be caused by relaxed muscles, such a treatment would seem to be clearly contraindicated. Surprisingly, however, it has been found that the expected worsening does not occur. In attempting to understand why the treatment is effective, the inventor has concluded that the conventionally accepted muscle relaxation theory of snoring has provided an incomplete explanation of what actually takes place. The inventor now hypothesizes that the cause of snoring is closely related to the balanced muscular structure of the human body, where every muscle or group of muscles is opposed by another muscle or group of muscles; such opposing muscles are known as "agonists" and "antagonists." In every such situation, the agonist tends to be stronger than the antagonist, or vice versa, depending on which of the two is normally exercised more. Muscles that work more strenuously tend to grow larger (hypertrophy) and become significantly stronger than those that work less. Since muscles perform their physiological function by contracting, the stronger muscle contracts more than the weaker during normal relaxation, in effect going into mild spasm. When a muscle relaxant is administered, however, the stronger muscle is relaxed to a state more nearly approximating that of the weaker muscle, tending to restore the normal balance, returning the structures on which they act to a neutral resting position.
Applying the discussion of the preceding paragraph to the problem of snoring, it is noted that the muscles involved are those used normally in swallowing. As one swallows, the soft palate is pulled back against the nasopharynx by the uppermost fibers of the superior pharyngeal constrictor. The levator palatini and the tensor palatini elevate the soft palate against the roof of the nasopharynx. The tongue, a bolus of food, or a volume of liquid pushing backward and upward also forces the soft palate backward and upward against the nasopharynx, thus sealing it off from the throat and preventing food or liquids from "going up the nose" during swallowing. Other pharyngeal constrictor muscles narrow the throat, forcing the food or liquid down the esophagus. Each of the muscles just discussed is opposed by other muscles, which do not ordinarily receive much exercise. Thus, for example, the muscles that move the soft palate downward and forward to open the nasopharynx to the oropharynx do not ordinarily do much work, except, perhaps during singing or yawning. Similarly, the genioglossus (the muscle that holds the tongue forward) is notoriously weak, receiving exercise essentially only during the socially unacceptable practice of " sticking out the tongue." During the relaxation accompanying sleep, the stronger swallowing muscles relax less than the weaker opposing muscles, encouraging the soft palate to move backward and upward, pulling the tongue backward, and constricting the throat. As a result, the entire airway is narrowed, the inspired air moves faster, and the relaxed tissues tend to vibrate; i.e., snoring occurs. If the nasopharynx becomes completely sealed off, air is inhaled through the mouth, and the narrowed oropharynx leads to a secondary form of snoring.
Administering a muscle relaxant tends to restore balance to the nasopharynx and oropharynx. No longer do the stronger muscles overpower the weaker ones. The tendency of the soft palate to move upward and backward is canceled out, the tendency of the tongue to fall back in the throat is counteracted, and constriction of the throat muscles is minimized. As a result, breathing occurs through airways maintained in substantially the same configuration during sleep as when awake, and snoring is eliminated. Equally important, obstructive sleep apnea is either drastically reduced or eliminated altogether.
Logical as the treatment taught by the invention might seem in retrospect, physicians and hosts of inventors have unsuccessfully sought an answer to the problem for centuries. The extraordinarily simple means of solving the problem--the use of muscle relaxants--has been available for decades, but no one has previously recognized it.
It is important to note that only those muscle relaxants that function at the spinal or supra-spinal level, i.e., above the neuro-muscular synapse, should be used in the treatment of snoring or obstructive sleep apnea. Muscle relaxants that act intrinsically in the muscle contraction per se or function as central nervous system depressants should be avoided because of their profound effect on the respiratory muscles, and consequently their potential jeopardizing of the respiratory function itself. Similarly, muscle relaxants known to be habit-forming should be avoided.

Method of inhibiting snoring and obstructive sleep apnea


A process for laminating an ink jet print having the steps of: a) providing an ink jet print having a support having thereon an ink jet image; b) contacting the imaged surface of the ink jet print with a transfer laminating element to form a composite, the transfer laminating element having a flexible, polymeric support having thereon a protection layer of a water-dispersible, hydrophobic polyester resin having the following general formula: In-P-Am wherein I is an ionic group; n is an integer from 1-3; P is a polyester backbone; A is an aliphatic group having a straight or branched chain fatty acid or triglyceride thereof halving from about 6 to about 24 carbon atoms; and m is an integer from 3-8; c) applying heat and pressure to the composite to transfer the layer on top of the ink jet image; d) allowing the composite to cool; and e) peeling the flexible, polymeric support of the transfer laminating element from the composite to form the laminated ink jet print.

This invention relates to an process for laminating ink jet prints with a transferable protection layer.
In a typical ink jet recording or printing system, ink droplets are ejected from a nozzle at high speed towards a recording element or medium to produce an image on the medium. The ink droplets, or recording liquid, generally comprise a recording agent, such as a dye or pigment, and a large amount of solvent. The solvent, or carrier liquid, typically is made up of water, an organic material such as a monohydric alcohol, a polyhydric alcohol or mixtures thereof.
An ink jet recording element typically comprises a support having on at least one surface thereof a base layer for absorbing fluid and an ink-receiving or image-forming layer, and includes those intended for reflection viewing, which have an opaque support, and those intended for viewing by transmitted light, which have a transparent support.
Ink jet prints prepared by printing onto ink jet recording elements are subject to environmental degradation such as water smearing and light fade. For example, since ink jet dyes are water-soluble, they can migrate from their location in the ink-receiving layer when water comes in contact with the recording element after imaging.
To reduce the vulnerability of prints to degradation and to enhance gloss, ink jet prints are often laminated. Typically, such conventional lamination is a process whereby a continuous polymeric film bearing an adhesive is brought into contact with the surface of the print. Heat and/or pressure is then used to affix the continuous polymeric film to the print surface. The continuous polymeric film then serves as a barrier layer that is impermeable to water and further acts to diminish the fading of the print image caused by light.
However, there is a problem with prior art laminating films since they are typically supplied in roll format and must be cut, or less desirably torn, to separate the laminated print from the continuous roll of laminating film. A requirement to cut adds expense to a laminator design that is required to run in a continuous mode.
U.S. Pat. No. 5,662,976 discloses an assembly for creating laminated cards which comprises a sheet of card stock with a release coating and a sheet of laminating film adhering to the release coating. A card form is cut into the sheet of card stock, and a lamination strip, which is sufficiently large to fold over so as to laminate both surfaces of the card, is cut into the lamination sheet. After printing, the card and the lamination strip are removed, and the lamination strip folded over to laminate the card. However, there is a problem with this laminating film in that expensive cutting and perforating steps are required to prepare the laminated card.
U.S. Pat. No. 5,387,573 discloses a dye-donor element for thermal dye transfer comprising a support and a transferable protection layer wherein the transferable protection layer is less than about 1 xcexc thick and contains particles in an amount of up to about 75% of the transferable protection layer. However, there is no disclosure in this patent that the protection layer can be used with ink jet prints.
U.S. Pat. No. 6,087,051 relates to an ink jet recording element containing a protective overcoat layer of an aqueous polyurethane resin or an aqueous polyacryl resin. In addition, there are comparison examples in that patent which use a polyester resin, and the aqueous polyurethane resin and polyacryl resins are said to have advantages over the polyester resin. However, there are problems using a polyurethane resin or an aqueous polyacryl resin in that these resins have to be synthesized from virgin raw materials and the resins cannot be recycled.
It is an object of the invention to provide a process for laminating ink jet prints wherein the protection layer is sufficiently thick to protect ink jet images from degradation by water, and yet can be employed without resort to expensive cutting steps. It is still another object to provide a process that allows for the direct visual distinction between laminated and unlaminated regions of the print.
These and other objects are provided by the present invention which comprises a process for laminating an ink jet print comprising:
a) providing an ink jet print comprising a support having thereon an ink jet image;
b) contacting the imaged surface of the ink jet print with a transfer laminating element to form a composite, the transfer laminating element comprising a flexible, polymeric support having thereon a protection layer of a water-dispersible, hydrophobic polyester resin having the following general formula:
In-P-Am
xe2x80x83wherein
I is an ionic group;
n is an integer from 1-3;
P is a polyester backbone;
A is an aliphatic group comprising a straight or branched chain fatty acid or triglyceride thereof having from about 6 to about 24 carbon atoms, and
m is an integer from 3-8;
c) applying heat and pressure to the composite to transfer the layer on top of the ink jet image;
d) allowing the composite to cool; and
e) peeling the flexible, polymeric support of the transfer laminating element from the composite to form the laminated ink jet print.
In using the process of the invention, the durability of an ink jet image is improved using the transfer layer described above.
The transfer laminating element employed in the invention comprises a flexible, polymeric support having thereon a protection layer of a water-dispersible, hydrophobic polyester resin as described above.
The flexible, polymeric support used in the invention can be, for example, various plastics including a polyester-type resin such as poly(ethylene terephthalate), poly(ethylene naphthalate), polycarbonate resins, polystyrene resins, polysulfone resins, methacrylic resins, cellophane, acetate plastics, cellulose diacetate, cellulose triacetate, vinyl chloride resins and polyester diacetate. The thickness of the support may be, for example, from about 6 to about 500 xcexcm, preferably from about 6 to about 50 xcexcm. In a preferred embodiment, the support is a transparent poly(ethylene terephthalate) film.
The water-dispersible, hydrophobic polyester resin in the protection layer has the formula as illustrated above. The ionic groups I in the above formula which provide the polymer with water-dispersibility are typically derived from a carboxylic acid group which is introduced into the resin by polyacid monomers such as trimellitic anhydride, trimellitic acid, or maleic anhydride or sulfonate groups which come from monomers such as dimethyl 5-sulfoisophthalate, dimethyl 5-sulfo, 1,3-benzenedicarboxylate, sulfoisophthalate ethylene glycol, dihydroxyethyl-5-sulfo-1,3-benzenedicarboxylate, or from sulfonated alkenically unsaturated end groups as described in U.S. Pat. No. 5,281,630, the disclosure of which is hereby incorporated by reference. The weight percent of ionic monomers in the resin is from 1% to 20%, but 1% to 10% is preferred.
The backbone P of the polymer in the above formula is composed of polyester groups. It can be any linear or branched polyester made using polyacids and polyalcohols. The weight percent of the polyester backbone ingredients range from 30-80% of the whole resin, with the most preferred being 50-60% by weight. Examples of aromatic dicarboxylic acids useful in the backbone polyester polymer, P, employed in the invention include, but are not limited to terephthalic, isophthalic, phthalic, and 2,6-naphthoic, succinic, glutaric, adipic, 1,4-cyclohexane dicarboxylic, maleic, fumaric, and azelaic. The polyalcohol component of the polyester can be virtually any dihydroxy functional compound. Aliphatic and alicyclic glycols would be the most useful. Useful glycols include, but are not limited to, ethylene glycol, 1,3-propylene glycol, 1,2-propylene glycol, dipropylene glycol, tripropylene glycol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, cyclohexanedimethanol, diethylene glycol, and triethylene glycol.
The backbone polyester consisting of any combination of the above polyacids and glycols may further directly include or incorporate by transesterification a multifunctional polyol selected from, but not limited to, glycerol, trimethylolpropane, erythritol, pentaerythritol, trimethylolethane, or a monosaccharide.
As noted above, A in the above formula is an aliphatic group comprising a straight or branched chain fatty acid or triglyceride thereof having from about 6 to about 24 carbon atoms, such as stearic, oleic, palmitic, lauric, linoleic, linolenic, behenic acid, or their mixtures. These can come from hydrogenated or unhydrogenated animal or vegetable oil, such as beef tallow, lard, corn oil, or soy bean oil. The weight percent of the aliphatic moiety can be 10-60%, with 20-40% by weight being the preferred amount.
In a preferred embodiment of the invention, the water-dispersible, hydrophobic polyester resin employed comprises a reaction product of 30-70% by weight of a poly(ethylene terephthalate) condensation polymer, 5-40% by weight of a hydroxy functional compound having at least two hydroxyl groups, 1-20% by weight of a carboxy functional compound having at least two carboxyl groups and 10-60% by weight of a C6-C24 straight chain or branched fatty acid or triglyceride. The resin is further characterized in that the hydroxy functional compound is present at 1-3 times the equivalents of the hydrophobic moiety. The preparation of such hydrophobic polyester resins is described in detail in U.S. Pat. No. 5,958,601, the disclosure of which is hereby incorporated by reference. In another preferred embodiment, the water-dispersible, hydrophobic polyester resin comprises water-dispersed transesterified polyester, e.g., poly(ethylene terephthalate) transesterified in the presence of stearic acid and trimellitic acid, or oleic acid and trimellitic acid.
In another preferred embodiment of the invention, the water-dispersible, hydrophobic polyester as described above is physically mixed with a thermoplastic or thermosetting polymer. The thermoplastic or thermosetting polymer lends added hydrophobicity to the layer, as well as enhanced coating flexibility and serves as a diluent to the polyester component to minimize cross-linking which would deleteriously alter coating properties.
Examples of such thermoplastic or thermosetting polymers useful in the invention include, but are not limited to, carboxylated styrene butadiene, styrene/acrylate or methacrylate ester compositions containing acrylic or methacrylic acids, hydrolyzed styrene maleic anhydride copolymers, styrene maleic acid salt copolymers, styrene maleic ester copolymers, styrene (meth)acrylate copolymers, styrene (meth)acrylate ester copolymers, styrene acrylate ester acrylonitrile terpolymers, acrylonitrile (meth)acrylate salt copolymers, polycarbonate-based polyurethanes, polyester-based polyurethanes, cellulose polymers, such as methyl cellulose and cellulose acetate butyrate, polyesters, polyamides, polyacetals, epoxy polymers, phenoxy polymers, etc. In a preferred embodiment, the water-dispersible, hydrophobic polyester resin and thermoplastic or thermosetting polymer is present in a ratio from about 1:4 to 4:1.
A preferred aqueous dispersion of a mixture of carboxylated styrene butadiene copolymer and a hydrophobic polyester of the composition generally described above is commercially available as EvCote(copyright) PWRHS-37 from EvCo Research Incorporated, Atlanta, Ga., USA.
In another preferred embodiment, the protection layer may also contain a synthetic or natural wax, such as an aqueous dispersion of high density polyethylene, Jon Wax 26(copyright) (S. C. Johnson Co.) or an aqueous dispersion of carnauba wax (Michelman Co.); and/or a microgel, such as a microgel of methyl methacrylate/ethylene glycol dimethacrylate/acrylic acid.
Since the transfer lamination element may come in contact with other image recording articles or the drive or transport mechanisms of laminating devices, additives such as surfactants, lubricants, matte particles and the like may be added to the element to the extent that they do not degrade the properties of interest.
The protection layer described above may be coated by conventional coating means onto the support such as wound wire rod coating, slot coating, slide hopper coating, gravure, curtain coating and the like.
Ink jet inks used to prepare the images to be protected using the invention are well-known in the art. The ink compositions used in ink jet printing typically are liquid compositions comprising a solvent or carrier liquid, dyes or pigments, humectants, organic solvents, detergents, thickeners, preservatives, and the like. The solvent or carrier liquid can be solely water or can be water mixed with other water-miscible solvents such as polyhydric alcohols. Inks in which organic materials such as polyhydric alcohols are the predominant carrier or solvent liquid may also be used. Particularly useful are mixed solvents of water and polyhydric alcohols. The dyes used in such compositions are typically water-soluble direct or acid type dyes. Such liquid compositions have been described extensively in the prior art including, for example, U.S. Pat. Nos. 4,381,946; 4,239,543 and 4,781,758, the disclosures of which are hereby incorporated by reference.
Any known ink jet image-receiving layer can be used in the present invention. For example, the image-receiving layer may consist primarily of inorganic oxide particles such as silicas, modified silicas, clays, aluminas, fusible beads such as beads comprised of thermoplastic or thermosetting polymers, non-fusible organic beads, or hydrophilic polymers such as naturally-occurring hydrophilic colloids and gums such as gelatin, albumin, guar, xantham, acacia, chitosan, starches and their derivatives, and the like; derivatives of natural polymers such as functionalized proteins, functionalized gums and starches, and cellulose ethers and their derivatives; and synthetic polymers such as polyvinyloxazoline, polyvinylmethyloxazoline, polyoxides, polyethers, poly(ethylene imine), poly(acrylic acid), poly(methacrylic acid), n-vinyl amides including polyacrylamide and polyvinylpyrrolidone, and poly(vinyl alcohol), its derivatives and copolymers; and combinations of these materials. Hydrophilic polymers, inorganic oxide particles, and organic beads may be present in one or more layers on the substrate and in various combinations within a layer.
A porous structure may be introduced into image-receiving layers comprised of hydrophilic polymers by the addition of ceramic or hard polymeric particulates, by foaming or blowing during coating, or by inducing phase separation in the layer through introduction of nonsolvent.
In practice, various additives may be employed in the image-receiving layer and protection overcoat. These additives include surface active agents surfactant(s) to improve coatability and to adjust the surface tension of the dried coating, acid or base to control the pH, antistatic agents, suspending agents, antioxidants, hardening agents to cross-link the coating, antioxidants, UV stabilizers, light stabilizers, and the like. In addition, a mordant may be added in small quantities (2%-10% by weight of the base layer) to improve waterfastness. Useful mordants are disclosed in U.S. Pat. No. 5,474,843.
The layers described above, including the image-receiving layer and the protection layer, may be coated by conventional coating means onto a transparent or opaque support material commonly used in this art. Coating methods may include, but are not limited to, wound wire rod coating, slot coating, slide hopper coating, gravure, curtain coating, and the like. Some of these methods allow for simultaneous coatings of both layers, which is preferred from a manufacturing economic perspective.
The image-receiving layer may be present in any amount which is effective for the intended purpose. In general, good results are obtained when it is present in an amount of from about 5 to about 30 g/m2, preferably from about 8 to about 15 g/m2, which corresponds to a dry thickness of about 5 to about 30 xcexcm, preferably about 8 to about 15 xcexcm.
The support for the ink jet recording element used in the invention can be any of those usually used for ink jet receivers, such as paper, resin-coated paper, poly(ethylene terephthalate), poly(ethylene naphthalate) and microporous materials such as poly polyethylene polymer-containing material sold by PPG Industries, Inc., Pittsburgh, Pa. under the trade name of Teslin(copyright), Tyvek(copyright) synthetic paper (DuPont Corp.), and OPPalyte(copyright) films (Mobil Chemical Co.) and other composite films listed in U.S. Pat. No. 5,244,861.
The receiver support used in the invention may have a thickness of from about 50 to about 500 xcexcm, preferably from about 75 to 300 xcexcm. Antioxidants, antistatic agents, plasticizers and other known additives may be incorporated into the support, if desired. In a preferred embodiment, either paper or poly(ethylene terephthalate) is employed.
In order to improve the adhesion of the image-receiving layer to the support, the surface of the support may be subjected to a corona-discharge-treatment prior to applying the image-receiving layer.
In addition, a subbing layer, such as a layer formed from a halogenated phenol or a partially hydrolyzed vinyl chloride-vinyl acetate copolymer can be applied to the surface of the support to increase adhesion of the image-receiving layer. If a subbing layer is used, it should have a thickness (i.e., a dry coat thickness) of less than about 2 xcexcm.
The following examples further illustrates the invention.

Process for laminating ink jet print with a water-dispersible, hydrophobic polyester resin


Devices for application of agents to the cutting mechanism of paper shredders are disclosed. Maintenance and lubrication can be performed by applying lubrication agents to the cutting mechanism of a paper shredder without requiring disassembly of the paper shredder. The agents can also include scented agents and decomposing agents or a combination thereof. Lubrication sheets are disclosed for applying agents to the cutting mechanism of paper shredders. A holder is disclosed for retaining lubrication sheets within proximity of a paper shredder. Paper shredders are also disclosed having various means for identifying when lubrication is needed and/or when the cutting mechanism of the paper shredder is lubricated by a lubrication sheet. Paper shredders having a reservoir of lubricant for delivery to the cutting mechanism of the paper shredder are also disclosed. Such reservoirs may be removable. Cap designs allowing use of a removable reservoir with the paper shredders are also disclosed.

1. The Field of the Invention
The present invention relates to the maintenance of paper shredders. More specifically, the present invention relates to delivery of lubricants and other agents to cutting mechanisms of paper shredders.
2. The Relevant Technology
In today's corporate world it is becoming increasingly popular to dispose of important business documents through paper shredding. The paper shredders come in various shapes and sizes, but they all have a similar goal; to shred important documents to protect privacy and business transactions and protect corporate know how.
To effectively shred documents, paper shredders require periodic maintenance and lubrication. Shredders are typically taken apart and manually oiled via a spray solution or liquid dispenser, which is problematic in several ways. Many owners and users of paper shredders fail to properly maintain and lubricate their machines because of the difficulty and inconvenience involved. This can lead to failure of the shredder and the expense of purchasing a new shredder. Those who do maintain their shredders often must hire service personnel to complete this task.
When owners or users of paper shredders personally dissemble and lubricate their shredders, they can be exposed to messy and potentially hazardous oils and inhalation of sprays. Moreover, inconsistent lubrication by consumers has resulted in injury as a result of lubricant combustion. Thus, while paper shredders are common in many homes and businesses, there is a need to improve the manner in which the paper shredders are maintained and serviced. There is also a need to improve the manner in which documents are destroyed by paper shredders so as to increase protection of information from unauthorized access.

Delivery of agents to the cutting mechanism of paper shredders