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At least 829 records · Page 46Linked to original sources
PubMed1993

Characterization by cDNA cloning of the mRNA of human ribosomal protein L8.

From a lambda UNI-ZAP XR cDNA library derived from poly(A)+RNA of human ovarian granulosa cells a cDNA clone pHG51 was isolated. Sequence analysis showed significant homology to the C-terminal region of rat ribosomal protein L8 cDNA. The 5'-end of the cDNA was completed by PCR with cloned total cDNA. Aligning of DNA sequences from PCR clones with the sequence of the pHG51 insert yielded the full-length cDNA. From the open reading frame of the cDNA an amino acid sequence for a polypeptide of 257 residues was derived, which was identical with rat ribosomal protein L8 and also possessed a high degree of identity to ribosomal proteins L8 of other species. It is therefore assumed that the characterized cDNA represents the mRNA of the human ribosomal protein L8. Southern analysis revealed that human ribosomal protein L8 is specified by multi copy genes.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of the human volume-sensitive chloride conductance regulatory protein, pICln, from ocular ciliary epithelium.

Chloride channels in the ocular ciliary epithelium are believed to play a key role in aqueous humor formation. We isolated a cDNA clone from a lambda Uni-ZAP cDNA library of human nonpigmented ciliary epithelial (NPE) cells encoding the swelling-induced chloride channel/channel regulator pICln. The human clone contains an open reading frame of 237 amino acids (M(r) 26,293). The deduced human amino-acid sequence shows 90.2% and 92.7% identity with counterparts isolated from rat kidney and the canine kidney epithelial cell line MDCK. Human NPE cell lines exhibited significant levels of pICln transcripts. Complementary perforated-patch, whole-cell patch clamping demonstrated that swelling activates Cl- channels of the NPE cells, as suggested by ruptured-patch measurements. The results document the molecular isolation and identification of a human cDNA clone of a Cl- conductance regulator from ocular cells displaying volume-activated Cl-channels.

Amino Acid Sequence↗
PubMed1995

Repression of p53-dependent sequence-specific transactivation by MEF2c.

Lambda ZAP cDNA library constructed from spleen of a p53-deficient mouse was screened by South-Western technique using Fragment A, a DNA sequence that p53 specifically binds to, as a probe. One (clone 2) of six clones isolated was identical to MEF2c, a MADS-family transcription factor. Transcripts of the mef2c gene was also detected in spleen where the expression has not been reported so far. Isolated clones except clone 2 had a growth-suppression activity on p53-deficient osteosarcoma cell line (Saos II). Clone 2 repressed the transactivation from Fragment A by p53, suggesting that MEF2c may act as a negative regulator of p53-responsive element.

Animals↗
PubMed1996

Heparinase III from Flavobacterium heparinum: cloning and recombinant expression in Escherichia coli.

Heparinase III (E.C. 4.2.2.8), formerly heparinase I, produced by Flavobacterium heparinum is an enzyme that specifically cleaves heparan sulfate-rich regions of acidic polysaccharides. In this study, we report the cloning of the heparinase III gene using polymerase chain reaction (PCR). Two degenerate oligonucleotides, based on amino acid sequences derived from tryptic peptides of purified heparinase III were used to generate a approximately 1100-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. The PCR-derived probe was used to screen a Flavobacterium genomic DNA library in lambda ZAP II. The open reading frame of the heparinase III gene is 1980 bp in length, encoding a precursor protein of 75,950 Da; 10 of the tryptic peptides mapped onto the open reading frame which corresponded to approximately 18% of the protein. Recombinant heparinase III was expressed in Escherichia coli using the T7 polymerase pET expression system. This is the first report of the cloning and recombinant expression of an enzyme primarily degrading heparan sulfate.

Amino Acid Sequence↗
PubMed1997

Isolation of a genetic locus associated with metronidazole resistance in Helicobacter pylori.

We examined the molecular mechanism of metronidazole resistance by constructing a lambda-Zap II phagemid expression library with genomic DNA from a metronidazole-resistance strain of Helicobacter pylori. Twenty-two clones were found to have elevated MTZ resistances in XLOLR strain of E. coli. Phagemids belonging to the twenty two clones were extracted and then retransformed into the XLOLR strain of E. coli. After MTZ selection, five clones could confer metronidazole resistance consistently. According to Southern hybridization and DNA sequencing, the five clones contained a same locus, recA. In addition, transforming the five clones into BL21 strain of E. coli produced a higher resistance to MTZ. Interestingly, electroporation of one of the five phagemid clones into two MTZ sensitive H. pylori yielded MTZ resistant strains. Comparing amino acid sequence in MTZ resistant with sensitive isolates revealed two point mutations at this locus. Above results suggest that mutation in recA may be associated with metronidazole resistance of H. pylori.

Base Sequence↗
PubMed1997

Identification and primary structure of five human NADH-ubiquinone oxidoreductase subunits.

The multi-subunit NADH-ubiquinone oxidoreductase (complex I) is the first enzyme complex in the electron transport chain of mitochondria. A small number of NADH-ubiquinone oxidoreductase subunits are the products of mitochondrial genes (subunits 1-7), while the remainder are nuclear encoded and imported from the cytoplasm. We have isolated and sequenced five subunits of the human complex I from a human heart lambda ZAP Express cDNA library. Comparison of the deduced amino acid sequences of the human subunits with the corresponding bovine sequences revealed greater than 80% amino acid identity. The high degree of similarity between human and bovine sequences suggests functional conservation of these subunits in the complex I. In silico Northern analysis revealed that two of the subunits were expressed ubiquitously while the remainder may have more restricted patterns of expression.

Amino Acid Sequence↗
PubMed1999

Regulation of phosphatidylserine synthesis in Jurkat T cell clones: caffeine bypasses CD3/TCR-induced protein tyrosine kinases and calcium signals.

Phosphatidylserine synthesis as measured by the incorporation of [(3)H]serine into phosphatidylserine (PtdSer) through the serine-base exchange enzyme system (serine-BEES) is markedly inhibited in Jurkat cells treated with caffeine. The caffeine-induced inhibition was compared to that observed in cells treated with either CD3 mAb or thapsigargin. While CD3- and thapsigargin-induced inhibition was related to the release of Ca(2+) from the endoplasmic reticulum (ER), a process that deprives the serine-BEES of its major cofactor, caffeine modified PtdSer synthesis in the absence of decreased Ca(2+) content of ER. Using Jurkat clones differing by the expression of cell surface markers or protein tyrosine kinases implicated in the CD3/TCR signal transmission pathway, we have shown that CD3 mAb-induced inhibition of PtdSer synthesis necessitates the expression of both the CD3/TCR and the protein tyrosine phosphatase CD45 at the cell surface as well as the presence of p56(lck) and ZAP-70 protein tyrosine kinases. By contrast, thapsigargin, a blocker of the Ca(2+)-ATPase of the ER, known for its Ca(2+) releasing properties, inhibited PtdSer synthesis in all the Jurkat clones tested, indicating that this compound bypasses the CD3/TCR-induced signals. Despite its lack of effect on Ca(2+) release from ER and on protein tyrosine phosphorylations, caffeine inhibited PtdSer synthesis in all the Jurkat clones. The use of several cAMP-inducing drugs and of others xanthine derivatives indicated that caffeine modify PtdSer synthesis either by a direct action on the serine-BEES or by a modification of the structure of the phospholipids used as substrate by the enzyme.

Antibodies, Monoclonal↗
PubMed1998

Decreased protein levels of the c-Cbl protooncogene in murine AIDS.

Murine acquired immunodeficiency syndrome (MAIDS) can be viewed as a lymphoproliferative disease which involves B cells as well as T cells from spleen and lymph nodes while thymus and Peyer's patches do not participate in the process. The 120-kDa protooncogene product c-Cbl was initially cloned from the murine Cas NS-1 B cell lymphoma. It is a main target of immunoreceptor (TCR and BCR)-mediated protein tyrosine kinase activity. Moreover, recent data suggest that c-Cbl might play a crucial role in the regulation of cell proliferation through regulation of GTP-binding proteins. Therefore, the involvement of c-Cbl was evaluated in the lymphoproliferative disease induced by the MAIDS virus. The expression of the c-Cbl protein was dramatically reduced in the lymph node of infected mice while it remained normal in the thymus. In contrast, the expression of actin, TCR-zeta chain, ZAP-70, and p59(fyn) remained similar in controls and infected mice. Identical results were obtained with sorted B cells and T cells. Surprisingly, a B cell lymphoma line derived from late stage MAIDS mice displayed a normal level of c-Cbl.

Animals↗
PubMed1994

Cloning and characterization of HLC-32, a 32-kDa protein component of the sea urchin extraembryonic matrix, the hyaline layer.

A 32-kDa protein (HLC-32) was purified from sea urchin embryos and characterized. In indirect immunofluorescence analysis this species was found to be distributed throughout the cytoplasm of the unfertilized egg but within 1 hr following fertilization was localized to the hyaline layer with some residual immunofluorescence remaining within the cytoplasm of the single cell embryo. By the early blastula stage the antigen was also detected in the basal lamina, indicating a bidirectional transport mode for this protein. A cDNA clone was isolated from a lambda Zap expression library. Sequence analysis revealed a protein rich in acidic (12.4 mole%) and basic (14.3 mole%) residues. Comparative sequence analysis revealed 41 and 47% amino acid sequence homology with two butanol-extractable proteins previously isolated from blastula stage embryos of the sea urchin Paracentrotus lividus. DNA gel blot analysis indicated that the gene encoding HLC-32 belonged to a small multigenic family, while in RNA gel blot analyses a 1.4-kB transcript was detected in ovaries but not gut, testes, mature eggs, or various stage embryos. At the early blastula stage of development this protein underwent a post-translational modification reaction which resulted in the loss of a 4-amino-acid peptide fragment from the amino-terminal end of this species.

Amino Acid Sequence↗
PubMed1995

Cloning of cDNAs for the precursor protein of a low-molecular-weight subunit of the inner layer of the egg envelope (chorion) of the fish Oryzias latipes.

cDNA clones for L-SF, the precursor of a low-molecular-weight subunit (ZI-3) of the inner layer of the Oryzias latipes egg envelope were isolated from Lambda ZAP cDNA libraries constructed from the poly(A)+ RNA of the liver of spawning female fish and estrogen-treated male fish. Among them, a clone, L-SF41, is 1473 bp long and contains an open reading frame encoding a signal peptide of 19 amino acids and L-SF protein of 420 amino acids. L-SF protein seems to be glycosylated, judging from the result of the glycanase digestion. L-SF protein contains a domain similar to ZP-domains in ZP3 of some mammalian species. Northern blot analysis employing XhoI-SmaI fragments of the cloned cDNA as probes revealed that expression of the L-SF gene occurred exclusively in the livers of spawning female fish and estrogen-treated male fish and that there was no mRNA encoding L-SF in the ovary of the spawning female fish.

Amino Acid Sequence↗
PubMed1993

Identification of a cDNA coding for a Ca(2+)-binding phosphoprotein (p90), calnexin, on melanosomes in normal and malignant human melanocytes.

In order to have a proper biosynthesis and secretion of the melanin-pigment granules (melanosomes) the melanocyte may require a melanosome-associated molecule that provides a signal for assembly and organization of melanogenic enzymes and proteins within the compartment of melanosomes. This study reports the presence of a Ca(2+)-binding phosphoprotein, p90, which can be engaged in such melanogenic function, located on the melanosomal membrane of human melanocytes. A human melanoma cDNA expression library in lambda Zap II was screened with a rabbit polyclonal antibody raised against human melanosomes isolated from cultured human melanoma cells, SK MEL 23. A cDNA encoding a melanosomal protein, M(r) 90 kDa, was identified through this immunoscreening. A partial sequencing of nucleotides (822 bp from the N-terminal domain) of this clone (3.8 kb) and predicted amino acids showed more than 90% homology with dog calnexin, a previously reported endoplasmic reticulum (ER) transmembrane protein. A fusion protein of this p90 with beta-galactosidase expressed in Escherichia coli revealed both the immuno-cross-reactivity with anti-dog calnexin and anti-human melanosome antibodies and the Ca(2+)-binding property. Upon immunohistochemistry, the anti-dog calnexin antibody revealed the positive immunoreactivities with both normal and malignant human melanocytes, showing a much higher expression of antigenic epitope than nonmelanocytic human cells. The laser scanning confocal immunofluorescence, using an antibody against a human melanosome-specific antigen (HMSA-5), and immunoelectron microscopy, using immunogold, confirmed the major localization of anti-dog calnexin antibody epitope on the melanosomes and ER.

Amino Acid Sequence↗
PubMed1997

Expression of 17 beta-hydroxysteroid dehydrogenase type IV in chick retinal pigment epithelium.

Retinal pigment epithelium (RPE) has essential roles that maintain functions of the neural retina. Previously, we described that the 3b5 monoclonal antibody (3b5 mAb) recognized the RPE cells in the chick embryonic eye. In the present study, a gene coding for the 3b5 antigen has been cloned and sequenced. A 10-day-old embryonic RPE cDNA library was constructed in lambda Uni-ZAP XR, and screened with the 3b5 mAb as a probe. The complete nucleotide sequence of the antigen recognized by the 3b5 mAb was obtained. The cDNA encodes a 735 amino acid protein with a calculated molecular mass of 80,147 Da. The deduced amino acid sequence shares 72% identity with the human 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) type IV. Northern blot analysis detected a 2.9 kb transcript. Immunohistochemical and in-situ hybridization studies revealed that 17 beta-HSD type IV was preferentially expressed in the RPE cells. The expression of steroid metabolizing enzymes in the RPE cells is able to create estrogenic environment, which may have a role of maintenance of the neural retina.

17-Hydroxysteroid Dehydrogenases↗
PubMed1994

The human tissue transglutaminase gene maps on chromosome 20q12 by in situ fluorescence hybridization.

A cDNA encoding for the human tissue transglutaminase gene has been used to identify the chromosomal localization of the corresponding structural gene. The precise chromosomal and subregional localizations have been established by using in situ fluorescence mapping with a recombinant lambda-Zap phage containing the full cDNA coding sequence. The study showed that the human tissue transglutaminase gene is localized on chromosome 20 and, more precisely, within the band 20q12. To date, this is the third member of the transglutaminase gene family to be mapped. Human factor XIIIa (plasma transglutaminase), human keratinocyte transglutaminase (type I), and human tissue transglutaminase (type II) genes, although codifying for homologous enzymes, are localized on three different chromosomes.

Chromosomes, Human, Pair 20↗
PubMed1996

Genomic structure and organization of the human rBAT gene (SLC3A1).

Cystinuria is an autosomal recessive disorder of amino acid transport, manifesting as three phenotypes (I, II, and III). An amino acid transport gene, rBAT, is responsible for cystinuria. Mutation and linkage analyses have demonstrated the disease to be heterogeneous, with rBAT being the defective gene in type I cystinuria. The genomic structure of the human rBAT gene (HGMW-approved symbol SLC 3A1) has been established via two strategies: (i) construction of two different genomic libraries by subcloning the Mega-YAC921B6 (CEPH), containing rBAT, in Lambda ZAP and screening using rBAT cDNA and different PCR products; and (ii) generation and sequencing of genomic fragments by long PCR using rBAT cDNA-derived primers. The rBAT gene spans approximately 45 kb and consists of 10 exons. The introns range from 500 to 13,000 bp. All splice sites conform to the GT/AG rule. The promoter region has been further analyzed, and a predicted TATA box 98 bp upstream of the first coding ATG was identified. In addition an Alu repeat has been detected 72 bp upstream of the predicted TATA box.

Amino Acid Transport Systems, Basic↗
PubMed1997

Identification of genes encoding zinc finger motifs in the cardiovascular system.

The Zn2+-finger DNA-binding domain has been identified in several developmental control proteins, transcription factors and gene products associated with diseases, as well as in several RNA-binding proteins. We applied library screening, expressed sequence tagging (EST sequencing), Zn2+-binding assays and Northern blot hybridization, in order to characterize novel cDNA clones of the human cardiovascular system which contain Zn2+-finger motifs. An embryonic (8-10 weeks gestation) heart lambda ZAP Express cDNA library was screened with an oligonucleotide probe deduced from a consensus amino acid sequence which is highly conserved for Zn2+-finger proteins, and approximately 350 positive clones were isolated from 1 x 10(4) plaque-forming units (pfu) initially plated. The isolated clones were classified as known and novel following single pass automated DNA sequencing. Analysis of Northern blot hybridization delineated the tissue specificity of these clones, as well as their association with cardiac growth and development. Existence of Zn2+-finger motifs in the novel clones was confirmed by Zn2+-binding assay. In this report, we present the characterization of eight novel clones, including the complete cDNA sequences of one of these clones (HHZ-123).

Amino Acid Sequence↗
PubMed1996

Attenuation of multiple organ dysfunction in a chronic sheep model by the 21-aminosteroid U74389G.

Animal studies have shown that 21-aminosteroids have beneficial effects on cell and organ functions in several acute models of traumatic, hemorrhagic, and septic shock. However, it is not known if the 21-aminosteroid U74389G has any beneficial effect on organ functions in a recently developed chronic sheep model of multiple organ dysfunction after trauma. Furthermore, it is not known whether this drug has any effect on in vivo leukocyte function in this animal model. To study this, anesthetized animals were subjected to hemorrhagic shock (2 hr at a mean arterial blood pressure of 50 mmHg) and femoral reaming at Day 0. The following 5 days, endotoxin (ET; 0.75 micrograms/kg BW) and zymosan-activated plasma (ZAP; 20 ml/animal) were given every 12 hr. During the third phase (Days 6-10), the animals were merely observed. This kind of model resulted in progressive organ dysfunction indicated by increased cardiac output, decreased systemic vascular resistance, an increase of plasma-sorbitoldehydrogenase, impaired bilirubin metabolism, and impaired renal and lung function in nontreated animals. Animals receiving U74389G (3 mg/kg BW) during resuscitation from hemorrhagic shock and each time before ET/ZAP administration showed less severe organ dysfunction. Furthermore, U74389G showed beneficial effects on lung function, although it had no effect on accumulation of leukocytes in the lung or on the chemiluminescence response of isolated leukocytes from bronchoalveolar lavage fluid. These results suggest that U74389G may be a useful therapeutic agent in the prevention of multiple organ dysfunction after hemorrhagic and traumatic shock.

Alanine Transaminase↗
PubMed1993

Protein profiles of Legionella pneumophila Philadelphia-1 grown in macrophages and characterization of a gene encoding a novel 24 kDa Legionella protein.

Legionella pneumophila Philadelphia-1 strain was grown in cultured macrophages of guinea pigs, hamsters, and A/J mice and the bacteria were purified by Percoll density gradient centrifugation without any detergent. Patterns of the bacterial proteins were compared by SDS-PAGE with those of organisms cultured in vitro. A 24 kDa protein was a major protein of intracellularly grown bacteria: its expression was about four times as much as a 24 kDa protein of agar-grown bacteria. At least three novel proteins (100, 65, and 16 kDa) that were not found on agar-grown bacteria were also observed. In this paper, we focused on the 24 kDa major Legionella protein expressed within macrophages. Western blot and N-terminal amino acid analysis revealed that this protein is a novel protein different from Legionella proteins previously reported, including a 24 kDa macrophage infectivity potentiator protein (Mip). On the basis of amino acid sequence (MQRIKKI and IANAQGK), two kinds of oligonucleotides were synthesized and radiolabeled. Using these oligonucleotides as DNA probes, a 7.2 kb EcoRI-digested DNA fragment encoding the 24 kDa protein was cloned into lambda ZAP II phage vector in Escherichia coli XL1-Blue. A 0.9 kb DNA fragment from the 7.2 kb fragment was further subcloned into pUC118 in E. coli CSR603 for maxicell analysis or XL1-Blue for DNA sequencing. Maxicells which carry recombinant plasmids consisting of the 0.9 kb DNA fragment and vector plasmid pUC118 expressed the 24 kDa protein. When the DNA fragment encoding the protein was sequenced, an open reading frame of 555 base pairs was identified. The inferred polypeptide had a molecular weight of 20 kDa and an estimated isoelectric point of 8.14. Both the nucleotide sequence and the deduced amino acid sequence were distinct from those of bacterial proteins reported previously, suggesting that the protein is a novel Legionella protein.

Amino Acid Sequence↗
PubMed1994

Altered expression of cytochrome P450 mRNA during chemical-induced hepatocarcinogenesis and following partial hepatectomy.

Levels of various cytochrome P450 proteins have been reported to be decreased to varying degrees in chemically induced hepatocyte nodules and following partial hepatectomy (PH). By screening a rat liver lambda ZAP cDNA expression library with antibodies raised against a partially purified preparation of cytochrome P450 isolated from untreated male Fischer 344 rats, we have isolated a 1.1-kb cDNA. This cDNA was sequenced for 139 bases from the 5' end of the sense strand and comparison of the resulting sequence with the sequences in Gene Man DNA data bank revealed 95% homology of the sequenced portion with male-specific rat cytochrome P450 (M-1, CYP IIC11). The 32P-labeled cDNA was used as a hybridization probe on RNA blots (Northern blots) prepared with total RNA from rat livers obtained post PH, from aflatoxin B1(AFB1)-induced rat liver tumors and from rat liver nodules induced with a combination of diethylnitrosamine/acetylaminofluorene/PH (DEN/AFF/PH). At 36 and 72 hr post PH, the mRNA level was decreased by > 93%. Relative to the corresponding control livers, the mRNA level was also decreased by 97% in the liver nodules and by 57% in AFB1-induced liver tumors. The RNA blots derived from the liver nodules and AFB1-induced liver tumors were also probed with a cDNA probe (R17) that recognizes other cytochromes P450 (CYP IIB1/CYP IIB2). The mRNA corresponding to CYP IIB1/CYP IIB2 was also depressed 92% in the nodules and 65% in the tumors. These results clearly indicate that the depression of both CYP IIC11 and IIB1/IIB2 in the hepatic nodules and the tumors is related to the inhibition of transcription and/or enhanced degradation of the mRNA.

Animals↗