A nonvolatile latch circuit includes: a first gate part controlling to load or intercept an input signal based on a gate signal; a first logic gate functioning as an inverter or a gate outputting a constant voltage in response to the first control signal; a second logic gate functioning as an inverter or a gate outputting the constant voltage in response to the first control signal; a second gate part controlling to load or intercept the output of the second logic gate based on an inverted signal of the gate signal and sends the output of the second logic gate to an first input terminal of the first logic gate; and first and second injection type MTJ elements provided between the driving power supply and the first and second logic gates and changing in resistance depending upon a current flow direction.

1. Field of the Invention
The present invention relates to a nonvolatile latch circuit and a nonvolatile flip-flop circuit.
2. Related Art
As the transistors become finer, not only the sub-threshold leak current but also the gate leak current increases and power dissipation caused by these leak currents occupies the greater part of the total power dissipation of the LSI. As for lowered powered dissipation at the circuit and system level, a technique of lowering the drive voltage and lowering the operation frequency as a basic principle has been proposed (see, for example, LongRun (January 2000) http://www.transmeta. com/index.html).
Aiming at further lowered power dissipation, a technique of dividing an LSI into several circuit blocks and intercepting power supply to blocks which are not in operation is proposed (see, for example, Shimizu, T.; Arakawa, F.; Kawahara, T.; VLSI Circuits, 2001. Digest of Technical Papers. 2001 Symposium on 14-16 Jun. 2001 Pages 55-56). Since this proposal cannot intercept the power supply for a block which is required to retain data, however, there is a problem that blocks to which the technique can be applied are restricted.
On the other hand, a technique of incorporating ferroelectric capacitors into a sequential circuit such as a flip-flop to form a nonvolatile sequential circuit is proposed (see, for example, JP-A 2000-124776, or Fujimori, Y.; Nakamura, T.; Takasu, H.; Technical Report of IEICE. ICD2002-10 Pages: 13-18). This nonvolatile sequential circuit stores data of 0 and 1 as a difference of remanent dielectric polarization of a ferroelectric capacitor before interception of the power supply. Even if the power supply is intercepted, the data is retained and the data can be read out after the power supply is connected again. If such a nonvolatile sequential circuit can be realized, the power supply can be intercepted inclusive of the sequential circuit at the time of non-operation and consequently a dramatic reduction of power dissipation can be anticipated. However, the nonvolatile sequential circuit has a problem of scalability that making the circuit fine reduces the readout margin because ferroelectric capacitors are used in storage elements.
Furthermore, in recent years, study of various nonvolatile memory elements using new materials and having a feature that the elements have two terminals and a silicon single crystal substrate is not needed is vigorously promoted. It is considered that those nonvolatile memory elements are small-sized and they can be incorporated into the wiring layer portion. Implementation of a small sized nonvolatile latch circuit using the nonvolatile memory element is proposed (see, for example, Keiko Abe, Shinobu Fujita, and Thomas H. Lee, EUROPEAN MICRO AND NANO SYSTEMS 2204, or U.S. Patent No. 2006/0083047). In Keiko Abe, Shinobu Fujita, and Thomas H. Lee, EUROPEAN MICRO AND NANO SYSTEMS 2004, a spin injection type MTJ (Magnetic Tunnel Junction) element which is excellent in scalability and high in endurance is mentioned as the nonvolatile memory element used for the nonvolatile latch.
When two nonvolatile latch circuits are connected to form a flip-flop, data is written into the storage element every clock period. If it is attempted to operate the flip-flop at an operation frequency of 1 GHz, the storage element is required to have high endurance amounting to 8.64×1013 times a day. A nonvolatile latch circuit capable of satisfying such high endurance is not known.

Nonvolatile latch circuit and nonvolatile flip-flop circuit


A stapler having a driver unit vertically separated from its clincher unit, which has a clincher base with a clincher. The clincher base is disposed to vertically reciprocate, and sheets inserted between the base and driver unit are clamped between the base and the driver unit when the base is reciprocated. The stapler has a first motor to operate the driver, and a second motor to reciprocate the clincher base and operate the clincher. In sequence, the second motor reciprocates the base and makes the base clamp the sheets, and then stops; the first motor makes the driver drive out the staple, and then stops; the second motor makes the clincher clinch the staple legs having passed the sheets, the base is returned, and the second motor stops; and after clinching, the first motor returns the driver to the initial position, and then stops.

There have been formerly known staplers in which a driver unit is vertically separated from a clincher unit (See JP-B 62-46324).
In such a stapler, the driver unit is arranged in a lower position, and the clincher unit is vertically movably arranged above the driver unit. The clincher unit is descended to clamp sheets placed on an upper face of the driver unit between the clincher unit and the driver unit. Then, a driver of the driver unit is operated to drive out a staple toward the sheets clamped. Then, a clincher of the clincher unit is operated to clinch leg portions of the stapler passing through the sheets.
However, according to the stapler, vertical movement of the driver unit and operations of the driver and the clincher are effected with a singe motor, using a cam mechanism, a link mechanism, etc. Thus, the structure of each of the mechanisms becomes complicated, unfavorably resulting in a large-scale stapler with these mechanisms. Further, since various operations are performed by the single motor, orientations of the driving unit and the clincher unit cannot be changed. Thus, the staple cannot be punched into the sheets in such a state that a back portion of the staple is oblique to the sheets.
It is an object of the present invention to provide a stapler which can simplify its mechanical structure and can change orientations of a driver unit and a clincher unit.

Motor-driven stapler having a driver and a cylinder unit that vertically reciprocates


A production method of 2-chloro-1,3,3,3-tetrafluoropropene (1224) according to the present invention includes bringing 2,3-dichloro-1,1,1,3-tetrafluoropropane (234da) into contact with an inorganic base having a pKa of 4.8 or greater in an aqueous medium in the presence of a phase transfer catalyst. Preferably, the inorganic base has a pKa of 10 or greater. Further, the phase transfer catalyst is preferably at least one selected from the group consisting of tetrabutylammonium bromide, methyltri-n-octylammonium chloride, benzyltrimethylammonium chloride and tetraethylammonium chloride. It is possible by this method to selectively produce 1224 from 234da.

In place of specific chlorofluorocarbons designated as strong ozone-depleting substances by the Montreal Protocol agreed in Canada in 1987, various alternative fluorocarbons with less fear of ozone depletion are synthesized and used. In recent years, the alternative chlorofluorocarbons are also required to show a shorter life in the air and less fear of global warming.
It is known that 2-chloro-1,3,3,3-tetrafluoropropene, which has a double bond in its molecule, gets quickly decomposed by reaction of the double bond with OH radial etc. in the air and thereby shows a low global warming potential and less fear of global warming. There exist trans and cis geometric isomers of 2-chloro-1,3,3,3-tetrafluoropropene. Hereinafter, the trans and cis isomers of 2-chloro-1,3,3,3-tetrafluoropropene is sometimes referred to as "1224E" and "1224Z", respectively; and 2-chloro-1,3,3,3-tetrafluoropropene is sometimes simply referred to as "1224" in the case where there is no need to distinguish the trans and cis isomers or in the case where it refers to a mixture of the trans and cis isomers. The boiling point of 1224E is 23° C., whereas the boiling point of 1224Z is 17° C. Both of 1224E and 1224Z have a boiling point in the vicinity of room temperature (about 20° C.) and can suitably be used as blowing agents, solvents, coolants, working fluids or the like.
In the case of using 1224 as a blowing agent for a heat insulating material, low-boiling 1224Z is suitably usable for a heat insulating material in a refrigerator; and high-boiling 1224E is suitably usable for a heat insulating material in a building because of its good handling properties.
As the boiling point of 1224 is in the vicinity of room temperature, 1224 can also suitably be used as a heating medium for a heat pump or a high-temperature working fluid. In the case of using 1224 as a high-temperature working fluid for a heat pump, the coefficient of performance (COP) of the heat pump, which indicates cooling/heating capacity per 1 kW of power consumption, and the heat transfer capacity of the heat pump in freezing cycles etc. vary with even a slight difference in boiling point of the working fluid. In the case of using 1224 as a high-temperature working fluid, the suitable boiling point of the working fluid varies depending on the conditions of thermal cycles. It is thus preferable to appropriately select and use either the trans isomer (1224E) or the cis isomer (1224Z) according to the purpose of use.
In the following Patent Documents 1 and 2 and Non-Patent Document 1, there are disclosed processes of forming 1224.
Patent Document 1 discloses a process for purifying a (E)-1-chloro-3,3,3-trifluoropropene composition, which contains at least hydrogen fluoride and 2-chloro-1,1,1,3,3-pentafluoropropane, by contact with a weak base. More specifically, this process includes the step of bringing trans-1-chloro-3,3,3-trifluoropropane (sometimes abbreviated as "1233E") containing hydrogen fluoride and 2-chloro-1,1,1,3,3-pentafluoropropane (sometimes abbreviated as "235da") as a trace impurity into contact with the weak base so as to remove hydrogen fluoride and 235da and obtain 1233E as a purified product without forming 1224.
Non-Patent Document 1 discloses, as a specific synthesis example of 1224, a method for forming 2,3-dichloro-1,1,1,3-tetrafluoropropane (sometimes referred to as "234da") by photochlorination of 1,3,3,3-tetrafluoropropene (sometimes referred to as "1234"), and then, forming 1224 by dehydrochlorination of 234da in a potassium hydroxide solution. Herein, 234da is a hydrochlorofluorocarbon represented by CF3CHClCHClF.
Further, Patent Document 2 discloses a method for forming 235da as a raw material of 1224.

Method for producing 2-chloro-1,3,3,3-tetrafluoropropene


A method for the selective killing of HIV-infected cells, via HIV-regulated expression of a toxin gene, is provided in the present invention. Specifically, the expression of the diphtheria toxin fragment A gene is subject to tight control by cis-acting HIV regulatory sequences and trans-acting regulatory factors. Also provided is a method of protecting a host from HIV infection by the stable transformation of target cells, those cells which can be infected with HIV, with an HIV-regulated toxin gene. When such a stably transformed cell becomes infected with HIV, induction of the toxin gene prevents the replication and spread of the virus.

The present invention is based on the surprising discovery that the diphtheria toxin fragment A can be selectively targeted to certain mammalian cell types by introduction into the cell of chimeric toxin genes in which expression of a toxin fragment A coding sequence is controlled by mammalian cell-specific regulatory sequences. The toxin fragment A coding sequence was selectively expressed in the target mammalian cell, inhibiting protein synthesis and resulting in cell death. Cell-specific expression of such chimeric toxin genes was sufficiently restricted to effect selective killing of targeted cells without elimination of non-targeted cells. It was surprising that selective lethality could be obtained using such chimeric toxin genes because there was evidence that the introduction of a single molecule of fragment A into a cell would be lethal (Yamaizumi et al. (1978) Cell 15:245-250) and it was not known, prior to the present invention, if cell-specific regulation, particularly of heterologous coding regions, would be restricted enough to cause selective lethality.
Attempts have been made to use the diphtheria toxin A fragment to selectively kill undesirable cells, such as malignant cells, without destroying healthy cells. Such attempts have concentrated on replacement of the natural fragment B protein delivery mechanism with alternate delivery mechanisms based on the specificity of certain proteins for cell surface molecules, for example by preparing toxin fragment A protein conjugates with antibodies (immunotoxins), hormones or plant lectins.
Diphtheria toxin is synthesized and secreted by strains of Corynebacterium diphtheriae which are lysogenic for bacteriophage .beta..sup.tox+. The naturally occurring toxin is a single polypeptide of about 58 kd (535 amino acids) which is highly toxic to many animal species. Diphtheria toxin inhibits protein synthesis in and is toxic to most eukaryotic cells that have been tested. The toxin is composed of two regions, separable by proteolytic cleavage, which are functionally distinct. Toxin activity is associated only with fragment A, the NH.sub.2 -terminal region of 193 amino acids. Fragment A functions by catalyzing the inactivation of elongation factor-2 (EF-2). The COOH-terminal 342 amino acid fragment B, is itself non-toxic, but functions to deliver the toxin fragment A to cells. Fragment A is non-toxic unless it is introduced into the cell cytoplasm. Purified toxin fragment A has been demonstrated to be highly toxic when introduced artificially into cells. A review of the structure and function of diphtheria toxin is provided in Pappenheimer (1977) Ann. Rev. Biochem. 46:69-94.
The diphtheria toxin (DT) gene, tox, is located on bacteriophage .beta.. The entire gene has been cloned and sequenced by separately cloning fragments having little or no toxic activity (Greenfield et al. (1983) Proc. Natl. Acad. Sci. USA 80:6853-6857). Several non-toxic mutant tox genes have also been cloned including tox45 (Leong et al. (1983) Science 220:515-517) which has a wild-type region A and non-functional B region, and tox228 (Kaczorek et al. (1983) Science 221:855-858) which carries mutations in both the A and B regions. Uchida et al. (1973) J. Biol. Chem. 248:3838-3844 and ibid. pp. 3845-3850 have identified several mutant DT proteins, designated CRM's (cross-reacting materials) which are non-toxic (CRM45, 197, 228) or have reduced toxicity (CRM176). The attenuated toxicity (about 90% of wild-type) of CRM176 results from a mutation in the A region which affects enzymatic activity of the tox176 fragment A. The coding sequence of the mature toxin is preceded by a signal sequence which presumably functions in secretion of tox gene product (Kaczorek et al. (1983)).
It is known that many differentiated eukaryotic cells synthesize proteins that are unique to a particular cell type. For example, it has been demonstrated that immunoglobulin kappa is specifically expressed in B lymphocyte cells, that interleukin-2 is selectively expressed in activated T-cells (Fujita et al. (1986) Cell 46:401-407), that gamma 2-crystallin is specifically expressed in the fiber cells of the ocular lens (i.e., Breitman et al. (1984) Proc. Natl. Acad. Sci. USA 81:7762), that elastase I is specifically expressed in pancreatic acinar cells (Ornitz et al. (1985) Nature 313:600-602), that insulin is specifically expressed in pancreatic endocrine .beta.-cells, and that chymotrypsin is specifically expressed in pancreatic exocrine cells (Walker et al. (1983) Nature 306:557-561). Additionally, there are examples of non-specific but preferential expression of transferrin (McKnight et al. (1983) Cell 34:335-341) and metallothionein in the liver. A particularly important type of cell-preferential expression occurs with certain retroviruses including human T-cell leukemia viruses, HTLV's (Sodroski et al. (1984) Science 225:381-385; Sodroski et al. ( 1985) ibid. 227:171-173) and bovine leukemia virus, BLV (Derse et al. (1985) Science 227:317-320; Rosen et al. (1985) ibid. 227:320-322). Markedly enhanced viral expression is observed in cells already infected with the virus. Selective transcription is stimulated by trans-acting regulatory factors produced in infected cells. These stimulatory factors appear to be unique for each virus. Sequences associated with control of stimulated expression have been localized to the long terminal repeat (LTR) sequence of both HTLV's and BLV. Heterologous genes placed under control of the LTR sequences are reported to be preferentially expressed in infected cells.
Chimeric genes in which a heterologous mammalian or viral structural gene is placed under the control of cell-specific regulatory elements have been reported to be successfully expressed in a cell-specific manner. An elastase-human growth hormone gene fusion was shown to be specifically expressed in pancreatic acinar cells of transgenic mice (Ornitz et al. (1985) supra). Oncogenes placed under the control of elastase and gamma A crystallin gene regulatory sequences have been shown to be specifically expressed (i.e., induce tumors) in the pancreas and ocular lens, respectively of transgenic mice (Ornitz et al. (1985) Cold Spring Harbor Symp. Quant. Biol. 50:389-409; Quaife et al. (1987) Cell 48:1023-1034; Mahon et al. (1987) Science 235:1622-1628).
A number of bacterial genes have been successfully expressed in mammalian cells under the control of mammalian promoters and regulatory sequences (see, for example, Gorman et al. (1982) Mol. Cell. Biol. 2:1044-1051; Southern and Berg (1982) J. Mol. Appl. Genet. 1:327-341). In fact, bacterial genes such as chloramphenicol acetyl transferase (CAT), aminoglycoside 3' phosphotransferase (neo), guanine phosphoribosyl transferase (gpt) and .beta.-galactosidase (lacZ) are often used as detectable or selectable markers in the study of mammalian expression systems. For example, the bacterial .beta.-galactosidase gene has been used to assess heat shock expression in Drosophila (Lis et al. (1983) Cell 35:403-410) and tissue-specific expression mediated by the gamma 2-crystallin promoter in the ocular lens of transgenic mice (Goring et al. (1987) Science 235:456-458).
Specific DNA sequences which function in cell-specific regulation have been isolated and identified in many cases. In most systems that have been studied, cell-specific expression is mediated by an enhancer, a cis-acting DNA sequence, which is believed to selectively activate expression in a target cell in response to tissue or cell-specific trans-acting factors. Immunoglobulin heavy chain (IgH) enhancers are selectively active in B-cells and are among the best characterized cell-specific expression elements (Gillies et al. (1983) Cell 33:717-728; Picard and Shaffner (1984) Nature 307:80-82; Ephrussi et al. (1985) Science 227:134-140). Enhancers are also reported to function in cell-selective expression of elastase (Hammer et al. (1987) Mol. Cell. Biol. 7:2956-2967), insulin (Edlund et al. (1985) 230:912-916) and interleukin-2 (Fujita et al. (1986)). It has recently been reported that cell-type specificity of immunoglobulin genes is conferred not only by the IgH enhancer but also by a 5'-upstream element associated with an immunoglobulin gene promoter (Mason et al. (1985) Cell 41:479-487; Foster et al. (1985) Nature 315:423-425). This upstream element apparently confers a level of cell-selective expression independent of the heavy chain enhancer. A similar 5'-upstream promoter associated element is reported to function in insulin gene regulation (Edlund et al. (1985)). In contrast, no such promoter associated element is believed to function in interleukin-2 regulation (Fujita et al. (1986)).
It has been reported (Maxwell et al. (1986) Cancer Research 46:4660-4664, which is incorporated by reference herein) that the diphtheria toxin A-chain (DT-A) gene is regulated in a cell-specific manner on transfection into human cells. This reference also reported selective killing of B-cells caused by expression of DT-A under the control of the immunoglobulin heavy chain enhancer. This reference also suggests that cell-specific regulatory mechanisms can be employed generally for selective cell killing by expression of a toxin gene and that such selective killing has application to cancer therapy. A second report (Maxwell et al. (1987) Mol. Cell. Biol. 7:1576-1579, which is incorporated by reference herein) describes the cloning and sequencing of the attenuated diphtheria toxin 176 and suggests the use of the tox176 coding region for selective cell killing.
Recently, it has been reported (Palmiter et al. (1987) Cell 50:435-443, which is incorporated by reference herein) that a chimeric diphtheria toxin fragment A coding sequence expressed under the regulatory control of an elastase I enhancer/promoter was selectively expressed in pancreatic acinar cells. Selective expression and selective lethality of the chimeric toxin gene was demonstrated by the production of transgenic mice lacking a normal pancreas. Similar results have also been obtained (Breitman et al. (1987) Science, 238:1553-1555) with diphtheria toxin fragment A under the control of gamma crystallin gene regulatory sequences, resulting in selective elimination of lens tissue in transgenic mice.
Some recently suggested approaches to therapy for Acquired Immune Deficiency Syndrome (AIDS) involve "intracellular immunization", a term coined by Baltimore ((1988) Nature 335:395-396) to describe the genetic modification of cells to render them incapable of supporting viral production. We have been exploring the use of regulated expression of a gene encoding a potent toxin, diphtheria toxin A fragment (DT-A) or an attenuated toxin fragment, to selectively kill cells infected with human immunodeficiency virus (HIV-1).
As described herein, we have placed expression of the reporter gene luciferase (luc), or of DT-A, under control of the HIV-1 trans-acting, essential Tat and Rev proteins. The Tat protein acts on a cis-acting element mapped to region +14 to +44 (referred to as the TAR region) of the HIV long terminal repeat (LTR) to increase viral expression from the LTR (Arya et al. (1985) Science 229:69-73; Rosen et al. (1985) supra; Sodroski et al. (1985) supra: Green et al. (1989) Cell 58:215-223). The Tat protein appears to exert an effect at both transcriptional (Peterlin et al. (1986) Proc. Natl. Acad. Sci. USA 83:9734-9738; Hauber et al. (1987) Proc. Natl. Acad. Sci. USA 84:6364-6368; Laspia et al. (1989) Cell 59:283-292) and post-transcriptional levels (Cullen (1986) Cell 46:973-982; Feinberg et al. (1986) Cell 46:807-817; Wright et al. (1986) Science 234:988-992; Braddock et al. (1989) Cell 58:269-279; Edery et al. (1989) Cell 56:303-312) and can stimulate expression of heterologous genes placed 3' to the TAR region (Tong-Starksen et al. (1987) Proc. Natl. Acad. Sci. USA 80:6845-6849; Felber and Pavlaskis (1988) Science 239:184-187). The Rev protein relieves the negative regulatory effect of cis-acting repressive sequences (crs) found in the env region of the HIV-1 genome (Rosen et al. (1988) Proc. Natl. Acad. Sci. USA 85:2071-2075; Hadzopoulou-Cladaras et al. (1989) J. Virol. 63:1265-1274) which repress the production of viral unspliced and singly spliced messenger RNAs (mRNAs). The Rev protein acts by binding to RNA at the Rev responsive element (RRE; Malim et al. (1989) Nature 338:254-257; Cochrane et al. (1990) Proc. Natl. Acad. Sci. USA 87:1198-1202), also localized to the env region; binding of the Rev protein to the RRE is essential for Rev function. Rev protein expression results in an increased accumulation of unspliced and singly spliced viral mRNAs, encoding structural proteins, in the cytoplasm (Felber et al. (1989) Proc. Natl. Acad. Sci. USA 86:1495-1499; Zapp and Green (1989) Cell 58:215-223). Thus, expression of the Rev protein promotes the transition from early or latent infection to productive infection. Like Tat, the Rev protein can also act in trans to activate expression of heterologous genes which contain the negative crs sequences and a correctly oriented RRE (Rosen et al. (1988) supra; Felber et al. (1989) supra). We demonstrate here that efficient regulation of both chimeric luc and chimeric DT-A expression by the Tat and Rev proteins can be achieved in transfected cells in vitro. Such regulation is applicable as a novel approach for treatment of AIDS, exploiting the extreme toxicity of DT-A to kill virus-infected cells.

Compositions and method for inhibition of HIV production


Methods are described for the identification and preparation of high-affinity nucleic acid ligands to cytokines. Included in the invention are specific nucleic acid ligands to IFN-gamma, IL-4, IL-10, TNF-alpha, and RANTES.

Cytokines are a diverse group of small proteins that mediate cell signaling/communication. They exert their biological functions through specific receptors expressed on the surface of target cells.
Cytokines can be subdivided into several groups, including the immune/hematopoietins, interferons, tumor necrosis factor (TNF)-related molecules, and the chemokines. Representative immune/hematopoietins include erythropoietin (EPO), granulocyte/macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), leukemia inhibition factor (LIF), oncostatin-M (OSM), ciliary neurotrophic factor (CNTF), growth hormone (GH), prolactin (PRL), interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, and IL-12. Representative interferons (IFN) include IFN.alpha., IFN.beta., and IFN-gamma. Representative TNF family members include TNF.alpha., interferon (IFN).beta., gp.sup.39 (CD40-L), CD27-L, CD30-L, and nerve growth factor (NGF). Representative chemokines include platelet factor (PF)4, platelet basic protein (PBP), gro.alpha., MIG, ENA-78, macrophage inflammatory protein (MIP)1.alpha., MIP1.beta., monocyte chemoattractant protein (MCP)-1, I-309, HC14, C10, Regulated on Activation, Normal T-cell Expressed, and Secreted (RANTES), and IL-8.
IFN-gamma
IFN-gamma was first described 30 years ago as an antiviral agent (Wheelock, 1965). Since that time the protein has been shown to be a remarkably pleiotropic cytokine which plays important roles in modulating virtually all phases of immune and inflammatory responses. The cDNAs for murine IFN-gamma (Gray and Goeddel, 1983) and human IFN-gamma (Gray and Goeddel, 1982) have been cloned, sequenced, and characterized.
IFN-gamma is a member of a family of proteins related by their ability to protect cells from viral infection. This family has been divided into three distinct classes based on a variety of criteria, IFN-alpha (originally known as Type I IFN or Leukocyte IFN), IFN-beta (also originally known as Type I IFN or Fibroblast IFN) and IFN-gamma (originally known as Type II IFN or Immune IFN). IFN-gamma is unrelated to the Type I interferons at both the genetic and protein levels (Gray, et al., 1982). The human and murine IFN-gamma proteins display a strict species specificity in their ability to bind to and activate human and murine cells. This is due at least in part to their modest homologies at both the cDNA and amino acid levels (60% and 40% respectively).
IFN-gamma is produced by a unique set of stimuli and only by T lymphocytes and natural killer (NK) cells. The human and murine genes for IFN-gamma are 6 kb in size, and each contain four exons and three introns. These genes have been localized to human chromosome 12 (12q24.1) and murine chromosome 10. Activation of the human gene leads to the transcription of a 1.2 kb mRNA that encodes a 166 amino acid polypeptide (Derynck, et al., 1982). The human protein contains a 23 residue amino terminal hydrophobic signal sequence which gets proteolytically removed, giving rise to a mature 143 residue positively charged polypeptide with a predicted molecular mass of 17 kDa. Variable post-translational enzymatic degradation of the positively charged carboxy terminus (Rinderknecht, et al., 1984) is responsible for the charge heterogeneity of the fully mature molecule. Proteins with six different carboxy termini have been detected for both natural and recombinant forms of IFN-gamma. Two polypeptides self-associate to form a homodimer with an apparent molecular mass of 34 kDa (Scahill, et al., 1983). The homodimer is the biologically active form of the protein. Mature human IFN-gamma contains no cysteine residues, thus the homodimer is held together entirely by noncovalent forces. This quaternary structure of the native protein explains its characteristic sensitivity to extremes of heat (protein denatured at temperatures above 56.degree. C.), and pH (activity rapidly lost at pH values less than 4.0 and greater than 9.0) (Mulkerrin and Wetzel, 1989).
The remarkable pleiotropic effects of IFN-gamma are mediated through binding to a single type of IFN-gamma receptor. The structure and function of murine and human IFN-gamma receptors have been described (Schreiber, et al., 1992). These receptor proteins are expressed on nearly all cells (except erythrocytes), and platelets (Anderson, et al., 1982). The receptor binds ligand with high affinity (Kd=10.sup.-9 -10.sup.-10 M) and is expressed on most cells at modest levels (200-25,000 sites/cell). Upon IFN-gamma binding to the receptor at the cell surface, the intracellular domain of the receptor is phosphorylated at serine and threonine residues (Hershey, et al., 1990).
One of the major physiologic roles of IFN-gamma is as a regulator of immune response induction, specifically its ability to regulate expression of class I and II major histocompatibility (MHC) antigens on a variety of immunologically important cell types (Trinchieri and Perussia, 1985) Functionally, IFN-gamma dependent upregulation of MHC gene expression is an important step in promoting antigen presentation during the inductive phase of immune responses and may play a role in antitumor activity of IFN-gamma (Buchmeier and Schreiber, 1985).
Another major physiologic role for IFN-gamma is its ability to activate human macrophage cytotoxicity (Schreiber and Celada, 1985). Therefore, IFN-gamma is the primary cytokine responsible for inducing nonspecific cell-mediated mechanisms of host defense toward a variety of intracellular and extracellular parasites and neoplastic cells (Bancroft, et al., 1987). This activation is a result of several distinct functions of IFN-gamma. IFN-gamma has been shown to effect the differentiation of immature myeloid precursors into mature monocytes (Adams and Hamilton, 1984). IFN-gamma promotes antigen presentation in macrophages, through the induction of MHC class II expression as described above, but also by increasing levels of several intracellular enzymes important for antigen processing (Allen and Unanue, 1987). Macrophage cell surface proteins such as ICAM-1 are upregulated by IFN-gamma, thus enhancing the functional results of the macrophage-T cell interaction during antigen presentation (Mantovani and Dejana, 1989). IFN-gamma activates the production of macrophage derived cytocidal compounds such as reactive oxygen- and reactive nitrogen-intermediates and tumor necrosis factor-a (TNF-a) (Ding, et al., 1988). IFN-gamma also reduces the susceptibility of macrophage populations to microbial infections (Bancroft, et al., 1989). Animal models have been used to study the role of IFN-gamma in the clearance of microbial pathogens. Neutralizing monoclonal antibodies to IFN-gamma were injected into mice before infecting them with sublethal doses of various microbial pathogens. These mice lost their ability to resolve the infection initiated with Listeria monocytogenes (Buchmeier and Schreiber, 1985), Toxoplasma gondii (Suzuki, et al., 1988), or Leishmania major (Green, et al., 1990).
Besides these nonspecific cell mediated cytocidal activities, IFN-gamma also enhances other macrophage immune response effector functions. IFN-gamma up-regulates expression of Fc receptors on monocytes/macrophages (FcgRI), thus enhancing the capacity of the macrophage for antibody dependent cell killing (Erbe, et al., 1990). IFN-gamma also promotes humoral immunity through enhancement of complement activity. It does this in two ways, i) by promoting the synthesis of a variety of complement proteins (ie., C2, C4, and Factor B) by macrophages and fibroblasts, and ii) by regulating the expression of complement receptors on the mononuclear phagocyte plasma membrane (Strunk, et al., 1985).
IFN-gamma also exerts its effects on other cells of the immune system. It regulates immunoglobulin isotype switching on B cells (Snapper and Paul, 1987). IFN-gamma plays a positive role in the generation of CD8.sup.+ cytolytic T cells (CTLs) (Landolfo, et al., 1985) and enhances NK cell activity. Recently, it has been unequivocally established that CD4.sup.+ T cells do not constitute a homogeneous class of cells. Indeed, a paradigm of lymphokine biology and of the function of CD4+ T cells has arisen, the so-called Th1/Th2 paradigm (for a review see Paul and Seder, 1994). The T.sub.H1 clones, through their production of IFN-gamma, are well suited to induce enhanced microbicidal and antitumor activity in macrophages as detailed above (enhanced cellular immunity), while the Th2 clones make products (IL-4, IL-5, IL-6, IL-10, IL-13) that are well adapted to act in helping B cells develop into antibody-producing cells (enhanced humoral immunity). The role played by IFN-gamma at this crucial nexus of T cell effector function is fundamental to the success or failure of the immune response.
IFN-gamma plays a major role in promoting inflammatory responses both directly, and indirectly through its ability to enhance TNF-.alpha. production. During an inflammatory response, cells leave the circulation and migrate to the point of infection. During this process they must first bind to and then extravasate through vascular endothelium. Both IFN-gamma and TNF-.alpha. can promote the expression of overlapping sets of cell adhesion molecules (ICAM-1, E-selectin, and others) that play an important role in this process (Pober, et al., 1986; Thornhill, et al., 1991). In fact, experiments have shown that these two cytokines exhibit synergistic effects in up-regulating cell adhesion molecules in vivo (Munro, et al., 1989). One can envision microbial infections in which the microorganism is already widespread at the time the immune response develops or in which the response does not quickly rid the host of the infectious agent. This results in continued T cell activation inducing both local inflammation and tissue damage with ensuing loss of normal function. Indeed, when the infectious agent is of little intrinsic pathogenicity, the disease induced by the infection may largely reflect the consequences of such a response.
Excessive production of IFN-gamma may play a role in autoimmune disorders (for review see Paul and Seder, 1994 and Steinman, 1993). The mechanism for this may involve excessive tissue damage due to aberrant or enhanced expression of class I and class II MHC molecules or the role of an excessive T.sub.H1 cellular response. A role for IFN-gamma and the tissue-damaging effects of immune responses mediated by T.sub.H1 -like cells has been suggested in autoimmune disorders such as rheumatoid arthritis (Feldmann, 1989), juvenile diabetes (Rapoport, et al., 1993), myasthenia gravis (Gu, et al., 1995), severe inflammatory bowel disease (Kuhn, et al., 1993), and multiple sclerosis (Traugott, 1988).
IL-4
Interleukin-4 (IL-4) is a remarkably pleiotropic cytokine first identified in 1982 as a B cell growth factor (BCGF) (Howard, et al., 1982). In that same year, IL-4 was identified as an IgG1 enhancing factor (Isakson, et al., 1982). Because of the effect IL-4 has on B cells, it was first called BCGF-1, later termed BSF-1 (B-cell stimulatory factor-1), and in 1986 it was given the name IL-4. The cDNAs for murine IL-4 (Noma, et al., 1986; Lee, et al., 1986) and human IL-4 (Yokota, et al., 1986) have been cloned, sequenced, and characterized.
IL-4 can be regarded as the prototypic member of a family of immune recognition-induced lymphokines designated the "IL-4 family" (for a review see Paul, 1991). This family consists of IL-4, IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). The properties shared by these proteins leads to this grouping and include, i) the linkage of the genes for the members of the family (van Leeuwen, et al., 1989), ii) the action of each member of the family as a hematopoietic growth factor in addition to any effects it may exert on lymphoid cells, iii) the receptors for these proteins are all members of the hematopoietin family of receptors (Bazan, 1990a), and iv) coexpression of these factors by a subpopulation of cloned CD4.sup.+ T cells (the so-called T.sub.H2 cells) (Mosmann, et al., 1989) and by mast cells (Plaut, et al., 1989).
The remarkable pleiotropic effects of IL-4 are mediated through binding to cell surface receptors (IL-4R). The murine IL-4R (Mosely, et al., 1989; Harada, et al., 1990), and the human IL-4R (Idzerda, et al., 1990; Galizzi, et al., 1990) have been cloned, sequenced, and characterized. IL-4R are present on a variety of hematopoietic (Park, et al., 1987) and nonhematopoietic cells (Lowenthal, et al., 1988). On both human and murine resting T and B cells, IL-4R are present in low numbers (<400) and are regulated by cytokines and other factors. The receptor binds IL-4 with high affinity (Kd=10.sup.-10 M). Now that most of the receptors for immunoregulatory and hematopoietic cytokines have been cloned, it is apparent that the majority of these receptors fall into a large family. This hematopoietic cytokine receptor superfamily includes receptors for IL-4, IL-2 (.beta. and .gamma. chains), IL-7, IL-9, and IL-13 which modulate the lymphoid system; and receptors for erythropoietin, granulocyte-colony stimulating factor (G-CSF), GM-CSF, IL-3, and IL-5 which modulate the hematopoietic system. The superfamily also includes receptors for factors believed to normally function outside the immune and hematopoietic systems, including receptors for growth hormone (GH), prolactin, leukemia inhibitory factor (LIF), IL-6, IL-11, and ciliary neurotrophic factor (CNF) (for a review see Kishimoto, et al., 1994).
A general first step in the signaling processes of immune and hematopoietic cytokines may be ligand-induced dimerization of receptor components whose cytoplasmic regions interact to initiate a downstream signaling cascade. The IL-4 receptor has a long putative intracellular domain (553 amino acids in mouse, 569 in human) with no known consensus sequences for kinase activity or for nucleotide-binding regions. The biochemical nature of signals induced by the binding of IL-4 to its receptor have not been elucidated. It does appear that the cytosolic domain of the receptor is essential for its signaling function (Mosely, et al., 1989). Ligand induced dimerization of the IL-4 receptor appears to be a critical first step in IL-4 mediated signal transduction.
One of the major physiologic roles of IL-4 is as a B lymphocyte activation and differentiation factor (Rabin, et al., 1985; Oliver, et al., 1985). The protein was first isolated based on this activity. In this regard, IL-4 activates production of IgG1 (Vitetta, et al., 1985), but is also responsible for isotype switching in B cells from production of IgG to IgE immunoglobulins (Coffman, et al., 1986; Lebman and Coffman, 1988, Del Prete, et al., 1988). The effect of IL-4 on the in vivo regulation of IgE has been clearly demonstrated. Neutralization of IL-4 by treatment with a monoclonal anti-IL-4 antibody (Finkelman, et al., 1986) or a monoclonal antibody to the IL-4 receptor (Finkelman, et al., 1990) will block the IgE response. A recombinant soluble IL-4 receptor has been shown to inhibit IgE production by up to 85% in vivo (Sato, et al., 1993). IL-4 deficient mice produced by gene-targeting in murine embryonic stem cells have normal B and T cell development, but serum levels of IgG1 and IgE are strongly reduced (Kuhn, et al., 1991). IL-4 augmented IgE production leads to an atopic state (allergy/asthma) (Finkelman, et al., 1989; Katona, et al., 1991).
The IL-4 mediated up-regulation of IgG1 may play a role in the inflammation cascade. IgG1 has recently been shown to form immune complexes which bind to the cellular receptors for the Fc domain of immunoglobulins (FcRs) leading to an inflammatory response (Sylvestre and Ravetch, 1994; Ravetch, 1994). IL-4 transgenic mice have been produced that hyperexpress IL-4 (Tepper, et al., 1990). These mice have elevated levels of serum IgG1 and IgE and they develop allergic inflammatory disease. These findings demonstrate the critical role IL-4 plays in the humoral immune response.
Another major physiologic role for IL-4 is as a T lymphocyte growth factor (Hu-Li, et al., 1987; Spits, et al., 1987). IL-4 enhances the proliferation of precursors of cytotoxic T cells (CTLs) and their differentiation into active CD8.sup.+ CTLs (Widmer and Grabstein, 1987; Trenn, 1988). IL-4 appears to augment the IL-2 driven induction of lymphokine-activated killer (LAK) cells (Higuchi, et al., 1989), which have been shown to lyse a variety of tumor cell targets without major histocompatibility complex (MHC) restriction. The role played by IL-4 at this crucial nexus of T cell effector function is fundamental to the success or failure of the immune response.
IL-4 has been shown to affect nonlymphoid hematopoietic cells in a variety of ways. IL-4 has been shown to modulate monocyte/macrophage growth (McInnes and Rennick, 1988; Jansen, et al., 1989) while enhancing their differentiation and cytotoxic activity for certain tumor cells (Crawford, et al., 1987; Te Velde, et al., 1988). IL-4 also has activity as a stimulant of mast cell growth (Mosmann, et al., 1986; Brown, et al., 1987), and increases production and recruitment of eosinophils (Tepper, et al., 1989).
IL-4 alone or in conjunction with other cytokines can promote the expression of a variety of cell-surface molecules on various cell types with diverse implications for disease. Specifically, IL-4 can interact with tumor necrosis factor (TNF) to selectively enhance vascular cell adhesion molecule-1 (VCAM-1) expression contributing to T cell extravasation at sites of inflammation (Briscoe, et al., 1992). IL-4 alone or in combination with TNF or IFN-gamma has been shown to increase both MHC antigen and tumor-associated antigen expression on a variety of neoplastic cells (Hoon, et al., 1991).
As detailed above, IgG1 immune complexes bind to the cellular receptors for the Fc domain of immunoglobulins (FcRs) leading to an inflammatory response. Inhibition of IL-4 and the subsequent reduction in IL-4 mediated IgG1 expression may prove efficacious against immune complex inflammatory disease states. Indeed, inhibitory ligands to IL-4 might also prevent the IL-4 mediated overexpression of VCAM-1, thus attenuating the ability of T cells to extravasate at sites of inflammation.
Inhibition of IL-4 activity with a monoclonal antibody, a recombinant soluble IL-4 receptor, or gene knock-out, results in a reduction of serum IgE levels. An inhibitory oligonucleotide ligand to IL-4 could be clinically effective against allergy and allergic asthma.
A recent report has described a disorder in bone homeostasis in transgenic mice that inappropriately express IL-4 under the direction of the lymphocyte-specific proximal promoter for the 1 ck gene (Lewis, et al., 1993). Bone disease in these mice resulted from markedly decreased bone formation by osteoblasts, features identical to those found in human osteoporosis. Inhibiting this IL-4 mediated reduction in osteoblast activity may prove beneficial against osteoporosis.
Graft-versus-host disease (GVHD) is a major complication of human tissue transplantation. GVHD does not exist as a single clinical manifestation but can involve immunologic abnormalities ranging from immunodeficiency to systemic autoimmunities (Ferrara, et al., 1991). These systemic autoimmunities include clinical and serological manifestations of human systemic lupus erythematosus (SLE). Several murine models of SLE have been developed (Gleichmann, et al., 1982; van Rappard-van Der Veen, et al., 1982), and the induction of systemic GVHD in mice has been described (Via, et al., 1988). Two recent studies have shown in vivo efficacy of a mouse monoclonal antibody to IL-4 in preventing GVHD and SLE in these murine model systems (Umland, et al., 1992; Ushiyama, et al., 1995). These observations suggest that an inhibitor of human IL-4 may be effective in treatment of chronic systemic autoimmunities such as SLE and GVHD.
A variety of microbicidal infections are characterized by depressed cellular but enhanced humoral immune responses, which suggests a T.sub.H2 type of response to infection. This T.sub.H2 phenotype is characterized by T cell secretion of IL-4, as detailed earlier. IL-4 blocks the microbicidal activity of IFN-gamma activated macrophages in fighting Leishmania major infection (Liew, et al., 1989; Leal, et al., 1993). Inhibition of IL-4 would enhance the T.sub.H1 effector arm of the immune response enhancing cellular immunity and leading to the resolution of infection. Neutralization of IL-4 in vivo allows mice otherwise susceptible to Leishmania major infection to fight off the parasite and clear the infection (Heinzel, et al., 1989). Several informative studies have looked at the T.sub.H1 /T.sub.H2 phenotypic distinction in infected mice, and suggest a T.sub.H1 dominated response being most effective in fighting microbial infection (for a review, see Sher and Coffman, 1992).
IL-10
IL-10 is a cytokine produced by the Th2 cells, but not Th1 cells, and inhibits synthesis of most of all cytokines produced by Th1 cells but not Th2 cells (Mosmann et al., 1991). In addition to the effect on CD4.sup.+ cells with Th1 phenotype, IL-10 also inhibits CD8.sup.+ T cells with "Th1-like" phenotype. IL-10 is a potent suppressor of macrophage activation. It can suppress the production of proinflammatory cytokines, including TNF.alpha., IL-1, IL-6, IL-8 and IFN-gamma. Overall, these results suggest that IL-10 is a potent macrophage deactivator and an effective anti-inflammatory reagent. In addition, IL-10 prevents the IFN-.gamma.-induced synthesis of nitric oxide, resulting in decreased resistance to intracellular parasites (Gazzinelli et al., 1992).
Both human and mouse (hIL-10 and mIL-10, respectively) have been cloned and expressed (Moore et al., 1990; Vieira et al., 1991). The two cDNAs exhibit high degree of nucleotide sequence homology (>80%) throughout and encode very similar open reading frames (73% amino acid homology). Both proteins are expressed as noncovalent homodimers that are acid labile (Moore et al., 1993). Whether monomers are equally bioactive is not clear yet. Based on the primary structure IL-10 has been categorized into the four a-helix bundle family of cytokines (Shanafelt et al., 1991). Possibly due to high degree of sequence homology and similar structure hIL-10 has been shown to be active on mouse cells (Moore et al., 1993) but not vice a versa. hIL-10 is an 18 kDa polypeptide with no detectable carbohydrate; however, in mIL-10 there is one N-linked glycosylation. The recombinant hIL-10 has been expressed in CHO cells, COS7 cells, mouse myeloma cells, the baculovirus expression system and E. coli. The rIL-10 expressed in these systems have indistinguishable biological behavior (Moore et al., 1993).
Parasitic infection often leads to polarized immune response of either Th1 or Th2 type which can mediate protection or susceptibility. The outcome of a parasitic infection depends on the nature of the parasite and the host. The best understood example is Leishmania major infection in mice. L. major is a protozoan parasite that establishes an intracellular infection in macrophages, where it is mainly localized in phagolysosomes. Activated macrophages can efficiently destroy the intracellular parasite and thus parasitic protection is achieved by macrophage activation. Nonactivated macrophages do not kill these organisms. As expected, activation of macrophages upon IFN-gamma treatment enhanced the protection, whereas IL-4 and IL-10 blocked the increased microbicidal activity induced by IFN-gamma (Liew et al., 1989). In most inbred strains (example, C57/BL6) cutaneous infection of L. major often leads to localized infection with spontaneous healing and confers resistance to reinfection. However, in BALB/c mice, L. major infection induces nonprotective immune response by producing IL-4. The antibody response mediated by IL-4 is ineffective and leads to death (Howard et al., 1980). In healing strains a strong Th1 response has been noticed with high level of IFN-.gamma., whereas in susceptible BALB/c mice a nonproductive Th2 response with significant levels of IL-4 was found (Heinzel et al., 1991). Further it was shown that a single injection of monoclonal anti-IFN-gamma antibody can convert a resistance into a susceptible mouse (Belosevic et al., 1989). As expected, the treatment of BALB/c mice with anti-IL-4 antibody led to the development of Th1 response and healing (Sher & Coffman, 1992). Thus, depending on the nature of the pathogen, changing the immune response to a T cell subset with a protective phenotype can lead to therapeutic intervention of the disease state. Understanding the regulation between the Th1 and Th2 phenotype mediated by cytokines will help in designing cytokine-antagonist in therapeutics. The production of IL-10 is strongly increased in mice infected with various pathogens such as Leishmania major, Schistosoma mansoni, Trypanosoma cruzi and Mycobacterium Leprae (Sher, et al, 1992; Salgame et al., 1991, Heinzel et al., 1991).
When designing immune therapy to facilitate mounting the right arm of defense mechanism toward pathogens, it is important to maintain a balance between the two arms also. Th2-type responses may be important in controlling the tissue damage mediated by Th1 cells during the response to an intracellular infectious agent. Keeping some Th1 cells functioning in a predominantly Th2 environment can help abrogate damaging effects of Th1 by secreting IL-10 and IL-4. One extreme of the spectrum of Th1/Th2 is reflected in transgenic mice lacking the IL-10 gene (Kuhn et al., 1993). The IL-10 deficient mouse is normal with respect to its development of T and B cell subsets. However these mice develop chronic enterocolitis (or inflammatory bowel disease) due to chronic inflammation via continuous overproduction of cytokines such as TNF.alpha. and IFN-gamma(Th1 response).
IL-12 can also induce the development of the Th1 subset. By using Lysteria monocytogen, an intracellular gram-positive bacterium, infection in antibody T cell receptor transgenic mice as a model it has been shown that IL-10 can block the production of IL-12 from macrophages (Hsieh et al., 1993) Thus an IL-10-antagonist will tip the Th1/Th2 population predominantly to Th2 type environment by 1, preventing the inhibition of the production of Th1 cytokines 2 by allowing the production of a cytokine that induces the development of Th1 subset.
With experimental evidence in hand it has been proposed that the resistance and/or progression to AIDS is dependent on a Th1/Th2 stage of an individual (Clerici & Shearer, 1993). This hypothesis is based on the findings that progression to AIDS is characterized by loss of IL-2 and IFN-gamma production (loss of Th1 response) with increase in IL-4 and IL-10 (acquired Th2 response). Many seronegatives (HIV-exposed individuals) generate a strong Th1-type response. It is important to note that after seroconversion both IL-4 and IL-10 levels go up at the expense of IL-4 and IFN-gamma. However, in full-blown AIDS patients, Th2 response seems to be mediated by high levels of IL-10 but not with IL-4, the level of which goes down to normal in these individuals. An anti-IL-10 reagent may serve as a potential therapeutic in shifting the Th2 response to Th1 in AIDS patients to offer protection.
TNF.alpha.
TNF.alpha. is an extracellular cytokine and a central mediator of the immune and inflammatory response (Beutler et al., 1989; Vassalli, 1992). It is a homo-trimer (Smith et al., 1987, Eck et al., 1988), and has a subunit size of 17 kD. It circulates at concentrations of less than 5 pg/ml in healthy individuals (Dinarello et al., 1993) and it can go as high as 1000 pg/ml in patients with sepsis syndrome (Casey et al., 1993). The human TNF.alpha. is nonglycosylated, whereas in some other species (notably the mouse) glycosylation occurs on a single N-linked site in the mature protein, but the sugar moiety is not essential for biological activity (Beutler et al., 1989). The human TNF.alpha. is acidic with a pH of 5.3 (Aggarwal et al., 1985). Each TNF.alpha. subunit consists of an anti parallel .beta.-sandwich and it participates in a trimer formation by an edge-to-face packing of .beta.-sheets. The structure of the TNF.alpha. trimer resembles the "jelly-roll" structural motif characteristic of viral coat proteins (Jones et al., 1989). TNF.alpha. is a relatively stable molecule and may be exposed to chaotropic agents such as urea, SDS, or guanidinium hydrochloride, and renatured with recovery of as much as 50% of the initial biological activity. The TNF.alpha. renaturability may reflect the limited number of internal disulfide bonds (one per monomer) required for maintenance of structure (Beutler et al., 1989).
Another related molecule, TNF.beta., has the same bioactivity as TNF.alpha.. The interspecies sequence identity within the TNF.alpha. and TNF.beta. families is 71% and 61%, respectively (Beutler et al., 1989). The sequence identity between hTNF-.alpha. and hTNF-.beta. is only 29% (Beutler et al., 1989). Despite their low similarity, both hTNF.alpha. and hTNF.beta. bind to the same receptors with comparable affinities.
TNF.alpha. mediates its bioactivity through binding to cell surface receptors. The TNF.alpha. receptors are found on the surface of virtually all somatic cells tested (Vassalli, 1992). Two distinct TNF.alpha. receptors have been characterized of apparent molecular weights 55 kD (p55 TNF.alpha.-R1) and 75 kD (p75 TNF.alpha.-R2) (Hohmann et al., 1989; Brockhaus et al., 1990; Loetscher et al., 1991). Both receptors bind TNF.alpha. and TNF.beta. with high affinities (Kd=0.3-0.6 nM) (Loetscher et al., 1990; Schall et al., 1990; Pennica et al., 1992).
TNF.alpha. has diverse activities, and thus is implicated in several diseases as follows:
Septic shock. Sepsis incidents have been increasing for the last 60 years and is the most common cause of death in intensive care units in the United States (Parrillo, 1991). The mortality of septic shock remains at approximately 50% despite the standard use of aggressive antibiotics and cardiovascular support for the past 10 years (Parrillo, 1991). The evidence implicating TNF.alpha. in sepsis is as follows. Pretreatment of mice or baboons with monoclonal antibodies to TNF.alpha. protects them from lethal doses of E. coli LPS (Beutler et al., 1985). Anti-TNF.alpha. antibodies protect primates against lethal endotoxin sepsis and against lethal S. aureus-induced shock (Fiedler et al., 1992; Hinshaw et al., 1992). Soluble-TNF.alpha.-receptor (p55)-IgG-Fc fusions (TNF.alpha. receptor immunoadhesin) were found to protect mice from endotoxic shock, even when administered lhr after endotoxin infusion. The same immunoadhesin was also effective against listeriosis in mice (Haak-Frendscho et al., 1994). Another immunoadhesin based on the p75 receptor was also shown to be effective in lethal endotoxemia and it was functioning simultaneously as both TNF.alpha. carrier and TNF.alpha. antagonist (Mohler et al., 1993).
Cachexia. In vivo administration of TNF.alpha. causes cachexia in mice (Oliff et al., 1987). Therefore, TNF.alpha. antagonists may protect cancer or AIDS infected patients from cachexia.
Cerebral malaria. High levels of TNF.alpha. are associated with poor prognosis in children with cerebral malaria, and antibodies to TNF.alpha. protect mice from cerebral complications of Plasmodium berghei infection (Grau et al., 1987).
Arthritis. Antibodies to TNF.alpha. reduce the production of the inflammatory cytokine, IL-1 in synovial cells (Brennan et al., 1989). TNF.alpha. is an inducer of collagenase, the major destructive protease in rheumatoid arthritis (Brennan et al., 1989). Anti-TNF.alpha. antibodies were found to ameliorate joint disease in murine collagen-induced arthritis (Williams et al., 1992). Transgenic mice carrying the hTNF.alpha. gene develop arthritis which can be prevented by in vivo administration of a monoclonal antibody against hTNF.alpha. (Keffer et al., 1991).
Graft Rejection and Graft versus Host Reaction (GVHR). TNF.alpha. has been implicated in the acute phase of graft-versus-host disease and in renal allograft rejection. Antagonists of TNF.alpha. may then be able to prevent these life-threatening conditions. Anti-TNF.alpha. antibodies have been found to delay graft rejection in experimental animals (Piguet, 1992). Also, injection of anti-TNF.alpha. antibodies during the acute phase of GVHR reduces mortality, and the severity of intestinal, epidermal, and alveolar lesions (Piguet, 1992). Clinical trials of the efficacy of anti-TNF.alpha. antibody in human bone marrow transplantation are underway.
AIDS. Studies of intracellular signal transduction pathways revealed that TNF.alpha. induces proteins that bind to kB-like enhancer elements and thus takes part in the control of NF-kB-inducible genes (Lenardo et al., 1989; Lowenthal et al., 1989; Osborn et al., 1989). The antiviral activity of TNF.alpha. at least in part is mediated by the interaction of NF-kB with a virus-inducible element in the .beta.-interferon gene (Goldfeld et al., 1989; Visvanathan et al., 1989). By an analogous mechanism, TNF.alpha. appears to activate human immunodeficiency virus type I (Duh et al., 1989; Folks et al., 1989). Therefore, TNF.alpha. antagonists may prove useful in delaying the activation of the AIDS virus and may work in conjunction with other treatments in the cure of AIDS.
Parkinson's disease. Recently, elevated TNF.alpha. levels have been found in the brain and the cerebrospinal fluid of Parkinsonian patients (Mogi et al., 1994). This report speculates that elevated TNF.alpha. levels may be related to neuronal degeneration associated with the disease.
RANTES
RANTES is a small (MW 8-kD) highly basic (pI.about.9.5) chemokine that belongs to the CC group (Schall, 1991; Baggiolini et al., 1994). It does not appear to be glycosylated (Schall, 1991) and is a chemoattractant for monocytes (Schall et al., 1990; Wang et al., 1993; Wiedermann et al., 1993), basophils (Bischoff et al., 1993; Kuna et al., 1993), eosinophils (Rot et al., 1992), and CD4.sup.+ /UCHL1.sup.+ T lymphocytes which are thought to be prestimulated or primed helper T cells involved in memory T cell function (Schall et al., 1990). RANTES is not only a chemoattractant but it also stimulates cells to release their effectors leading to tissue damage. For example, RANTES causes histamine release from basophils (Kuna et al., 1992; Kuna et al., 1993; Alam et al., 1993). It also causes the secretion of eosinophil basic peptide (Alam et al., 1993) and the production of oxygen free radicals (Rot et al., 1992) by eosinophils.
Initially, it was thought that RANTES was synthesized by activated T cells but recently other cells were found to synthesize it very fast upon stimulation. RANTES mRNA is expressed late (3 to 5 days) after activation of resting T cells, whereas in fibroblasts, renal epithelial and mesangial cells, RANTES mRNA is quickly up-regulated by TNF.alpha. stimulation (Nelson et al., 1993).
Receptors for RANTES have been identified. There is a promiscuous receptor on the surface of erythrocytes that binds all chemokines with a Kd=5 nM (Horuk et al., 1993; Neote et al., 1993). This receptor is thought to be a sink for chemokines to help in the establishment of chemotactic gradients. Signal transducing receptors have also been identified and cloned (Gao et al., 1993; Neote et al., 1993; Van-Riper et al., 1993; Wang et al., 1993). Monocytes carry a G-protein coupled receptor that binds RANTES with estimated Kd of 400 pM, but also MCAF and MIP-1a with lower affinities (estimated Kd of 6 and 1.6 nM respectively) (Wang et al., 1993). A receptor molecule has been cloned from neutrophils that can bind RANTES with a lower affinity of about 50 nM (Gao et al., 1993).
Disease State. RANTES antagonists may have therapeutic application in inflammation. Blockage of the chemoattractant and effector cell activation properties of RANTES would block local inflammation and tissue damage. The mechanism of action of the RANTES antagonist will be the inhibition of RANTES binding to cell surface receptors.
RANTES is chemoattractant for monocytes, basophils, eosinophils and memory lymphocytes. Basophils are the major source of mediators such as histamine and peptido-leukotrienes, and are an essential element of the late-phase responses to allergens in hypersensitivity diseases. These cells are also involved in other inflammatory pathologies, including certain autoimmune reactions, parasitic infections and inflammatory bowel diseases. In these conditions, basophil recruitment and activation is independent of IgE. Numerous reports have accumulated over the years that describe the effects of a group of elusive stimuli operationally called "histamine-releasing factors." A large number of these elusive stimuli may well be contributed by RANTES.
Eosinophils also are important in allergic inflamation, and together with lymphocytes, form prominent infiltrates in the bronchial mucosa of patients with asthma. They are believed to be the cause of epithelial damage and the characteristic airway hyper-reactivity. The recruitment of lymphocytes of the Th2 type, which comigrate with eosinophils into sites of late-phase reactions, is an important source of other chemoattractant cytokines and growth factors that prime eosinophils.
RANTES, with its effects on monocytes, basophils, eosinophils and lymphocytes appears to be a potent stimulator of effector-cell accumulation and activation in chronic inflammatory diseases and in particular, allergic inflammation.
The recruitment system of inflammatory cells has some redundancy built into it. However, RANTES has some unique properties. It is a more potent chemoattractant than MCP-1 and MIP-1.alpha., while MCP-1 is more potent stimulator of histamine release from basophils (Baggiolini et al., 1994). RANTES causes the production of oxygen radicals by eosinophiles while MIP-1.alpha. cannot (Rot et al., 1992). RANTES is as potent as C5a in the recruitment of eosinphils, but not as potent a trigger of the eosinophil oxidation burst (Rot et al., 1992). C5a is a very potent chemoattractant: however, it lacks the specificity of RANTES. It attracts not only basophils and eosinophils but also neutrophils. Since the eosinophils, but not the neutrophils, are important in the pathophysiology of some inflammatory conditions, such as the allergen-induced late-phase reaction and asthma, specific chemoattractants such as RANTES are expected to be involved.
Using in situ hybridization, RANTES expression has been found in interstitial mononuclear cells and proximal tubular epithelial cells in human kidney transplants undergoing rejection. Antibody staining revealed the presence of RANTES not only within the interstitial infiltrate and renal tubular epithelial cells but also in high abundance in inflamed endothelium (Wiedermann et al., 1993). Based on these results a haptotactic mechanism was postulated. Haptotaxis is defined as cell migration induced by surface-bound gradients. The haptotactic mechanism was supported by in vitro experiments and anti-RANTES antibodies have been found to prevent that in vitro haptotaxis.
Human rheumatoid synovial fibroblasts express mRNA for RANTES and IL-8 after stimulation with TNF.alpha. and IL-1.beta. (Rathanaswami et al., 1993). There is a differential regulation of expression of IL-8 and RANTES mRNA. Cycloheximide enhanced the mRNA levels for IL-8 and RANTES after stimulation with IL-1.beta. but reduced the levels of RANTES mRNA after stimulation with TNF.alpha.. Also, IL-4 down-regulates and IFN-gamma enhances the TNF.alpha. and IL-1.beta. induced increase in RANTES mRNA, whereas the induction of IL-8 mRNA by TNF.alpha. or IL-1.beta. was inhibited by IFN-gamma and augmented by IL-4. Moreover, the combination of TNF.alpha. and IL-1.beta. synergistically increased the level of IL-8 mRNA, whereas under the same conditions, the levels of RANTES mRNA were less than those induced with TNF.alpha. alone. These studies suggest that the synovial fibroblasts may participate in the ongoing inflammatory process in rheumatoid arthritis, and RANTES might be one of the participating effectors. The observed differential regulation of IL-8 and RANTES indicates that the type of cellular infiltrate and the progress of the inflammatory disease is likely to depend on the relative levels of stimulatory and inhibitory cytokines.
RANTES has also been implicated in atherosclerosis and possibly in postangioplasty restenosis (Schall, 1991). The participation of MCP-1 in atherosclerosis has been studied to a greater extent. Recently mRNAs for RANTES, MIP-1.alpha. and MIP-1.beta. have been detected in in situ in normal carotid plaque and heart transplant atherosclerosis. RANTES mRNA is not detected in the same cells expressing MIP-1.alpha. and MIP-1.beta., but it is expressed in lymphocytes and macrophages typically more proximal to the lumen. The data argue for positive feed-back mechanisms for the CC chemokines and possible differential expression of these chemokines at various stages in the progression of arterial disease.
Finally, elevated RANTES levels have been correlated with endometriosis (Khorram et al., 1993). RANTES levels were elevated in pelvic fluids from women with endometriosis, and these levels correlate with the severity of the disease.
Protein Homology between Human and Animal. The murine RANTES has been cloned (Schall et al., 1992). Sequence analysis revealed 85% amino acid identity between the human and mouse proteins. The human and murine RANTES exhibit immune crossreactivity. Boyden chamber chemotaxis experiments reveal some lack of species specificity in monocyte chemoattractant potential, as recombinant muRANTES attracts human monocytes in a dose-dependent fashion in vitro. Also, hRANTES transfection into mouse tumor cell lines produce tumors in which the secretion of hRANTES by those tumors correlates with increased murine monocyte infiltration in vivo (Schall et al., 1992).
SELEX
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 07/536,428, entitled "Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled "Nucleic Acid Ligands," now issued as U.S. Pat. No. 5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled "Methods for Identifying Nucleic Acid Ligands," now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure, now abandoned (see U.S. Pat. No. 5,707,796" describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic Acid Ligands, now abandoned," describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine, now abandoned (see U.S. Pat. No. 5,580,737)," describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,", now abandoned (see U.S. Pat. No. 5,683,867) describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled "Nucleic Acids Ligands to HIV-RT and HIV-1 Rev" now issued as U.S. Pat. No. 5,496,938 describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX,", now U.S. Pat. No. 5,705,337, describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,", now abandoned (see U.S. Pat. No. 5,660,985) that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method of Preparation of Known and Novel 2' Modified Nucleosides by Intramolecular Nucleophilic Displacement," describes oligonucleotides containing various 2'-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX", now U.S. Pat. No. 5,647,459, and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX,", now U.S. Pat. No. 5,683,867, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.

High affinity nucleic acid ligands of cytokines


A hot part (450) of a gas turbine includes a planar member having a gas path surface (451) that faces at least one of an inner side and an outer side in a radial direction of the rotor in a combustion gas flow path. A cooling flow path is formed in an interior of the planar member along an outer peripheral surface (452) that intersects the gas path surface (451). The planar member has an outer peripheral surface side turbulator (482) that protrudes from an inner surface on an outer peripheral surface (452) side of the cooling flow path.

In a gas turbine, air pressurized in a compressor is mixed with a fuel in a combustor, generating a combustion gas which is a high-temperature fluid. The combustion gas is introduced into a combustion gas flow path of a turbine in which vanes and blades are alternately disposed. In the turbine, the blades and a rotor are rotated by the combustion gas that flows through the combustion gas flow path. As a result, the turbine converts the energy of the combustion gas into rotational energy, and applies a rotational driving force to the compressor and a power generator.
The combustion gas flow path through which the combustion gas flows is surrounded by a wall portion on an inner peripheral side and an outer peripheral side, and forms a ring shape. The wall portion of the combustion gas flow path is formed by arranging planar hot parts in a circumferential direction and an axial direction. Specifically, on the inner peripheral side of the combustion gas flow path, inner shrouds attached to vane base ends are arranged in the circumferential direction in a ring shape. In addition, on the inner peripheral side of the combustion gas flow path, platforms attached to blade base ends are arranged in the circumferential direction in a ring shape, in a position adjacent to the inner shrouds in the axial direction along the combustion gas flow path. Accordingly, ring-shaped bodies of the inner shrouds and the platforms are alternately arranged in the axial direction of the rotor, and constitute the wall portion on the inner peripheral side. On the outer peripheral side of the combustion gas flow path, outer shrouds attached to vane leading ends are arranged in the circumferential direction in a ring shape. In addition, on the outer peripheral side of the combustion gas flow path, ring segments curved in an arc shape are arranged in the circumferential direction in a ring shape, in a position adjacent to the outer shrouds in the axial direction along the combustion gas flow path. Accordingly, the ring-shaped bodies of the outer shrouds and the ring segments are alternately arranged in the axial direction, and constitute the wall portion on the outer peripheral side.
The hot parts that constitute such a combustion gas flow path and come into contact with the high-temperature combustion gas are cooled utilizing a portion of the compressed air as cooling air. Examples of a structure for cooling the hot parts of the gas turbine include a cooling structure disclosed in Patent Literature 1. In this cooling structure, a cooling air flow path that extends in the axial direction is formed in an end surface of a shroud of a vane. Specifically, a channel that extends in the axial direction is formed in the end surface of the shroud of the vane. An opening of this channel is blocked by a plug, thereby forming the cooling air flow path that extends in the axial direction.

Hot part of gas turbine, gas turbine including the same, and manufacturing method of hot part of gas turbine


Method and arrangement for continuous filtering of particles out of a contaminated liquid by means of a number of separate filtering units (14) in order continuously to feed purified liquid to a consumer. Each filtering unit (14) comprises a rotatable, perforated drum (16) with a particle-catching filter means (18) on the outer surface of the drum. A first predetermined degree of coating with particles is created on the filter means of the drums, and purified liquid inside all the drums of the filtering units (14) except at least one, which is kept inactive in a standby state by shutting off the associated pump (26), is then fed to a consumer. The feed of purified liquid from a drum to the consumer is interrupted when a second predetermined, higher degree of coating with particles has been reached on the filter means on an individual drum, at least one filtering unit kept in standby state being activated as replacement for the filtering unit(s) from which the feed of purified liquid has been interrupted. The filter means of each filtering unit shut down is cleaned, and liquid in the tank is made to pass through the cleaned filter means and to circulate back to the tank until the first predetermined degree of coating with particles has been reached on the filter means of the drum. The cleaned shut-down filtering unit is put in a standby state by shutting off its pump. The second, higher degree of coating is detected by means of a respective pressure sensor coordinated with each filtrate outlet pipe (24).

It is previously known when certain process liquids are filtered to filter particles which are smaller than the filter meshes of a filter element by first building up a layer of particles on the filter surface which are larger than the filter meshes and in this way creating a three-dimensional deep filter layer of particles on the filter surface which itself functions as a filter for smaller particles which would otherwise pass through the filter element. Such "coating filters" are therefore suitable for liquid suspensions containing very small particles which can be caught therein.
Conventional coating filters can be built up on a filter element and consist of a suspension containing an aid addition of, for example, cellulose powder to the process liquid to be purified by filtering. This suspension is filtered through the filter element first in order to build up a coating layer on it, after which the process liquid itself can be filtered through the coating. When the coating layer has become saturated (clogged) with particles and the filtrate flow has decreased to too low a level, filtering is interrupted and the filter is cleaned. This leads to a marked reduction in production. This method also requires consumption of filter aid and gives rise to a large quantity of costly waste, which results in heavy system investments and high operating costs.
SE 514 311 C2 describes a method of the kind referred to in the introduction, in which a coating layer of particles originating from the process liquid itself is built up on the outside of a filter element on a perforated drum. A number of filtering units of this kind can work in parallel in a common tank in order to bring about high filtering production, that is to say a large filtrate flow. When the filtrate flow from the filtering units has decreased to a minimum value, which has resulted in a rise in level of the suspension in the tank which is sensed by a liquid level monitor common to the filtering units, filtering is interrupted in all the filtering units for the purpose of cleaning the filter elements by slow rotation of the drums against a scraping device on the outside of the filter elements or simply by rapid rotation of the drum. The result is an intermittent operating procedure and thus a reduction in production when the filter units are to be cleaned.

Method and arrangement for continuous filtering of particles out of a liquid


A terminal block device to which a first wire and a second wire are to be connected includes a terminal block body made of an insulating material, a cover that is made of an insulating material and covers the terminal block body, a shaft member at which a terminal of the first wire and a terminal of the second wire are engaged, a cylindrical relay terminal through which the shaft member is inserted and which is inserted through the cover, a fastening member for fastening the terminal of the first wire and the terminal of the second wire to the relay terminal. The relay terminal includes a first seat surface that is contactable with the terminal of the first wire between the terminal block body and the cover and a second seat surface that is contactable with the terminal of the second wire outside the cover.

The present invention relates to a terminal block device. More particularly, the invention relates to a terminal block device to which a plurality of first wires and a plurality of second wires electrically are to be connected.
Japanese Unexamined Patent Application Publication No. 2012-199191 discloses a terminal block device that includes a resin support and an insert bolt that is partially embedded in the resin support. The insert bolt includes a bolt, a nut that is screwed onto the bolt. The bolt has a head portion, a threaded portion, and a flange portion that has a shape of a collar formed around an edge of the head portion on the threaded portion side. The nut as screwed onto the threaded portion of the bolt has on the side thereof adjacent to the head portion of the bolt a first end surface that contacts with the head portion of the bolt and on the opposite side thereof a second end surface from which part of the threaded portion of the bolt extends out. With the nut screwed on the bolt, the head portion and the flange portion of the bolt and the nut are embedded in the resin support with the second end surface of the nut exposed above the surface of the support.
According to the terminal block device disclosed in Japanese Unexamined Patent Application Publication No. 2012-199191, mounting a crimp terminal to the terminal block device is performed by firstly placing the crimp terminal onto the second end surface of the nut that is exposed above the surface of the resin support and then fastening tight the crimp terminal by a fixing nut until the crimp terminal firmly contacts with the second end surface of the nut. The fastened crimp terminal is pressed against the second end surface of the nut, so that the second end surface of the nut is subjected to a pressure. Therefore, no pressure is applied from the fixing nut to the support and no creep or cold flow occurs in the resin support, and re-tightening of the fixing nut is unnecessary.
Japanese Unexamined Patent Application Publication No. 2005-318742 discloses an electric motor that includes a motor body. The motor body includes a rotor fixed on a shaft, a cylindrical stator disposed around the rotor, an end bracket that covers one end of the stator, and another end bracket that covers the other end of the stator. The stator has a stator winding and a plurality of external connection terminals each including a lead wire is connected to the stator winding. The electric motor further includes a plurality of terminal blocks to which the respective external connection terminals are connected, a terminal cover that covers the external connection terminals and the terminal blocks, and a mounting bracket for mounting the terminal blocks and the terminal cover to the motor body. The external connection terminals are disposed in an L-shape extending from the stator winding radially outwardly and then along the stator in an axial direction of the electric motor.
Japanese Unexamined Patent Application Publication No. 2010-74958 discloses a method for manufacturing a motor, and the motor. The motor disclosed in the above Publication includes a cylindrical frame and a first end bracket that is connected to the rear end of the cylindrical frame. The first end bracket substantially closes an entire opening of the frame at the rear end thereof. An opening is formed between the frame and a first end bracket in upper part thereof. A terminal block is disposed in the opening and fixed to the upper outer surface of the frame. The terminal block includes a terminal block body that has a cuboid shape and is made of a resin and a plurality of bolts that are inserted through the resin terminal block body. The bolts are inserted such that ends of the bolts extend out from or above the upper surface of the terminal block body. The terminal block body is disposed such that the rear surface of the terminal block body faces toward the first end bracket through the terminal block accommodating opening.
The stator includes an iron core having first and second coil ends and a winding wound around the iron core, and a plurality of lead wires of different phases extends out from the first coil end. A round crimp terminal as a connection terminal is fixed to an end of each lead wire. The lead wires extend radially outwardly from the first coil end and bent in an arc shape so as to be directed to the terminal blocks. The round crimp terminals for the respective lead wires are fixed to the terminal blocks by a bolt. Each lead wire is electrically connected to its corresponding terminal block through the round crimp terminal.
However, the terminal block disclosed in Japanese Unexamined Patent Application Publication No. 2012-1991191 has a plurality of terminals connected thereto and the terminals are isolated from one another by partition members for insulation of the wires. Such configuration of the terminal block allows the wires to be arranged or oriented only in one direction. Furthermore, in the terminal block disclosed in the same Publication, a part of the bolt and the nut that is screwed onto the bolt need to be fixed within the molded resin support. Although some terminal blocks allow selection of the orienting direction of wires out of two directions, there still remain problems associated with the restriction of orienting direction of wires for connection to the terminal block, thus limiting the freedom in the wire connection.
In Japanese Unexamined Patent Application Publications No. 2005-318742 and No. 2010-74958, the lead wires of different phases are extended out from the stator, bent into an L-shape or an arc shape, and connected to the respective terminal blocks. Therefore, depending on the manner of bending, the wires may be bent to such an extent that no sufficient insulation distance is provided between the wires and the end bracket. In order to secure a sufficient insulation distance between the wires and the end bracket, the degree of bending of the wire needs to be determined accurately for connecting the wires to the terminal blocks, which makes the connecting work troublesome and time consuming.
The present invention has been made in view of the above problems and it is a first object of the invention to provide a terminal block device that allows an enhanced freedom in the orienting direction of wires for connection to the terminal block device, requires no resin support that is molded with conductive members embedded therein and therefore is capable of preventing creep in the material of the support.
It is a second object of the present invention to provide a terminal block device that allows connection of wires to a terminal block irrespective of the degree of bending of the wires.


Methods for producing metal/metalloid oxide particles comprise rare earth metals herein include reacting a reactant stream in a gas flow. The reactant stream includes a rare earth metal precursor and an oxygen source. A collection of particles comprising metal/metalloid oxide have an average particle size from about 15 nm to about 1 micron. The metal/metalloid oxide comprises a non-rare earth metal oxide wherein less than about 25 percent of a non-rare earth metal is substituted with a rare earth metal. The metal/metalloid oxide particles can be reacted with H2S or C2S to form corresponding metal/metalloid sulfide particles. The metal/metalloid sulfide particles can be doped with rare earth metals. The particles are useful as phosphors, for example for use in displays.

The invention relates to method for synthesizing metal/metalloid oxide particles with rare earth metals, especially metal/metalloid oxides with fluorescent properties. In addition, the invention relates to submicron metal/metalloid oxide particles with rare earth metals and displays produced from these particles.
Electronic displays often use phosphor materials, which emit visible light in response to interaction with electrons. Phosphor materials can be applied to substrates to produce cathode ray tubes, flat panel displays and the like. Improvements in display devices place stringent demands on the phosphor materials, for example, due to decreases in electron velocity and increases in display resolution. Electron velocity is reduced in order to reduce power demands. In particular, flat panel displays generally require phosphors that are responsive to low velocity electrons or low voltages.
In addition, a desire for color display requires the use of materials or combinations of materials that emit light at different wavelengths at positions in the display that can be selectively excited. A variety of materials have been used as phosphors. In order to obtain materials that emit at desired wavelengths of light, activators have been doped into phosphor material. Alternatively, multiple phosphors can be mixed to obtain the desired emission. Furthermore, the phosphor materials must show sufficient luminescence.
In addition, technological advances have increased the demand for improved material processing with strict tolerances on processing parameters. As miniaturization continues even further, material parameters will need to fall within stricter tolerances. Current integrated circuit technology already requires tolerances on processing dimensions on a submicron scale.
Various metal compositions exhibit desired phosphorescent properties upon excitation. Specifically, various metal oxides, including rare earth metal oxides exhibit fluorescence. In addition, doping of rare earth metals into non-rare earth metal oxides can be used to adjust the wavelength and luminosity of the phosphor particles.

High luminescence phosphor particles and methods for producing the particles


The invention relates to medicine and veterinary. The device for magnetotherapy comprises a variable generator (1) of current pulses, an energy accumulator (2) and a circuit (3) for discharge of the energy accumulator (2), including a controlled switching element (5) and an inductor (4) capable of producing a therapeutic pulsed magnetic field, and also a unit (7) for controlling the duration of pulses of the magnetic field produced by the inductor (4) and a controller (10) of the amplitude of pulses of the magnetic field. According to the invention, the discharge circuit (3) of the energy accumulator (2) includes a unit (6) for controlling the slope of the leading edge of pulses of the magnetic field produced by the inductor (4). The device is predominantly intended for arresting and relieving acute pain syndromes, phantom limb pain and attacks of bronchial asthma, and also for treating mastities of cattle.

There is known a device for magnetotherapy, comprising a series connection of a generator of current pulses, an energy accumulator and an accumulator discharge circuit including a controlled switching element and an inductor capable of producing a therapeutic pulsed magnetic field. The device further comprises a unit for controlling the duration of pulses of the magnetic field produced by the inductor, having its output connected to the input of the controlled switching element (SU, A, 1321426).
The known device is intended for relieving the pain in the cases of radiculitis, plexitis and other diseases, and also for controlling the progress of X-ray contrast media in the organism of a patient. In the operation of the known device, the physician selects the patient-dependent amplitude and duration of pulses of the therapeutic magnetic field produced by the inductor, and also the quantity of sessions of treating the patient with the magnetic field. In this, the time required for selecting the parameters of magnetic pulses optimized for the patient in question is typically at least 3 to 7 minutes. Furthermore, as a magnetotherapy session is in progress, the eventually varying characteristics of the units of the device, e.g. on account of their heating, may cause leaps of the amplitude of pulses of the magnetic field produced, the leaps exceeding the permissible value not being positively precluded.
There is further known a device for magnetotherapy, comprising a series connection of a variable generator of current pulses, an energy accumulator and a discharge circuit of the energy accumulator, including a controlled switching element and an inductor capable of producing a therapeutic pulsed magnetic field, and also a unit for controlling the duration of pulses of the magnetic field produced by the inductor, having its output connected to the control input of the switching element, and a controller of the amplitude of pulses of the magnetic field, having its output connected to the input of the generator of current pulses (EP, B, 0048451). The amplitude controller of this device limits the peak values of the amplitude of pulses of the magnetic field. However, the selection of patient-optimized parameters of pulses of the therapeutic magnetic field in operation of this device is performed by way of time-consuming variation of the amplitude and duration of pulses, which, apart from being time-consuming, is not always adequately effective. Thus, this device has been found to be poorly manageable in the selection of the optimized parameters for relieving an acute pain syndrome, phantom limb pains, bronchial asthma attacks.

ulitsa Zaslavskogo, II, korpus 2, kv. 58.