A polynucleotide (hpa) encoding a polypeptide having heparanase activity, vectors including same, genetically modified cells expressing heparanase, a recombinant protein having heparanase activity and antisense oligonucleotides and constructs for modulating heparanase expression.

The present invention relates to a polynucleotide, referred to hereinbelow as hpa, encoding a polypeptide having heparanase activity, vectors (nucleic acid constructs) including same and genetically modified cells expressing heparanase. The invention further relates to a recombinant protein having heparanase activity and to antisense oligonucleotides, constructs and ribozymes for down regulating heparanase activity. In addition, the invention relates to heparanase promoter sequences and their uses.
Heparan sulfate proteoglycans: Heparan sulfate proteoglycans (HSPG) are ubiquitous macromolecules associated with the cell surface and extra cellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (1-4). The basic HSPG structure includes a protein core to which several linear heparan sulfate chains are covalently attached. These polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups (1 -4). Studies on the involvement of ECM molecules in cell attachment, growth and differentiation revealed a central role of HSPG in embryonic morphogenesis, angiogenesis, neurite outgrowth and tissue repair (1-5). HSPG are prominent components of blood vessels (3). In large blood vessels they are concentrated mostly in the intima and inner media, whereas in capillaries they are found mainly in the subendothelial basement membrane where they support proliferating and migrating endothelial cells and stabilize the structure of the capillary wall. The ability of HSPG to interact with ECM macromolecules such as collagen, laminin and fibronectin, and with different attachment sites on plasma membranes suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion. Cleavage of the heparan sulfate (HS) chains may therefore result in degradation of the subendothelial ECM and hence may play a decisive role in extravasation of blood-borne cells. HS catabolism is observed in inflammation, wound repair, diabetes, and cancer metastasis, suggesting that enzymes which degrade HS play important roles in pathologic processes. Heparanase activity has been described in activated immune system cells and highly metastatic cancer cells (6-8), but research has been handicapped by the lack of biologic tools to explore potential causative roles of heparanase in disease conditions.
Involvement of Heparanase in Tumor Cell Invasion and Metastasis: Circulating tumor cells arrested in the capillary beds of different organs must invade the endothelial cell lining and degrade its underlying basement membrane (BM) in order to invade into the extravascular tissue(s) where they establish metastasis (9, 10). Metastatic tumor cells often attach at or near the intercellular junctions between adjacent endothelial cells. Such attachment of the metastatic cells is followed by rupture of the junctions, retraction of the endothelial cell borders and migration through the breach in the endothelium toward the exposed underlying BM (9). Once located between endothelial cells and the BM, the invading cells must degrade the subendothelial glycoproteins and proteoglycans of the BM in order to migrate out of the vascular compartment. Several cellular enzymes (e.g., collagenase IV, plasminogen activator, cathepsin B, elastase, etc.) are thought to be involved in degradation of BM (10). Among these enzymes is an endo-β-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites (6, 8, 11). Expression of a HS degrading heparanase was found to correlate with the metastatic potential of mouse lymphoma (11), fibrosarcoma and melanoma (8) cells. Moreover, elevated levels of heparanase were detected in sera from metastatic tumor bearing animals and melanoma patients (8) and in tumor biopsies of cancer patients (12).
The control of cell proliferation and tumor progression by the local microenvironment, focusing on the interaction of cells with the extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells, was investigated previously by the present inventors. This cultured ECM closely resembles the subendothelium in vivo in its morphological appearance and molecular composition. It contains collagens (mostly type III and IV, with smaller amounts of types I and V), proteoglycans (mostly heparan sulfate- and dermatan sulfate-proteoglycans, with smaller amounts of chondroitin sulfate proteoglycans), laminin, fibronectin, entactin and elastin (13, 14). The ability of cells to degrade HS in the cultured ECM was studied by allowing cells to interact with a metabolically sulfate labeled ECM, followed by gel filtration (Sepharose 6B) analysis of degradation products released into the culture medium (11). While intact HSPG are eluted next to the void volume of the column (Kav<0.2, Mr˜0.5×106), labeled degradation fragments of HS side chains are eluted more toward the Vt of the column (0.5<kav<0.8, Mr=5-7×103) (11).
The heparanase inhibitory effect of various non-anticoagulant species of heparin that might be of potential use in preventing extravasation of blood-borne cells was also investigated by the present inventors. Inhibition of heparanase was best achieved by heparin species containing 16 sugar units or more and having sulfate groups at both the N and O positions. While O-desulfation abolished the heparanase inhibiting effect of heparin, O-sulfated, N-acetylated heparin retained a high inhibitory activity, provided that the N-substituted molecules had a molecular size of about 4,000 daltons or more (7). Treatment of experimental animals with heparanase inhibitors (e.g., non-anticoagulant species of heparin) markedly reduced (>90%) the incidence of lung metastases induced by B 16 melanoma, Lewis lung carcinoma and mammary adenocarcinoma cells (7, 8, 16). Heparin fractions with high and low affinity to anti-thrombin III exhibited a comparable high anti-metastatic activity, indicating that the heparanase inhibiting activity of heparin, rather than its anticoagulant activity, plays a role in the anti-metastatic properties of the polysaccharide (7).
Heparanase activity in the urine of cancer patients: In an attempt to further elucidate the involvement of heparanase in tumor progression and its relevance to human cancer, urine samples for heparanase activity were screened (16a). Heparanase activity was detected in the urine of some, but not all, cancer patients. High levels of heparanase activity were determined in the urine of patients with an aggressive metastatic disease and there was no detectable activity in the urine of healthy donors.
Heparanase activity was also found in the urine of 20% of normal and microalbuminuric insulin dependent diabetes mellitus (IDDM) patients, most likely due to diabetic nephropathy, the most important single disorder leading to renal failure in adults.
Possible involvement of heparanase in tumor angiogenesis: Fibroblast growth factors are a family of structurally related polypeptides characterized by high affinity to heparin (17). They are highly mitogenic for vascular endothelial cells and are among the most potent inducers of neovascularization (17, 18). Basic fibroblast growth factor (bFGF) has been extracted from the subendothelial ECM produced in vitro (19) and from basement membranes of the cornea (20), suggesting that ECM may serve as a reservoir for bFGF. Immunohistochemical staining revealed the localization of bFGF in basement membranes of diverse tissues and blood vessels (21). Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Studies on the interaction of bFGF with ECM revealed that bFGF binds to HSPG in the ECM and can be released in an active form by HS degrading enzymes (15, 20, 22). It was demonstrated that heparanase activity expressed by platelets, mast cells, neutrophils, and lymphoma cells is involved in release of active bFGF from ECM and basement membranes (23), suggesting that heparanase activity may not only function in cell migration and invasion, but may also elicit an indirect neovascular response. These results suggest that the ECM HSPG provides a natural storage depot for bFGF and possibly other heparin-binding growth promoting factors (24, 25). Displacement of bFGF from its storage within basement membranes and ECM may therefore provide a novel mechanism for induction of neovascularization in normal and pathological situations.
Recent studies indicate that heparin and HS are involved in binding of bFGF to high affinity cell surface receptors and in bFGF cell signaling (26, 27). Moreover, the size of HS required for optimal effect was similar to that of HS fragments released by heparanase (28). Similar results were obtained with vascular endothelial cells growth factor (VEGF) (29), suggesting the operation of a dual receptor mechanism involving HS in cell interaction with heparin-binding growth factors. It is therefore proposed that restriction of endothelial cell growth factors in ECM prevents their systemic action on the vascular endothelium, thus maintaining a very low rate of endothelial cells turnover and vessel growth. On the other hand, release of bFGF from storage in ECM as a complex with HS fragment, may elicit localized endothelial cell proliferation and neovascularization in processes such as wound healing, inflammation and tumor development (24, 25).
Expression of heparanase by cells of the immune system: Heparanase activity correlates with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses. Interaction of platelets, granulocytes, T and B lymphocytes, macrophages and mast cells with the subendothelial ECM is associated with degradation of HS by a specific heparanase activity (6). The enzyme is released from intracellular compartments (e.g., lysosomes, specific granules, etc.) in response to various activation signals (e.g., thrombin, calcium ionophore, immune complexes, antigens, mitogens, etc.), suggesting its regulated involvement in inflammation and cellular immunity.
Some of the observations regarding the heparanase enzyme were reviewed in reference No. 6 and are listed hereinbelow:
First, a proteolytic activity (plasminogen activator) and heparanase participate synergistically in sequential degradation of the ECM HSPG by inflammatory leukocytes and malignant cells.
Second, a large proportion of the platelet heparanase exists in a latent form, probably as a complex with chondroitin sulfate. The latent enzyme is activated by tumor cell-derived factor(s) and may then facilitate cell invasion through the vascular endothelium in the process of tumor metastasis.
Third, release of the platelet heparanase from α-granules is induced by a strong stimulant (i.e., thrombin), but not in response to platelet activation on ECM.
Fourth, the neutrophil heparanase is preferentially and readily released in response to a threshold activation and upon incubation of the cells on ECM.
Fifth, contact of neutrophils with ECM inhibited release of noxious enzymes (proteases, lysozyme) and oxygen radicals, but not of enzymes (heparanase, gelatinase) which may enable diapedesis. This protective role of the subendothelial ECM was observed when the cells were stimulated with soluble factors but not with phagocytosable stimulants.
Sixth, intracellular heparanase is secreted within minutes after exposure of T cell lines to specific antigens.
Seventh, mitogens (Con A, LPS) induce synthesis and secretion of heparanase by normal T and B lymphocytes maintained in vitro. T lymphocyte heparanase is also induced by immunization with antigen in vivo.
Eighth, heparanase activity is expressed by pre-B lymphomas and B-lymphomas, but not by plasmacytomas and resting normal B lymphocytes.
Ninth, heparanase activity is expressed by activated macrophages during incubation with ECM, but there was little or no release of the enzyme into the incubation medium. Similar results were obtained with human myeloid leukemia cells induced to differentiate to mature macrophages.
Tenth, T-cell mediated delayed type hypersensitivity and experimental autoimmunity are suppressed by low doses of heparanase inhibiting non-anticoagulant species of heparin (30).
Eleventh, heparanase activity expressed by platelets, neutrophils and metastatic tumor cells releases active bFGF from ECM and basement membranes. Release of bFGF from storage in ECM may elicit a localized neovascular response in processes such as wound healing, inflammation and tumor development.
Twelfth, among the breakdown products of the ECM generated by heparanase is a tri-sulfated disaccharide that can inhibit T-cell mediated inflammation in vivo (31). This inhibition was associated with an inhibitory effect of the disaccharide on the production of biologically active TNFα by activated T cells in vitro (31).
Other potential therapeutic applications: Apart from its involvement in tumor cell metastasis, inflammation and autoimmunity, mammalian heparanase may be applied to modulate: bioavailability of heparin-binding growth factors (15); cellular responses to heparin-binding growth factors (e.g., bFGF, VEGF) and cytokines (IL-8) (31a, 29); cell interaction with plasma lipoproteins (32); cellular susceptibility to certain viral and some bacterial and protozoa infections (33, 33a, 33b); and disintegration of amyloid plaques (34). Heparanase may thus prove useful for conditions such as wound healing, angiogenesis, restenosis, atherosclerosis, inflammation, neurodegenerative diseases and viral infections. Mammalian heparanase can be used to neutralize plasma heparin, as a potential replacement of protamine. Anti-heparanase antibodies may be applied for immunodetection and diagnosis of micrometastases, autoimmune lesions and renal failure in biopsy specimens, plasma samples, and body fluids. Common use in basic research is expected.
The identification of the hpa gene encoding for heparanase enzyme will enable the production of a recombinant enzyme in heterologous expression systems. Availability of the recombinant protein will pave the way for solving the protein structure function relationship and will provide a tool for developing new inhibitors.
Viral Infection: The presence of heparan sulfate on cell surfaces have been shown to be the principal requirement for the binding of Herpes Simplex (33) and Dengue (33a) viruses to cells and for subsequent infection of the cells. Removal of the cell surface heparan sulfate by heparanase may therefore abolish virus infection. In fact, treatment of cells with bacterial heparitinase (degrading heparan sulfate) or heparinase (degrading heparan) reduced the binding of two related animal herpes viruses to cells and rendered the cells at least partially resistant to virus infection (33). There are some indications that the cell surface heparan sulfate is also involved in HIV infection (33b).
Neurodegenerative diseases: Heparan sulfate proteoglycans were identified in the prion protein amyloid plaques of Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease and Scrape (34). Heparanase may disintegrate these amyloid plaques which are also thought to play a role in the pathogenesis of Alzheimer's disease.
Restenosis and Atherosclerosis: Proliferation of arterial smooth muscle cells (SMCs) in response to endothelial injury and accumulation of cholesterol rich lipoproteins are basic events in the pathogenesis of atherosclerosis and restenosis (35). Apart from its involvement in SMC proliferation (i.e., low affinity receptors for heparin-binding growth factors), HS is also involved in lipoprotein binding, retention and uptake (36). It was demonstrated that HSPG and lipoprotein lipase participate in a novel catabolic pathway that may allow substantial cellular and interstitial accumulation of cholesterol rich lipoproteins (32). The latter pathway is expected to be highly atherogenic by promoting accumulation of apoB and apoE rich lipoproteins (i.e. LDL, VLDL, chylomicrons), independent of feed back inhibition by the cellular sterol content. Removal of SMC HS by heparanase is therefore expected to inhibit both SMC proliferation and lipid accumulation and thus may halt the progression of restenosis and atherosclerosis.
Gene Therapy:
The ultimate goal in the management of inherited as well as acquired diseases is a rational therapy with the aim to eliminate the underlying biochemical defects associated with the disease rather then symptomatic treatment. Gene therapy is a promising candidate to meet these objectives. Initially it was developed for treatment of genetic disorders, however, the consensus view today is that it offers the prospect of providing therapy for a variety of acquired diseases, including cancer, viral infections, vascular diseases and neurodegenerative disorders.
The gene-based therapeutic can act either intracellularly, affecting only the cells to which it is delivered, or extracellularly, using the recipient cells as local endogenous factories for the therapeutic product(s). The application of gene therapy may follow any of the following strategies: (i) prophylactic gene therapy, such as using gene transfer to protect cells against viral infection; (ii) cytotoxic gene therapy, such as cancer therapy, where genes encode cytotoxic products to render the target cells vulnerable to attack by the normal immune response; (iii) biochemical correction, primarily for the treatment of single gene defects, where a normal copy of the gene is added to the affected or other cells.
To allow efficient transfer of the therapeutic genes, a variety of gene delivery techniques have been developed based on viral and non-viral vector systems. The most widely used and most efficient systems for delivering genetic material into target cells are viral vectors. So far, 329 clinical studies (phase I, I/II and II) with over 2,500 patients have been initiated Worldwide since 1989 (50).
The approach of gene addition pose serious barriers. The expression of many genes is tightly regulated and context dependent, so achieving the correct balance and function of expression is challenging. The gene itself is often quite large, containing many exons and introns. The delivery vector is usually a virus, which can infect with a high efficiency but may, on the other hand, induce immunological response and consequently decreases effectiveness, especially upon secondary administration. Most of the current expression vector-based gene therapy protocols fail to achieve clinically significant transgene expression required for treating genetic diseases. Apparently, it is difficult to deliver enough virus to the right cell type to elicit an effective and therapeutic effect (51).
Homologous recombination, which was initially considered to be of limited use for gene therapy because of its low frequency in mammalian cells, has recently emerged as a potential strategy for developing gene therapy. Different approaches have been used to study homologous recombination in mammalian cells; some involve DNA repair mechanisms. These studies aimed at either gene disruption or gene correction and include RNA/DNA chimeric oligonucleotides, small or large homologous DNA fragments, or adeno-associated viral vectors. Most of these studies show a reasonable frequency of homologous recombination, which warrants further in vivo testing (52). Homologous recombination-based gene therapy has the potential to develop into a powerful therapeutic modality for genetic diseases. It can offer permanent expression and normal regulation of corrected genes in appropriate cells or organs and probably can be used for treating dominantly inherited diseases such as polycystic kidney disease.
Genomic Sequences Function in Regulation of Gene Expression:
The efficient expression of therapeutic genes in target cells or tissues is an important component of efficient and safe gene therapy. The expression of genes is driven by the promoter region upstream of the coding sequence, although regulation of expression may be supplemented by farther upstream or downstream DNA sequences or DNA in the introns of the gene. Since this important information is embedded in the DNA, the description of gene structure is crucial to the analysis of gene regulation. Characterization of cell specific or tissue specific promoters, as well as other tissue specific regulatory elements enables the use of such sequences to direct efficient cell specific, or developmental stage specific gene expression. This information provides the basis for targeting individual genes and for control of their expression by exogenous agents, such as drugs. Identification of transcription factors and other regulatory proteins required for proper gene expression will point at new potential targets for modulating gene expression, when so desired or required.
Efficient expression of many mammalian genes depends on the presence of at least one intron. The expression of mouse thymidylate synthase (TS) gene, for example, is greatly influenced by intron sequences. The addition of almost any of the introns from the mouse TS gene to an intronless TS minigene leads to a large increase in expression (42). The involvement of intron 1 in the regulation of expression was demonstrated for many other genes. In human factor IX (hFIX), intron 1 is able to increase the expression level about 3 fold mare as compared to that of the hFIX cDNA (43). The expression enhancing activity of intron 1 is due to efficient functional splicing sequences, present in the precursor mRNA. By being efficiently assembled into spliceosome complexes, transcripts with splicing sequences may be better protected in the nucleus from random degradations, than those without such sequences (44).
A forward-inserted intronl-carrying HFIX expression cassette suggested to be useful for directed gene transfer, while for retroviral-mediated gene transfer system, reversely-inserted intron 1-carrying HFIX expression cassette was considered (43).
A highly conserved cis-acting sequence element was identified in the first intron of the mouse and rat c-Ha-ras, and in the first exon of Ha- and Ki-ras genes of human, mouse and rat. This cis-acting regulatory sequence confers strong transcription enhancer activity that is differentially modulated by steroid hormones in metastatic and nonmetastatic subpopulations. Perturbations in the regulatory activities of such cis-acting sequences may play an important role in governing oncogenic potency of Ha-ras through transcriptional control mechanisms (45).
Intron sequences affect tissue specific, as well as inducible gene expression. A 182 bp intron 1 DNA segment of the mouse Co12a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression on a reporter gene in intact mouse embryos, while Co12a1 promoter sequences are dispensable for chondrocyte expression (46). In Co11A1 gene the intron plays little or no role in constitutive expression of collagen in the skin, and in cultured cells derived from the skin, however, in the lungs of young mice, intron deletion results in decrease of expression to less than 50% (47).
A classical enhancer activity was shown in the 2 kb intron fragment in bovine beta-casein gene. The enhancer activity was largely dependent on the lactogenic hormones, especially prolactin. It was suggested that several elements in the intron-1 of the bovine beta-casein gene cooperatively interact not only with each other but also with its promoter for hormonal induction (48).
Identification and characterization of regulatory elements in genomic non-coding sequences, such as introns, provides a tool for designing and constructing novel vectors for tissue specific, hormone regulated or any other defined expression pattern, for gene therapy. Such an expression cassette was developed, utilizing regulatory elements from the human cytokeratin 18 (K18) gene, including 5′ genomic sequences and one of its introns. This cassette efficiently expresses reporter genes, as well as the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, in cultured lung epithelial cells (49).
Alternative Splicing:
Alternative splicing of pre mRNA is a powerful and versatile regulatory mechanism that can effect quantitative control of gene expression and functional diversification of proteins. It contributes to major developmental decisions and also to a fine-tuning of gene function. Genetic and biochemical approaches have identified cis-acting regulatory elements and trans-acting factors that control alternative splicing of specific mRNAs. This mechanism results in the generation of variant isoforms of various proteins from a single gene. These include cell surface molecules such as CD44, receptors, cytokines such as VEGF and enzymes. Products of alternatively spliced transcripts differ in their expression pattern, substrate specificity and other biological parameters.
The FGF receptor RNA undergoes alternative splicing which results in the production of several isoforms, which exhibit different ligand binding specificities. The alternative splicing is regulated in a cell specific manner (53).
Alternative spliced mRNAs are often correlated with malignancy. An increase in specific splice variant of tyrosinase was identified in murine melanomas (54). Multiple splicing variants of estrogen receptor are present in individual human breast tumors. CD44 has various isoform, some are characteristic of malignant tissues.
Identification of tumor specific alternative splice variants provide new tool for cancer diagnostics. CD44 variants have been used for detection of malignancy in urine samples from patients with urothelial cancer by competitive RT-PCR (55). CD44 exon 6 was suggested as prognostic indicator of metastasis in breast cancer (56).
Different enzymes or polypeptides generated by alternative splicing may have different function or catalytic specificity. The identification and characterization of the enzyme forms, which are involved in pathological processes, is crucial for the design of appropriate and efficient drugs.
Modulation of Gene Expression—Antisense Technology:
An antisense oligonucleotide (e.g., antisense oligodeoxyribonucleotide) may bind its target nucleic acid either by Watson-Crick base pairing or Hoogsteen and anti-Hoogsteen base pairing (64). According to the Watson-Crick base pairing, heterocyclic bases of the antisense oligonucleotide form hydrogen bonds with the heterocyclic bases of target single-stranded nucleic acids (RNA or single-stranded DNA), whereas according to the Hoogsteen base pairing, the heterocyclic bases of the target nucleic acid are double-stranded DNA, wherein a third strand is accommodated in the major groove of the B-form DNA duplex by Hoogsteen and anti-Hoogsteen base pairing to form a triple helix structure.
According to both the Watson-Crick and the Hoogsteen base pairing models, antisense oligonucleotides have the potential to regulate gene expression and to disrupt the essential functions of the nucleic acids in cells. Therefore, antisense oligonucleotides have possible uses in modulating a wide range of diseases in which gene expression is altered.
Since the development of effective methods for chemically synthesizing oligonucleotides, these molecules have been extensively used in biochemistry and biological research and have the potential use in medicine, since carefully devised oligonucleotides can be used to control gene expression by regulating levels of transcription, transcripts and/or translation.
Oligodeoxyribonucleotides as long as 100 base pairs (bp) are routinely synthesized by solid phase methods using commercially available, fully automated synthesis machines. The chemical synthesis of oligoribonucleotides, however, is far less routine. Oligoribonucleotides are also much less stable than oligodeoxyribonucleotides, a fact which has contributed to the more prevalent use of oligodeoxyribonucleotides in medical and biological research, directed at, for example, the regulation of transcription or translation levels.
Gene expression involves few distinct and well regulated steps. The first major step of gene expression involves transcription of a messenger RNA (mRNA) which is an RNA sequence complementary to the antisense (i.e., −) DNA strand, or, in other words, identical in sequence to the DNA sense (i.e., +) strand, composing the gene. In eukaryotes, transcription occurs in the cell nucleus.
The second major step of gene expression involves translation of a protein (e.g., enzymes, structural proteins, secreted proteins, gene expression factors, etc.) in which the MRNA interacts with ribosomal RNA complexes (ribosomes) and amino acid activated transfer RNAs (tRNAs) to direct the synthesis of the protein coded for by the MRNA sequence.
Initiation of transcription requires specific recognition of a promoter DNA sequence located upstream to the coding sequence of a gene by an RNA-synthesizing enzyme—RNA polymerase. This recognition is preceded by sequence-specific binding of one or more transcription factors to the promoter sequence. Additional proteins which bind at or close to the promoter sequence may trans upregulate transcription via cis elements known as enhancer sequences. Other proteins which bind to or close to the promoter, but whose binding prohibits the action of RNA polymerase, are known as repressors.
There are also evidence that in some cases gene expression is downregulated by endogenous antisense RNA repressors that bind a complementary mRNA transcript and thereby prevent its translation into a functional protein.
Thus, gene expression is typically upregulated by transcription factors and enhancers and downregulated by repressors.
However, in many disease situation gene expression is impaired. In many cases, such as different types of cancer, for various reasons the expression of a specific endogenous or exogenous (e.g., of a pathogen such as a virus) gene is upregulated. Furthermore, in infectious diseases caused by pathogens such as parasites, bacteria or viruses, the disease progression depends on expression of the pathogen genes, this phenomenon may also be considered as far as the patient is concerned as upregulation of exogenous genes.
Most conventional drugs function by interaction with and modulation of one or more targeted endogenous or exogenous proteins, e.g., enzymes. Such drugs, however, typically are not specific for targeted proteins but interact with other proteins as well. Thus, a relatively large dose of drug must be used to effectively modulate a targeted protein.
Typical daily doses of drugs are from 10−5-10−1 millimoles per kilogram of body weight or 10−3-10 millimoles for a 100 kilogram person. If this modulation instead could be effected by interaction with and inactivation of mRNA, a dramatic reduction in the necessary amount of drug could likely be achieved, along with a corresponding reduction in side effects. Further reductions could be effected if such interaction could be rendered site-specific. Given that a functioning gene continually produces mRNA, it would thus be even more advantageous if gene transcription could be arrested in its entirety.
Given these facts, it would be advantageous if gene expression could be arrested or downmodulated at the transcription level.
The ability of chemically synthesizing oligonucleotides and analogs thereof having a selected predetermined sequence offers means for downmodulating gene expression. Three types of gene expression modulation strategies may be considered.
At the transcription level, antisense or sense oligonucleotides or analogs that bind to the genomic DNA by strand displacement or the formation of a triple helix, may prevent transcription (64).
At the transcript level, antisense oligonucleotides or analogs that bind target mRNA molecules lead to the enzymatic cleavage of the hybrid by intracellular RNase H (65). In this case, by hybridizing to the targeted mRNA, the oligonucleotides or oligonucleotide analogs provide a duplex hybrid recognized and destroyed by the RNase H enzyme. Alternatively, such hybrid formation may lead to interference with correct splicing (66). As a result, in both cases, the number of the target mRNA intact transcripts ready for translation is reduced or eliminated.
At the translation level, antisense oligonucleotides or analogs that bind target mRNA molecules prevent, by steric hindrance, binding of essential translation factors (ribosomes), to the target mRNA, a phenomenon known in the art as hybridization arrest, disabling the translation of such mRNAs (67).
Thus, antisense sequences, which as described hereinabove may arrest the expression of any endogenous and/or exogenous gene depending on their specific sequence, attracted much attention by scientists and pharmacologists who were devoted at developing the antisense approach into a new pharmacological tool (68).
For example, several antisense oligonucleotides have been shown to arrest hematopoietic cell proliferation (69), growth (70), entry into the S phase of the cell cycle (71), reduced survival (72) and prevent receptor mediated responses (73). For use of antisense oligonucleotides as antiviral agents the reader is referred to reference 74.
For efficient in vivo inhibition of gene expression using antisense oligonucleotides or analogs, the oligonucleotides or analogs must fulfill the following requirements (i) sufficient specificity in binding to the target sequence; (ii) solubility in water; (iii) stability against intra- and extracellular nucleases; (iv) capability of penetration through the cell membrane; and (v) when used to treat an organism, low toxicity.
Unmodified oligonucleotides are impractical for use as antisense sequences since they have short in vivo half-lives, during which they are degraded rapidly by nucleases. Furthermore, they are difficult to prepare in more than milligram quantities. In addition, such oligonucleotides are poor cell membrane penetraters (75).
Thus it is apparent that in order to meet all the above listed requirements, oligonucleotide analogs need to be devised in a suitable manner. Therefore, an extensive search for modified oligonucleotides has been initiated.
For example, problems arising in connection with double-stranded DNA (dsDNA) recognition through triple helix formation have been diminished by a clever "switch back" chemical linking, whereby a sequence of polypurine on one strand is recognized, and by "switching back", a homopurine sequence on the other strand can be recognized. Also, good helix formation has been obtained by using artificial bases, thereby improving binding conditions with regard to ionic strength and pH.
In addition, in order to improve half-life as well as membrane penetration, a large number of variations in polynucleotide backbones have been done, nevertheless with little success.
Oligonucleotides can be modified either in the base, the sugar or the phosphate moiety. These modifications include, for example, the use of methylphosphonates, monothiophosphates, dithiophosphates, phosphoramidates, phosphate esters, bridged phosphorothioates, bridged phosphoramidates, bridged methylenephosphonates, dephospho intemucleotide analogs with siloxane bridges, carbonate bridges, carboxymethyl ester bridges, carbonate bridges, carboxymethyl ester bridges, acetamide bridges, carbamate bridges, thioether bridges, sulfoxy bridges, sulfono bridges, various "plastic" DNAs, α-anomeric bridges and borane derivatives. For further details the reader is referred to reference 76.
International patent application WO 89/12060 discloses various building blocks for synthesizing oligonucleotide analogs, as well as oligonucleotide analogs formed by joining such building blocks in a defined sequence. The building blocks may be either "rigid" (i.e., containing a ring structure) or "flexible" (i.e., lacking a ring structure). In both cases, the building blocks contain a hydroxy group and a mercapto group, through which the building blocks are said to join to form oligonucleotide analogs. The linking moiety in the oligonucleotide analogs is selected from the group consisting of sulfide (—S—), sulfoxide (—SO—), and sulfone (—SO2—). However, the application provides no data supporting the specific binding of an oligonucleotide analog to a target oligonucleotide.
International patent application WO 92/20702 describe an acyclic oligonucleotide which includes a peptide backbone on which any selected chemical nucleobases or analogs are stringed and serve as coding characters as they do in natural DNA or RNA. These new compounds, known as peptide nucleic acids (PNAs), are not only more stable in cells than their natural counterparts, but also bind natural DNA and RNA 50 to 100 times more tightly than the natural nucleic acids cling to each other (77). PNA oligomers can be synthesized from the four protected monomers containing thymine, cytosine, adenine and guanine by Merrifield solid-phase peptide synthesis. In order to increase solubility in water and to prevent aggregation, a lysine amide group is placed at the C-terminal.
Thus, antisense technology requires pairing of messenger RNA with an oligonucleotide to form a double helix that inhibits translation. The concept of antisense-mediated gene therapy was already introduced in 1978 for cancer therapy. This approach was based on certain genes that are crucial in cell division and growth of cancer cells. Synthetic fragments of genetic substance DNA can achieve this goal. Such molecules bind to the targeted gene molecules in RNA of tumor cells, thereby inhibiting the translation of the genes and resulting in dysfunctional growth of these cells. Other mechanisms has also been proposed. These strategies have been used, with some success in treatment of cancers, as well as other illnesses, including viral and other infectious diseases. Antisense oligonucleotides are typically synthesized in lengths of 13-30 nucleotides. The life span of oligonucleotide molecules in blood is rather short. Thus, they have to be chemically modified to prevent destruction by ubiquitous nucleases present in the body. Phosphorothioates are very widely used modification in antisense oligonucleotide ongoing clinical trials (57). A new generation of antisense molecules consist of hybrid antisense oligonucleotide with a central portion of synthetic DNA while four bases on each end have been modified with 2′O-methyl ribose to resemble RNA. In preclinical studies in laboratory animals, such compounds have demonstrated greater stability to metabolism in body tissues and an improved safety profile when compared with the first-generation unmodified phosphorothioate (Hybridon Inc. news). Dosens of other nucleotide analogs have also been tested in antisense technology.
RNA oligonucleotides may also be used for antisense inhibition as they form a stable RNA-RNA duplex with the target, suggesting efficient inhibition. However, due to their low stability RNA oligonucleotides are typically expressed inside the cells using vectors designed for this purpose. This approach is favored when attempting to target a MRNA that encodes an abundant and long-lived protein (57).
Recent scientific publications have validated the efficacy of antisense compounds in animal models of hepatitis, cancers, coronary artery restenosis and other diseases. The first antisense drug was recently approved by the FDA. This drug Fomivirsen, developed by Isis, is indicated for local treatment of cytomegalovirus in patients with AIDS who are intolerant of or have a contraindication to other treatments for CMV retinitis or who were insufficiently responsive to previous treatments for CMV retinitis (Pharmacotherapy News Network).
Several antisense compounds are now in clinical trials in the United States. These include locally administered antivirals, systemic cancer therapeutics. Antisense therapeutics has the potential to treat many life-threatening diseases with a number of advantages over traditional drugs. Traditional drugs intervene after a disease-causing protein is formed. Antisense therapeutics, however, block mRNA transcription/translation and intervene before a protein is formed, and since antisense therapeutics target only one specific mRNA, they should be more effective with fewer side effects than current protein-inhibiting therapy.
A second option for disrupting gene expression at the level of transcription uses synthetic oligonucleotides capable of hybridizing with double stranded DNA. A triple helix is formed. Such oligonucleotides may prevent binding of transcription factors to the gene's promoter and therefore inhibit transcription. Alternatively, they may prevent duplex unwinding and, therefore, transcription of genes within the triple helical structure.
Another approach is the use of specific nucleic acid sequences to act as decoys for transcription factors. Since transcription factors bind specific DNA sequences it is possible to synthesize oligonucleotides that will effectively compete with the native DNA sequences for available transcription factors in vivo. This approach requires the identification of gene specific transcription factor (57).
Indirect inhibition of gene expression was demonstrated for matrix metalloproteinase genes (MMP-1,-3, and -9), which are associated with invasive potential of human cancer cells. E1AF is a transcription activator of MMP genes. Expression of E1AF antisense RNA in HSC3AS cells showed decrease in mRNA and protein levels of MMP-1,-3, and -9. Moreover, HSC3AS showed lower invasive potential in vitro and in vivo. These results imply that transfection of antisense inhibits tumor invasion by down-regulating MMP genes (58).
Ribozymes:
Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials (62). More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied in human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated—WEB home page).
Gene Disruption in Animal Models:
The emergence of gene inactivation by homologous recombination methodology in embryonic stem cells has revolutionized the field of mouse genetics. The availability of a rapidly growing number of mouse null mutants has represented an invaluable source of knowledge on mammalian development, cellular biology and physiology, and has provided many models for human inherited diseases. Animal models are required for an effective drug delivery development program and evaluation of gene therapy approach. The improvement of the original knockout strategy, as well as exploitation of exogenous enzymatic systems that are active in the recombination process, has been considerably extended the range of genetic manipulations that can be produced. Additional methods have been developed to provide versatile research tools: Double replacement method, sequential gene targeting, conditional cell type specific gene targeting, single copy integration method, inducible gene targeting, gene disruption by viral delivery, replacing one gene with another, the so called knock-in method and the induction of specific balanced chromosomal translocation. It is now possible to introduce a point mutation as a unique change in the entire genome, therefore allowing very fine dissection of gene function in vivo. Furthermore, the advent of methods allowing conditional gene targeting opens the way for analysis of consequence of a particular mutation in a defined organ and at a specific time during the life of the experimental animal (59).
DNA Vaccination:
Observations in the early 1990s that plasmid DNA could directly transfect animal cells in vivo sparked exploration of the use of DNA plasmids to induce immune response by direct injection into animal of DNA encoding antigenic protein. When a DNA vaccine plasmid enters the eukaryotic cell, the protein it encodes is transcribed and translated within the cell. In the case of pathogens, these proteins are presented to the immune system in their native form, mimicking the presentation of antigens during a natural infection. DNA vaccination is particularly useful for the induction of T cell activation. It was applied for viral and bacterial infectious diseases, as well as for allergy and for cancer. The central hypothesis behind active specific immunotherapy for cancer is that tumor cells express unique antigens that should stimulate the immune system. The first DNA vaccine against tumor was carcino-embrionic antigen (CEA). DNA vaccinated animals expressed immunoprotection and immunotherapy of human CEA-expressing syngeneic mouse colon and breast carcinoma (61). In a mouse model of neuroblastoma, DNA immunization with HuD resulted in tumor growth inhibition with no neurological disease (60). Immunity to the brown locus protein, gp75 tyrosinase-related protein-1, associated with melanoma, was investigated in a syngeneic mouse model. Priming with human gp75 DNA broke tolerance to mouse gp75. Immunity against mouse gp75 provided significant tumor protection (60).
Glycosyl Hydrolases:
Glycosyl hydrolases are a widespread group of enzymes that hydrolyze the o-glycosidic bond between two or more carbohydrates or between a carbohydrate and a noncarbohydrate moiety. The enzymatic hydrolysis of glycosidic bond occurs by using major one or two mechanisms leading to overall retention or inversion of the anomeric configuration. In both mechanisms catalysis involves two residues: a proton donor and a nucleophile. Glycosyl hydrolyses have been classified into 58 families based on amino acid similarities. The glycosyl hydrolyses from families 1, 2, 5, 10, 17, 30, 35, 39 and 42 act on a large variety of substrates, however, they all hydrolyze the glycosidic bond in a general acid catalysis mechanism, with retention of the anomeric configuration. The mechanism involves two glutamic acid residues, which are the proton donors and the nucleophile, with an aspargine always preceding the proton donor. Analyses of a set of known 3D structures from this group revealed that their catalytic domains, despite the low level of sequence identity, adopt a similar (α/β) 8 fold with the proton donor and the nucleophile located at the C-terminal ends of strands β4 and β7, respectively. Mutations in the functional conserved amino acids of lysosomal glycosyl hydrolases were identified in lysosomal storage diseases.
Lysosomal glycosyl hydrolases including β-glucuronidase, β-manosidase, β-glucocerebrosidase, β-galactosidase and α-L iduronidase, are all exo-glycosyl hydrolases, belong to the GH-A clan and share a similar catalytic site. However, many endo-glucanases from various organisms, such as bacterial and fungal xylenases and cellulases share this catalytic domain.
Genomic Sequence of hpa Gene and its Implications:
It is well established that heparanase activity is correlated with cancer metastasis. This correlation was demonstrated at the level of enzymatic activity as well as the levels of protein and hpa cDNA expression in highly metastatic cancer cells as compared with non-metastatic cells. As such, inhibition of heparanase activity is desirable, and has been attempted by several means. The genomic region, encoding the hpa gene and the surrounding, provides a new powerful tool for regulation of heparanase activity at the level of gene expression. Regulatory sequences may reside in noncoding regions both upstream and downstream the transcribed region as well as in intron sequences. A DNA sequence upstream of the transcription start site contains the promoter region and potential regulatory elements. Regulatory factors, which interact with the promoter region may be identified and be used as potential drugs for inhibition of cancer, metastasis and inflammation. The promoter region can be used to screen for inhibitors of heparanase gene expression. Furthermore, the hpa promoter can be used to direct cell specific, particularly cancer cell specific, expression of foreign genes, such as cytotoxic or apoptotic genes, in order to specifically destroy cancer cells.
Cancer and yet unknown related genetic disorders may involve rearrangements and mutations in the heparanase gene, either in coding or non-coding regions. Such mutations may affect expression level or enzymatic activity. The genomic sequence of hpa enables the amplification of specific genomic DNA fragments, identification and diagnosis of mutations.
There is thus a widely recognized need for, and it would be highly advantageous to have genomic, cDNA and composite polynucleotides encoding a polypeptide having heparanase activity, vectors including same, genetically modified cells expressing heparanase and a recombinant protein having heparanase activity, as well as antisense oligonucleotides, constructs and ribozymes which can be used for down regulation heparanase activity.

Hadasit Medical Research Services and Development Ltd.


A communication system communication system includes a transmitter and a receiver. The transmitter inverts multiple bits of first transmission data, serializes the first transmission data to generate second transmission data, and provides the second transmission data to communication channels, in response to an error signal. The receiver inverts multiple bits of first reception data provided from the communication channels, parallelizes the first reception data to generate second reception data, and generates the error signal based on the second reception data, in response to the error signal.

1. Field of the Invention
The present invention relates to a data communication system, and more particularly, to a system and method for communicating data over communications channels.
2. Description of the Related Art
FIG. 1 is a diagram illustrating a conventional communication system. Referring to FIG. 1, the communication system 5 includes a transmitter 10, a receiver 20, and communication channels CH1 through CH4. Data DO00 through DO03 of the transmitter 10 are transmitted to the receiver 20 through the communication channels CH1 through CH4.
FIG. 2 is a schematic diagram illustrating an environment in which simultaneous switching noise (SSN) occurs during data communication between two semiconductor memory devices. Referring to FIG. 2, a first semiconductor memory device 30 (memory device 1) operates as a transmitter, and a second semiconductor memory device 40 (memory device 2) operates as a receiver. The first semiconductor memory device 30 includes output drivers 31 through 34 operating at a high power supply voltage VDDQ and a low power supply voltage VSSQ. The second semiconductor memory device 40 includes input drivers 41 through 44 operating at the high power supply voltage VDDQ and the low power supply voltage VSSQ.
FIG. 3 is a schematic diagram illustrating an environment in which inter-symbol interference (ISI) occurs during the data communication between the two semiconductor memory devices. In FIG. 3, the communication channel CH2 of the communication channels CH1 through CH4 in FIG. 2 is illustrated, and data D1 passes through the communication channel CH2. Parasitic capacitance CCH exists between the ground and the communication channel CH2 disposed outside of a semiconductor chip. The parasitic capacitances CCH also exist between the ground and the communication channels CH1, CH3 and CH4.
FIG. 4 is a diagram illustrating data affected by ISI and SSN during transmission through four communication channels, and the data include bits highly susceptible to errors. Referring to FIGS. 2 and 4, each of the four data D0-D3 includes nine bits, which are sequentially transmitted through the communication channels CH1 through CH4. The data D0 is transmitted through the communication channel CH1, the data D1 is transmitted through the communication channel CH2, the data D2 is transmitted through the communication channel CH3, and the data D3 is transmitted through the communication channel DH4, respectively. A reference numeral 51 corresponds to a data bit susceptible to errors affected by ISI. A reference numeral 52 corresponds to data bits susceptible to errors affected by SSN.
Referring to FIGS. 3 and 4, a state of the data D1 changes from a second bit "0" to a third bit "1", and then from the third bit "1" to a fourth bit "0" as indicated by the reference numeral 51. A voltage of the third bit "1" is not high enough to be recognized as a logic high when the state of the data D1 changes from 0 to 1 to 0 because the communication channels have the parasitic capacitances, as indicated in FIG. 3. Also, a voltage of "0" is not low enough to be recognized as a logic low when the state of the data D1 changes from 1 to 0 to 1. This phenomenon is referred to as ISI.
Referring to FIGS. 2 and 4, a state of the data D0 changes from "1" to "0" while states of the data D1 through D3 change from "0" to "1", as indicated by the reference numeral 52. The output driver 31 connected to the communication channel CH1 may output "1" instead of "0" in a first row shown by the reference number 52, when the data D0 transmitted through the communication channel CH1 changes from "1" to "0" and the states of the data D1 through D3 transmitted through the communication channels CH2 through CH4 change from "0" to "1" in the second through fourth rows, as indicated by the reference numeral 52. This phenomenon is referred to as SSN.
Further Bit Error Rate (BER) during transmission through communication channels between semiconductor chips increases as a communication between the semiconductor chips becomes faster. Accordingly, there exists a probability of error occurrence caused by ISI and SSN.

System and method for communicating data over communication channels


Thermal insulation is provided for use in applications requiring continuous resistance to temperatures of 1260° C. without reaction with alumino-silicate firebricks, the insulation comprises fibers having a composition in wt %65%<SiO2<86%MgO<10%14%<CaO<28%Al2O3<2%ZrO2<3%B2O3<5%P2O5<5%72%<SiO2+ZrO2+B2O3+5*P2O5 95%<SiO2+CaO+MgO+Al2O3+ZrO2+B2O3+P2O5

This invention relates to saline soluble, non-metallic, amorphous, inorganic oxide, refractory fibrous materials. The invention particularly relates to glassy fibres having silica as their principal constituent.
Inorganic fibrous materials are well known and widely used for many purposes (e.g. as thermal or acoustic insulation in bulk, mat, or blanket form, as vacuum formed shapes, as vacuum formed boards and papers, and as ropes, yarns or textiles; as a reinforcing fibre for building materials; as a constituent of brake blocks for vehicles). In most of these applications the properties for which inorganic fibrous materials are used require resistance to heat, and often resistance to aggressive chemical environments.
Inorganic fibrous materials can be either glassy or crystalline. Asbestos is an inorganic fibrous material one form of which has been strongly implicated in respiratory disease.
It is still not clear what the causative mechanism is that relates some asbestos with disease but some researchers believe that the mechanism is mechanical and size related. Asbestos of a critical size can pierce cells in the body and so, through long and repeated cell injury, have a bad effect on health. Whether this mechanism is true or not regulatory agencies have indicated a desire to categorise any inorganic fibre product that has a respiratory fraction as hazardous, regardless of whether there is any evidence to support such categorisation. Unfortunately for many of the applications for which inorganic fibres are used, there are no realistic substitutes.
Accordingly there is a demand for inorganic fibres that will pose as little risk as possible (if any) and for which there are objective grounds to believe them safe.
A line of study has proposed that if inorganic fibres were made that were sufficiently soluble in physiological fluids that their residence time in the human body was short; then damage would not occur or at least be minimised. As the risk of asbestos linked disease appears to depend very much on the length of exposure this idea appears reasonable. Asbestos is extremely insoluble.
As intercellular fluid is saline in nature the importance of fibre solubility in saline solution has long been recognised. If fibres are soluble in physiological saline solution then, provided the dissolved components are not toxic, the fibres should be safer than fibres which are not so soluble. The shorter the time a fibre is resident in the body the less damage it can do. H. Förster in 'The behaviour of mineral fibres in physiological solutions' (Proceedings of 1982 WHO IARC Conference, Copenhagen, Volume 2, pages 27-55 (1988)) discussed the behaviour of commercially produced mineral fibres in physiological saline solutions. Fibres of widely varying solubility were discussed.
International Patent Application No. WO87/05007 disclosed that fibres comprising magnesia, silica, calcia and less than 10 wt % alumina are soluble in saline solution. The solubilities of the fibres disclosed were in terms of parts per million of silicon (extracted from the silica containing material of the fibre) present in a saline solution after 5 hours of exposure. The highest value revealed in the examples had a silicon level of 67 ppm. In contrast, and adjusted to the same regime of measurement, the highest level disclosed in the Förster paper was equivalent to approximately 1 ppm. Conversely if the highest value revealed in the International Patent Application was converted to the same measurement regime as the Förster paper it would have an extraction rate of 901,500 mg Si/kg fibre—i.e. some 69 times higher than any of the fibres Förster tested, and the fibres that had the highest extraction rate in the Förster test were glass fibres which had high alkali contents and so would have a low melting point. This is convincingly better performance even taking into account factors such as differences in test solutions and duration of experiment.
International Patent Application No. WO89/12032 disclosed additional fibres soluble in saline solution and discusses some of the constituents that may be present in such fibres.
European Patent Application No. 0399320 disclosed glass fibres having a high physiological solubility.
Further patent specifications disclosing selection of fibres for their saline solubility include for example European 0412878 and 0459897, French 2662687 and 2662688, PCT WO86/04807, WO90/02713, WO92/09536, WO93/22251, WO94/15883, WO97/16386 and U.S. Pat. No. 5,250,488.
The refractoriness of the fibres disclosed in these various prior art documents varies considerably and for these alkaline earth silicate materials the properties are critically dependent upon composition.
WO94/15883 disclosed a number of fibres that are usable as refractory insulation at temperatures of up to 1260° C. or more. These fibres comprised CaO, MgO, SiO2, and optionally ZrO2 as principal constituents. Such fibres are frequently known as CMS (calcium magnesium silicate) or CMZS ((calcium magnesium zirconium silicate) fibres. WO94/15883 required that any alumina present only be in small quantities.
A drawback found in use of these fibres, is that at temperatures between about 1300° C. and 1350° C. the fibres undergo a considerable increase in shrinkage. Typically, shrinkages increase from about 1-3% at 1200° C.; to, say, 5% or more at 1300° C.; to >20% at 1350° C. This means that, for example, a temperature overrun on a furnace can result in damage to the insulation and hence to the furnace itself.
A further drawback is that calcium magnesium silicate fibres can react with, and stick to, alumina containing materials due to formation of a eutectic composition. Since aluminosilicate materials are widely used this is a major problem.
WO97/16386 disclosed fibres that are usable as refractory insulation at temperatures of up to 1260° C. or more. These fibres comprised MgO, SiO2, and optionally ZrO2 as principal constituents. As with WO94/15883, this patent required that any alumina present only be in small quantities.
While these fibres do not show the dramatic change in shrinkage evident in the fibres of WO94/15883, they do show a significantly higher shrinkage at normal use temperatures typically having a shrinkage of 3-6% over the range 1200° C.-1450° C. These fibres do not appear to have the drawback of reacting with and sticking to alumina containing materials, however they tend to be difficult to make.

This patent is subject to a terminal disclaimer.

Abstract: Addition of elements selected from the group Sc, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Y or mixtures thereof improves fiber quality and the strength of blankets made from the fibers.


This invention relates to a graphite material for negative electrode of lithium secondary battery. This graphite material is obtained by carbonizing the pulverized green coke to yield coke, adding one kind or more of boron or a compound thereof to the coke, graphitizing the resulting mixture and controlling the particle size of the product graphite. This graphite material for negative electrode is characterized by showing a tap density of 0.95 g/cm3 or more after tapping 20 times or 1.15 g/cm3 or more after tapping 300 times and a BET specific surface area of 1.5 m2/g or less. A lithium secondary battery made by the use of this graphite material for negative electrode has a large discharge capacity and suffers little loss during charging and discharging.

A lithium secondary battery using graphite as a negative electrode has been studied extensively because it is free from the old problem accompanying the use of metallic lithium or the problem of dendrite growth at the time of charging, it generates high electromotive force and it gives a highly reliable performance.
Carbonaceous materials classified as graphite are diversified in shape, structure and texture and the differences in these factors are reflected in the performance of electrodes. A secondary battery in which flake graphite such as natural graphite is used as a graphite material is known to have a higher discharge capacity than the one in which synthetic graphite obtained by graphitizing mesophase microbeads, pitch-derived carbon fibers, pitch coke and the like is used because of a highly developed crystal structure of flake graphite. However, one problem with flake graphite is a high initial irreversible capacity of a battery made therefrom. In addition, it appears difficult to obtain a high discharge capacity when flake graphite is used under the conditions of large charge and discharge current. Further problems associated with flake graphite are high bulk density of particles and difficulty in preparing a slurry for coating use. For the purpose of improving the maneuverability and particle characteristics of flake graphite, processes consisting of blending flake graphite with an organic binder or coating the surface of flake graphite with an organic binder followed by granulation and heat treatment are proposed in JP10-36108 A and elsewhere. According to these processes, however, a part of the binder component remains behind thereby reducing the discharge capacity.
When mesophase microbeads collected at the stage in which an optically anisotropic phase forms spherically in mesophase pitch are used as a raw material for graphite, the growth of carbon layer after graphitization is inferior to that in natural graphite and the discharge capacity thereby obtained amounts to merely 80–85% of the theoretical discharge capacity.
It is reported in J. Electrochem. Soc., Vol. 142, No. 8, 2564 (1995) that pitch-based carbon fibers possess a variety of properties as synthetic graphite and yield electrodes of excellent heavy load characteristics. Being a fiber, however, raises problems such as the following; the growth of crystal structure is retarded to keep the discharge capacity from increasing beyond a moderate level and the necessity of installing a fiber-making step and the like incurs an extra manufacturing cost.
Pitch coke belongs to a class of readily graphitizable materials and, upon graphitization in an ultra-high temperature range, may form carbon network layers whose interlayer distance is close to that of natural graphite; however, in the case where pitch coke has not much of optically anisotropic textures, graphitization does not progress to a degree comparable to natural graphite and the carbon network layers are not preferentially oriented in the specified direction in the crystal structure. In consequence, graphite of this kind is free of the restriction on the current density such as seen in the case of natural graphite like flake graphite and is an extremely promising material for negative electrode of lithium secondary battery as evidenced by a large number of researches made on it up to the present (for example, JP63-121257A, JP1-204361A and JP4-206276A). However, a material obtained by calcining ordinary pitch coke at an ultra-high temperature (2000–3000° C.) shows a lower discharge capacity (<300 mAh/g) than the theoretical discharge capacity (372 mAh/g).
An attempt has been made to provide graphite with a high discharge capacity by adding boron to carbon particles derived from pitch coke or pitch, for example, in JP10-223223A This method is effective for raising the discharge capacity and reducing the irreversible capacity and provides a technique effective for improving the performance of graphite materials for negative electrodes. On the other hand, this method requires a pulverizing operation in the step for particle size control and may develop the possibility of deteriorating the particle characteristics such as reducing the bulk density and tap density and increasing the specific surface area. In addition, there remain practical problems in need of solution such as lack of stability in manufacture of electrode foils and insufficient particle characteristics relating to cycle performance of materials for negative electrodes.

Graphite material for negative pole of lithium secondary battery, method of manufacturing the graphite material, and lithium secondary battery


A throttle body assembly, and method of manufacturing such, is disclosed having a unitary shaft and blade component injection molded in situ within a throttle body housing to mechanically entrap the shaft and blade component, yet still allowing for it to partially rotate, within the throttle body housing. The throttle body housing defines an interior passage, a shaft through-hole, and a shaft socket opposite the shaft through-hole, both aligned along a transverse axis to the interior passage. The shaft and blade component is a single unitary piece injection molded in situ within the throttle body housing, such that it has a blade section disposed within the interior passage, first and second shaft sections extending from the blade section opposite each other partially disposed within the shaft socket and shaft through-hole, respectively, and a pivotable axis generally coaxial with the transverse axis.

1. Field
The invention relates to throttle body assemblies for internal combustion engines, and in particular, the injection molding of a shaft and blade component in situ within the throttle body housing.
2. Background Art
A throttle is the mechanism that constricts or obstructs the flow of a fluid. An engine's power may be increased or decreased by the restriction of inlet gases by the use of a throttle. In a fuel-injected engine, the throttle is typically placed on the entrance of the intake manifold, or housed in a throttle body. In a carbureted engine, it is found in the carburetor.
A fuel-injected engine's throttle is typically a butterfly valve located within the interior passage of a throttle body assembly. When the blade of the butterfly valve is wide open (in-line with fluid flow), the engine may run at full power. When the blade of the butterfly valve is partially closed (at an angle to the fluid flow), the engine's power is decreased.
Butterfly valves within throttle body assemblies may be controlled by an operator using a pedal or lever with a direct mechanical linkage, or with an Engine Control Unit (ECU). ECUs receive information from sensors (or the operator), process the information, and in turn control the position of the butterfly valve. Often the position of the butterfly valve, as controlled by an ECU, is based on the desired engine outputs as programmed into the ECU. The ECU sends a signal to rotate the blade of the butterfly valve inside the throttle body, opening the throttle passage to allow more air into the intake manifold increasing the engine's power.
Examples of throttle body assemblies and ECUs used with internal combustion engines may be found in U.S. Pat. Nos. 6,804,997 and 6,955,081 to Schwulst, and U.S. Pat. No. 7,225,793 to Schwulst et al., all of which are hereby incorporated by reference in their entirety.
Traditionally throttle body assemblies have been made of metal with a multi-component pin and blade sub-assembly assembled into the interior passage of the throttle body housing. Some improvements to this traditional design have included injection molded throttle body housings with blades injection molded in the throttle body housing around, or connected to, an inserted pin (or pins) to support and rotate the blade. These throttle body assemblies require additional complexity of bushings and seals to manage the support of the pin(s) and allow rotation of the blade.
The following references were considered in conjunction with preparing this application: U.S. Pat. No. 7,846,364 to Arai et al., U.S. Pat. No. 7,790,083 to Lorenz et al., U.S. Pat. No. 7,328,507 to Arai et al., U.S. Pat. No. 7,107,679 to Arai et al., U.S. Pat. No. 7,107,678 to Arai et al., U.S. Pat. No. 7,089,663 to Arai et al., U.S. Pat. No. 7,069,902 to Arai et al., U.S. Pat. No. 5,421,718 to Karlsson et al., U.S. Pat. No. 5,304,336 to Karlsson et al., and U.S. P.G. Pub. No. 2010/0090148 to Deiss et al.

Throttle body with blade and shaft injection molded within the body


An aberration adjusting method of a charged particle beam optical system. The method includes an aberration measuring step of measuring N aberrations of the charged particle beam optical system, an aberration sensitivity acquiring step of changing M control amounts to control optical elements included in the charged particle beam optical system, obtaining variations of the N aberrations by executing the aberration measuring step, and obtaining aberration sensitivities of the M control amounts. The method further includes a control amount deciding step of deciding the M control amounts on the basis of the N aberrations and the aberration sensitivities of the M control amounts to set the N aberrations to target aberrations. The aberration is a displacement of each image height of charged particles beams. The control amount is an amount for controlling a position of the charged particle beams, and M<N, in which M and N are positive integers.

Conventional electron beam lithography machines include machines of a point beam type, which use a spot-shaped beam and machines of a variable rectangular beam type, which use a beam having a rectangular section with a variable size.
An electron beam lithography machine of a point beam type performs drawing by using a single electron beam. Hence, this machine has a low throughput and is used only for research and development. The throughput of an electron beam lithography machine of a variable rectangular beam type is higher than that of a point beam type by one or two orders of magnitude. However, this type also basically executes drawing by using a single electron beam, so there is still a problem of throughput in exposing a pattern in which fine patterns of about 01. μm are integrated at a high density.
As a machine to solve this problem, there is an electron beam lithography machine of a stencil mask type. In this machine, a pattern to be drawn is formed as pattern conduction pores in a stencil mask. Then, the pattern to be drawn is transferred to the sample surface through a reduced electron optical system by irradiating the stencil mask with an electron beam. As another method, a multi-electron beam lithography machine is used. A substrate having a plurality of apertures is irradiated with an electron beam to irradiate a sample surface with a plurality of electron beams from the plurality of apertures through a reduced electron optical system. The sample surface is scanned by deflecting the plurality of electron beams. The plurality of electron beams are individually turned on/off in accordance with the pattern to be drawn, thereby drawing the pattern. In all methods described above, the throughput can be improved because the area to be exposed at once, i.e., the exposure area, is wider than a conventional one.
The reduced electron optical system of an electron beam lithography machine of a stencil mask type or a multi-electron beam lithography machine must be adjusted to suppress aberration in balance in the entire exposure field wider than usual. To do this, it is necessary to separately measure spherical aberration, coma, astigmatism, displacement, and focus position at each image height in the entire exposure field and execute adjustment on the basis of the measurement result. However, in conventional aberration measurement, defocus of a beam and its position are only measured so aberration cannot be suppressed in balance in the entire exposure field.
Japanese Patent Laid-Open No. 2004-153245 (patent reference 1) proposes an astigmatism correction sensitivity deciding method in a charged particle beam lithography machine and an exposure method using the method.

Aberration adjusting method, device fabrication method, and charged particle beam lithography machine


A controlled device executes processing for device authentication and processing for device registration, receives a response of registration completion from a direct-access management server, and sets an automatic connection flag indicating the registration is completed. When the automatic connection flag is not set, the controlled device establishes an always-on connection session with the direct-access management server after executing the processing for the device authentication and the processing for the device registration. When the automatic connection flag is set, the controlled device skips the processing for the device authentication and the processing for the device registration to enter processing for establishing an always-on connection session. Thus, a controlled device whose registration has been completed can automatically enter the state of an always-on connection with the direct-access management server without performing the processing for the device authentication and the processing for the device registration.

As methods for accessing from a controlling device on the Internet to a controlled device in a home, a DDNS (dynamic domain name system) and a mail system are available.
The DDNS is a scheme in which an in-home controlled device registers its own address with a DDNS server and receives a specified domain name and port number from an internet-side controlling device to allow the access of the controlling device.
As a protocol for realizing real-time communication, an XMPP (eXtensible Messaging and Presence Protocol) is available (e.g., refer to "Extensible Messaging and Presence Protocol (XMPP): Core", RFC3920, The Internet Engineering Task Force (IETF), October 2004). One real-time communication scheme using the XMPP is instant messaging (IM) (e.g., refer to "Extensible Messaging and Presence Protocol (XMPP): Instant Messaging and Presence", RFC3921, The Internet Engineering Task Force (IETF), October 2004). In the instant messaging (IM), it is checked whether or not an IM client (a friend who can perform IM exchange) is online on the Internet and, when he/she is online, chatting, file exchange, and so on can be performed with the IM client.
Also, as a protocol for calling up data or a service existing on another computer, a SOAP (simple object access protocol) is available (e.g., refer to "SOAP Version 1.2", W3C Recommendation, World Wide Web Consortium (W3C), June 2003). In communications based on the SOAP, messages in which auxiliary information called envelopes are attached to XML (eXtensible Markup Language) documents are exchanged using a protocol such as an HTTP (hyper text protocol). Both a client using a service and a server providing the service have SOAP generation/interpretation engines to allow an object to be called up between different environments.

Processing device, method for establishing processing device communication session, program, and recording medium


A trailer device for engaging and towing vehicles has, a rigid, longitudinal first frame having front and rear ends and extending substantially horizontally between them, the front end providing attachment to a fifth-wheel type truck tractor for hauling. A vehicle support surface is mounted substantially horizontally on the first frame, having right and left longitudinal portions, each formed of a plurality of segments along its length, at least one segment of each such longitudinal portion of the vehicle support surface being transversely divided into a plurality of subsegments, at least one subsegment of each transversely divided segment being movable. Pneumatic cylinders can move a subsegment of each transversely divided segment of each right and left longitudinal portion of the vehicle support surface. A jacking mechanism is mounted beneath the front end of the rigid longitudinal first frame. Wheels support the rear end of the rigid longitudinal first frame. A vehicle engagement assembly is positioned along a central longitudinal axis of the first frame on the rear end thereof and pivotally attached thereto for securely retaining in position a vehicle loaded at least in part onto the device. A winch at the forward end of the platform also allows even a heavy vehicle, e.g. truck tractor, to be pulled forwardly onto the vehicle support surface for disabled vehicle recovery and/or towing.

This invention relates to recovering and towing vehicles, such as wreckers and the like for pulling cars, trucks and other vehicles which have been disabled and, more particularly, to a towing vehicle of novel universal type for recovery, loading thereon and towing a disabled vehicle for purposes of moving it.
There are a variety of so-called wreckers in both the patented prior art and in commercial practice, which date back over many decades. By the term "wrecker" is meant a towing vehicle of self-powered, self-contained type typically having a truck-like frame and/or body, and typically supported by dual or tandem dual wheels, the body or frame having a crane, winch or hydraulically extensible structure for engaging a disabled vehicle, lifting it and towing it. Because such a wrecker is itself a powered vehicle, it must be licensed and must comply with all requirements of motor vehicles, including insurance, payment of taxes and the like. Thus, wreckers are expensive to acquire, maintain and operate.
It has previously been proposed to use trailers instead of wreckers for transporting large vehicles, and there are long-bed trailer arrangements for this purpose as proposed, for example, in Schramm U.S. Pat. No. 2,838,191 which proposed an elevatable bed vehicle which is of the type now known as "low-boy", or other conventional flatbed trailer used for transporting, in particular, heavy equipment. Such a flatbed trailer is not suited for routine recovery and transporting of disabled vehicles as loading (off pound hauling) of such vehicles is very cumbersome.
Similarly, Bills, Jr. U.S. Pat. No. 4,568,235 discloses a low-angle tilt trailer having a bed which can be positioned hydraulically to a rearward position in which it engages the ground for facilitating the loading and unloading of equipment. Again, this patent is representative of a type of specialized trailer used for heavy machinery, and wherein the machinery is transported in its entirety (hauled) upon the surface of the trailer.
Mauldin U.S. Pat. No. 3,635,492, discloses a trailer including a hydraulically operated mechanism for elevating or lowering the frame of the trailer, being a light duty construction suited only for transporting of very light loads, as for example, boats and intended to facilitate the launching and recovery of boats. Thus, such patent is typical of other types of trailers suited for pulling loads on and off the vehicle, but such boat trailers and the like are not suited for carrying of heavy vehicles, and particularly disabled vehicles.
For purposes of recovery of a disabled truck tractor, or truck unit Karlik U.S. Pat. No. 4,493,491 discloses an apparatus in the form of a trailer, including a rear deck portion which has a sloping ramp section, there being a winch at the forward portion of the trailer to enable a truck or the like to be pulled onto the ramp section. However, such a trailer is not suited for recovery of a newer type of truck, particularly heavy duty truck tractors, which have air "dams" and other aerodynamic structure at the front of the truck which make them extremely difficult to be engaged by any kind of conventional wrecker, and which structure interferes with the recovery of such a truck by a trailer of the type disclosed in Karlik.
Further, certain trucks, such as fire trucks, which have a very long overhang, present special difficulties for recovery by wreckers and other vehicles of the type identified above.
It is to be appreciated that many trucks, because of their configuration at the front of the vehicle, are only recoverable with difficulty. Although there are heavy duty wreckers which are available for pulling such vehicles, recovery of a disabled vehicle of such a specialized type often requires the removal of bumpers, air dams and other structure, or presents extreme difficulty when recovering, particularly in the case of such fire trucks. Further, because of the extreme cost, for purchase and maintenance as well as for insurance, tax and licensing of such specialized wreckers, few business entities can afford practically to keep such wreckers at ready disposal. Thus, a need exists for a relatively low-cost recovery vehicle, particularly such a vehicle which is of the trailer type, so that high insurance, licensing and other costs of self-contained vehicles can be avoided. A need also exists for such a vehicle which can recover the many types of specialized trucks such as those discussed above which have structure, such as a long front overhang, which interferes with their recovery. By recovery is, of course, meant the engagement of the vehicle to be recovered (whether damaged, disabled, stopped or otherwise inoperable) by the recovering vehicle as for purposes of loading or towing (pulling with some of the towed vehicle's wheels on ground) the vehicle to be recovered.

Universal vehicular recovery and towing platform


A method and an apparatus for making a multilayer confectionery product provided. The method and apparatus include at least two sets of forming drums that form and laminate confectionery sheets.

Some confectionery products available in the market include multiple different confectionery compositions, but not all of the different compositions of the confectionery products are visible to a consumer. An example of such confectionery product includes a chewing gum center-filled candy that includes a hard candy outer-shell encasing the chewing gum center. Unfortunately, such confectionery products do not provide the consumer with visual benefits of being provided with two different confectionery compositions at the same time.
Recently, the assignee of the present application has launched a multilayer confectionery product including a chewy candy layer and two chewing gum layers. This multilayer confectionery product presents three visibly distinct confectionery layers and allows the consumer to simultaneously enjoy the flavor and sensorial benefits of the different confectionery compositions.
However, manufacturing of such multilayer confectionery products poses unique challenges. For example, when laminating several layers of different confectionery compositions having different characteristics, the layer having a lower viscosity than other layers may ooze, or the layers may not adhere to each other sufficiently and may slide against each other.
Further, manufacturing of a multilayer confectionery product including a chewing gum layer can introduce additional processing challenges as the gum making process is typically time-consuming and involves a significant amount of machinery. The conventional gum making/forming can include mixing and producing a finished gum as a non-uniform output, extruding and forming the finished gum into loaves, conditioning the loaves of the finished gum, extruding the loaves into a continuous thin sheet of the finished gum, rolling the continuous sheet through a series of rollers to a uniform reduced thickness, scoring and dividing sheets into individual scored sheets, and conditioning the individual sheets in a conditioning room. Such processes of making and forming gum products are disclosed in U.S. Pat. Nos. 4,882,175; 6,254,373 and 7,112,345; which are assigned to the predecessor of interest of the present assignee; and U.S. patent application Ser. No. 12/352,110 assigned to the present assignee; the teachings and disclosures of which are hereby incorporated by reference in their entireties to the extent not inconsistent with the present disclosure.
Traditional gum sizing machineries include a sizing extruder that forces the chewing gum through a small rectangular orifice (e.g. a rectangular orifice having dimensions of about 25 mm by 457 mm). A relatively sizeable amount of force is required as the orifice size gets smaller. Typically, the product exiting the sizing extruder is still much too thick. As a result, many prior systems will typically employ a series of sizing rollers arranged in sequence over a conveyor belt to progressively reduce the thickness of gum from around 25 millimeters to typically about 2-6 millimeters, such as the multiple sizing rollers in traditional rolling and scoring systems. Further, to prevent sticking of gum to the rollers, dusting with a suitable powder agent is typically employed. Thereafter, a scoring roll and dividing roll may be used to generate thin sticks, or somewhat shorter and fatter slabs of gum, or pellets. Such traditional lines also typically will necessitate a fair amount of subsequent cooling and/or conditioning prior to packaging as warm pliable product does not package well. Further, cleaning of such traditional lines is labor intensive and can last hours, for example, up to 10 hours.
The present invention therefore relates to improvements in systems and methods of forming and/or cooling multilayer confectionery products.

System and method of forming multilayer confectionery