Cloning and expression of human Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase

Here we report the cDNA sequence of human Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase. The cDNA was isolated from a human placental cDNA library by screening with a 150bp probe generated by PCR using degenerate primers based on the sequences found in the sialyl motif. Comparative analysis of this cDNA with the rat liver alpha 2,3-sialyltransferase sequence indicates 91% nucleotide similarity between the two sequence in the predicted coding region. On the amino acid level, the degree of conservation is 97%. Surprisingly, Northern analysis indicated that the gene is expressed at low levels in human placenta but is abundantly expressed in skeletal muscle and fetal tissues.     

Cloning and expression of human endothelial-monocyte-activating polypeptide 2 (EMAP-2) and identification of its putative precursor

Endothelial-monocyte-activating polypeptide 2 (EMAP-2) modulates a range of properties of endothelial cells, monocytes and neutrophils in vitro, and induces an acute inflammatory reaction and tumour regression in vivo. We generated the full-length human cDNA sequences of EMAP-2 and its putative precursor pro-EMAP-2 as PCR products. These were cloned into the pCR3 vector and subcloned into pGEX-2T for expression as fusion products with glutathione-S-transferase (GST). Recombinant EMAP-2 (rEMAP-2) was isolated by thrombin cleavage of the fusion protein, followed by affinity chromatography. rEMAP-2 retained biological activity, which was blocked by polyclonal antibodies raised against GST-EMAP-2. By Western blotting, a 34-kDa product corresponding to the predicted precursor proEMAP-2 was detected in lysates of the U937 monocytic cell line, while supernatants contained higher levels of the mature 22-kDa molecule.     

Cloning and expression of human deoxyguanosine kinase cDNA

A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.     

Cloning and squencing of human thrombopoietin (hTPO) cDNA and it''s expression in COS-7 cells]

Viliuisk encephalomyelitis (VE) is an unique neurological disease occurring in the Iakut (Sakha) people of Siberia. Evolution of the disease follows one of three broad clinical forms: subacute, slowly progressive or chronic. Death occurs within 3 to 6 months in subacute cases and within 6 years in the slowly progressive cases. Chronic cases lack a subacute phase but show a slowly progressive dementia associated with bradykinesia, dysarthria and spastic paraparesis that stabilizes late in the disease process. In subacute and slowly progressive cases, focal necrotizing encephalomyelitis is seen at necropsy. Chronic cases show multifocal areas of lysis with a gliotic margin, predominantly within grey matter, lacking associated chronic inflammatory changes seen in the other forms of the disease. Epidemiological studies are consistent with a disease of low-grade communicability, but laboratory studies have so far failed to reveal an infectious organism. The spectrum of neuropathological changes are reviewed in this examination of 11 cases. Although the aetiology of VE remains obscure, further studies are warranted since it may represent a novel disease process.     

Cloning and expression of the human galanin receptor GalR2

We have previously shown that the novel anticonvulsant levetiracetam exerts potent anticonvulsant activity against both focal and secondarily generalized seizures in fully amygdala-kindled rats, i.e. , a model of temporal lobe epilepsy. We examined whether levetiracetam also exhibits antiepileptogenic activity, i.e., prevents or retards acquisition or development of amygdala-kindling in rats. Before the experiments with chronic administration of levetiracetam at different doses, we determined the pharmacokinetics of the drug after i.p. injection. Levetiracetam had a relatively short half-life (about 2-3 hr) in rats, so that any lasting effects of the drug after chronic administration were certainly not due to drug accumulation. When rats were treated with levetiracetam during kindling acquisition at daily i.p. doses of 13, 27 or 54 mg/kg, the drug dose-dependently suppressed the increase in seizure severity and duration induced by repeated amygdala stimulation. After termination of daily treatment with 54 mg/kg, duration of behavioral seizures and of afterdischarges recorded from the amygdala remained to be significantly shorter compared to vehicle controls, although amygdala stimulations were continued in the absence of drug. These data thus indicate that levetiracetam not simply masked the expression of kindled seizures through an anticonvulsant action, but exerted a true antiepileptogenic effect. Adverse effects were not observed at any dose of levetiracetam tested in kindled rats. The powerful antiepileptogenic activity of levetiracetam in the kindling model indicates that levetiracetam is not only an interesting novel drug for symptomatic treatment of epilepsy but might be suited for pharmacological prevention of this disease in patients with a high prospective risk of the development of epilepsy.     

Cloning and expression of human carboxypeptidase Z, a novel metallocarboxypeptidase

A novel cDNA, designated carboxypeptidase Z (CPZ), was identified based on its homology to known metallocarboxypeptidases. Northern blot analysis shows bands of 2.1 and/or 2.6 kilobases in all tissues examined. The major form of CPZ mRNA in human salivary gland encodes a protein with an open reading frame of 641 amino acids. In addition, three variants were found that presumably arise due to alternative intron splicing. The 641-amino acid protein contains an 18-residue signal peptide-like sequence, a 120-residue region that shows 23-29% amino acid identity with a Cys-rich domain found in frizzled proteins and in type XVIII collagen, and then a 390-residue carboxypeptidase domain with 49% amino acid identity to carboxypeptidases E and N. The 641-amino acid form of CPZ expressed in the baculovirus system cleaves 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Phe-Ala-Arg, although the level of enzyme activity was approximately 10-fold lower than either carboxypeptidase E or D expressed using the same viral system. The CPZ activity is more active at neutral pH than at pH 5.5 and is inhibited by active site-directed inhibitors of metallocarboxypeptidases. In summary, CPZ is a novel metallocarboxypeptidase that is active toward substrates with C-terminal basic amino acids.     

Cloning, expression, and chromosome mapping of human galectin-7

The galectins are a family of beta-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. Here we report the cloning and expression of a novel member of this family (galectin-7) that correspond to IEF (isoelectric focusing) 17 (12,700 Da; pI, 7.6) in the human keratinocyte protein data base, and that is strikingly down-regulated in SV40 transformed keratinocytes (K14). The cDNA was cloned from a lambda gt11 cDNA expression library using degenerated oligodeoxyribonucleotides back-translated from an IEF 17 peptide sequence. The protein encoded by the galectin-7 clone comigrated with IEF 17 as determined by two-dimensional (two-dimensional gel electrophoresis) analysis of proteins expressed by transiently transfected COS-1 cells, and bound lactose. Alignment of the amino acid sequences with other members of the family showed that the amino acids central to the beta-galactoside interaction are conserved. Galectin-7 was partially externalized to the medium by keratinocytes although it has no typical secretion signal peptide. Immunoblotting as well as immunofluorescence analysis of human tissues with a specific galectin-7 antibody revealed a narrow distribution of the protein which was found mainly in stratified squamous epithelium. The antigen localized to basal keratinocytes, although it was also found, albeit at lower levels, in the suprabasal layers where it concentrated to areas of cell to cell contact. Both, its cellular localization as well as its striking down-regulation in K14 keratinocytes imply a role in cell-cell and/or cell-matrix interactions necessary for normal growth control. The galectin-7 gene was mapped to chromosome 19.     

Cloning, characterization, and chromosomal localization of human superillin (SVIL)

The specific aim of this study was to determine the response of alveolar bone after it was augmented vertically using distraction osteogenesis and subsequently loaded with implant restorations. Four dogs each had four implants placed horizontally into an edentulous mandibular quadrant and, after integration, a distraction osteogenesis device was fabricated in the laboratory. An osteotomy was made to allow the crest of the alveolar ridge to be distracted vertically. After 10 mm of vertical distraction, the device was stabilized with light cured resin. Following bone fill confirmation of the distraction gap at 10 weeks, two implants were placed into the ridges, one in distracted bone and one in nondistracted bone. After 4 months for implant integration, freestanding prostheses were fabricated. Crestal bone levels were evaluated throughout the period of function. Animals were sacrificed after 1 year of loading, for histologic evaluation of the bone. The vertical ridge augmentation averaged 8.85 +/- 1.05 mm after 10 weeks of healing following distraction, without change over 1 year of implant loading. Histologic examination showed that bone had formed between the distracted segments, creating an augmented ridge. The average thickness of the labial cortex in the distraction gap was significantly thinner than the lingual cortex in distracted bone and the lingual and labial nondistracted cortical bone. The presence of the dental implant did not significantly affect cortical bone thickness. Serial sections showed that implants remained integrated and functional without soft tissue inflammation. Dental implants placed into alveolar ridges augmented with the technique of distraction osteogenesis maintained bone and were functional for the length of this study.     

Cloning and functional expression of a human liver organic cation transporter

OBJECTIVE: The triglyceride-lowering effects of omega-3 fats and HDL cholesterol-raising effects of exercise may be appropriate management for dyslipidemia in NIDDM. However, fish oil may impair glycemic control in NIDDM. The present study examined the effects of moderate aerobic exercise and the incorporation of fish into a low-fat (30% total energy) diet on serum lipids and glycemic control in dyslipidemic NIDDM patients. RESEARCH DESIGN AND METHODS: In a controlled, 8-week intervention, 55 sedentary NIDDM subjects with serum triglycerides > 1.8 mmol/l and/or HDL cholesterol < 1.0 mmol/l were randomly assigned to a low-fat diet (30% daily energy intake) with or without one fish meal daily (3.6 g omega-3/day) and further randomized to a moderate (55-65% VO2max) or light (heart rate < 100 bpm) exercise program. An oral glucose tolerance test (75 g), fasting serum glucose, insulin, lipids, and GHb were measured before and after intervention. Self-monitoring of blood glucose was performed throughout. RESULTS: In the 49 subjects who completed the study, moderate exercise improved aerobic fitness (VO2max) by 12% (from 1.87 to 2.07 l/min, P = 0.0001). Fish consumption reduced triglycerides (0.80 mmol/l, P = 0.03) and HDL3 cholesterol (0.05 mmol/l, P = 0.02) and increased HDL2 cholesterol (0.06 mmol/l, P = 0.01). After adjustment for age, sex, and changes in body weight, fish diets were associated with increases in GHb (0.50%, P = 0.05) and self-monitored glucose (0.57 mmol/l, P = 0.0002), which were prevented by moderate exercise. CONCLUSIONS: A reduced fat diet incorporating one daily fish meal reduces serum triglycerides and increases HDL2 cholesterol in dyslipidemic NIDDM patients. Associated deterioration in glycemic control can be prevented by a concomitant program of moderate exercise.     

Cloning, subcellular localization and functional expression of human RNase HII

Recently we showed that the major mammalian RNase H, RNase HI, is evolutionarily related to prokaryotic RNase HII (Frank et al., FEBS-Lett. 421, 23-26, 1998), an enzyme described to be a minor activity in E. coli. As a consequence we addressed the question of whether a human RNase H exists, sharing homology with the main E. coli enzyme, RNase HI. Employing sequence analysis of expressed sequence tags, followed by specific PCR amplification of human cDNA, we cloned, sequenced and expressed a human open reading frame, coding for a 32 kDa protein. Purification of the recombinant His(6)-tagged protein from E. coli extracts using Ni(2+)-chelating chromatography and subsequent renaturation gel assay proved that it is an active RNase H. The properties of this enzyme suggest that it is identical with the human RNase HII, previously purified by one of us (Frank et al., Nucleic Acids Res. 22, 5247-5254, 1994). Studies using a green fluorescent protein-fusion construct reveal that this protein is located in the nucleus.     

Cloning and expression of the red visual pigment gene of goat (Capra hircus)

Cerebral cortical maps in adult primates reorganize within minutes-hours after peripheral injuries, but subcortical versus intracortical contributions to this rapid reorganization remain controversial. The present results show that injury of nerves to the hands of adult monkeys triggers rapid (minutes-hours) changes in maps of the hand in the brainstem main cuneate nucleus. These findings suggest that peripheral injury causes an initial concurrent reorganization of brainstem and cortical substrates and that early sensory changes emerge from reorganization involving multiple central levels.     

Cloning and expression of the gene encoding flavodoxin from Desulfovibrio vulgaris (Miyazaki F)

Blocking CD28-B7 T cell costimulatory activation by the fusion protein CTLA4Ig prevents rejection and induces long-term graft acceptance in various experimental transplant models. There are reported differences in the efficacy of CTLA4Ig in renal and cardiac rodent allograft models, but it is not clear whether these are due to the strain or species differences investigated in the different studies reported. This study investigates the effect of blocking CD28-B7 T cell costimulation with murine CTLA4Ig in rat models of acute renal and cardiac allograft rejection models, using the same complete major histocompatibility complex-incompatible strain combination. A single injection of murine CTLA4Ig 2 d after engraftment was able to induce long-term graft acceptance (> 100 d) in 54% of Lewis rat recipients of Wistar-Furth kidneys. Transferring this protocol into the acute Wistar-Furth to Lewis heart allograft model resulted in a mean graft survival time of 24.7+/-16.9 d, and all grafts were ultimately rejected. Only concomitant injection of donor cells (4 x 10(7) splenocytes) plus a single injection of CTLA4Ig on the day of transplant could induce long-term graft acceptance in 50% of animals. In both the cardiac and renal transplant models, the thymus and spleen were required for induction of tolerance. The maintenance phase of tolerance, however, did not require an intact thymus but did require the presence of a spleen. These data have important clinical applicability because human studies with T cell costimulatory blockade are being planned.     

Cloning, expression and chromosome mapping of adducin-like 70 (ADDL), a human cDNA highly homologous to human erythrocyte adducin

From a human fetal-brain cDNA library we isolated a novel human cDNA, termed human adducin-like 70 (gene symbol ADDL), whose predicted amino acid sequence showed a high degree of homology to adducins. This cDNA clone (ADDL), which contained an open reading frame of 2,022 nucleotides encoding 674 amino acids, revealed 54%, 53%, and 59% identity in predicted amino acid sequence with alpha and beta components of human adducin and rat adducin 63, respectively. Human adducin-like 70 is likely to play an important role in the skeletal organization of the cell membrane. Northern blot analysis indicated ubiquitous expression of this gene in adult human tissues. We localized the gene to chromosome bands 10q24.2-->q24.3 by fluorescence in situ hybridization (FISH).     

Granular-lattice (Avellino) corneal dystrophy

Smooth muscle differentiation in the ovary is rare, and its histopathologic spectrum, including ovarian smooth muscle metaplasia (SMM), has not been well described. The clinicopathologic findings in 48 ovaries with SMM from 40 women are reported. The average age of women with ovarian SMM was 55.6 years (range, 34 to 86 years). Foci of SMM were semiquantitatively characterized as 1+ in 46% (1 to 3 foci), 2+ in 37% (4 to 6 foci), and 3+ in 17% (> 6 foci). SMM was bilateral in 8 (23%) of the 35 patients who had bilateral oophorectomies. SMM was intimately associated with another ovarian process in 28 (58%) cases, including ovarian cysts (11), endometriosis (3), granulosa cell tumors (3), extensive stromal luteinization (1), ovarian fibroma (1), adhesions (1), and folliculogenesis (8). Ovaries with 2+ to 3+ SMM were associated with another ovarian lesion significantly more often than those with 1+ SMM (p < 0.01). Most women with ovarian SMM (86%) also had uterine leiomyomas. Significant endometrial pathology was present in 13 (37%) of 35 simultaneously removed uteri. In conclusion, SMM occurs most often in perimenopausal or postmenopausal women, most of whom also have uterine leiomyomas. Ovarian SMM is usually confined to a few microscopic fields, is bilateral in < 25% of patients, and is often associated with other ovarian lesions.     

Cloning and structure of the gene encoding the human N-methyl-D-aspartate receptor (NMDAR1)

The complete gene encoding the human N-methyl-D-aspartate receptor subunit NR1 (NMDAR1) has been isolated on a single cosmid clone. The gene is composed of 21 exons distributed over a total length of about 31 kb. More than 24 kb were sequenced. Exons 4, 20 and 21 are identical in their amino-acid sequence to those exons that are subject to alternative splicing in rat, indicating that all eight NMDAR1 isoforms found in rat will also be expressed in the human brain. Computer analysis of the pre-mRNA sequence revealed no secondary structures stable enough to explain alternative splicing. We suggest that cell-specific factors control expression of different isoforms. The promoter region contains two perfect copies of the recognition sequence for the Drosophila even-skipped protein, indicating that the developmentally regulated expression of NMDAR1 is controlled by a homeobox protein. The complete cosmid clone covering NMDAR1 was mapped to chromosome 9q34.3-qter by fluorescent in situ hybridization (FISH). The telomeric location is supported by an imperfect (CA)n repeat homologous to a subtelomeric repeat on chromosome 16p.