Cloning of cDNAs encoding the human BAG1 protein and localization of the human BAG1 gene to chromosome 9p12

cDNAs encoding the human homolog of BAG1, a Bcl-2-binding protein with anti-apoptotic function, were cloned. DNA sequence analysis of human BAG1 cDNAs predicts a protein with an additional 55 amino acids at its NH2-terminus compared to the mouse protein. Immunoblot assays using monoclonal antibodies raised against bacterially produced h-BAG1 protein confirmed the larger size of the human protein (approximately 34 kDa) compared to mouse. PCR analysis of DNA from human x rodent somatic cell hybrids using human BAG1-specific primers localized the gene to human chromosome 9. Cosmid clones of h-BAG1 were obtained and used for fluorescence in situ hybridization analysis of normal metaphase chromosomes, thus localizing h-BAG1 to 9p12, a region associated with hereditary disorders that may involve developmental dysregulation of programmed cell death.     

Cloning and sequencing of a gene from the archaeon Pyrococcus furiosus with high homology to a gene encoding phosphoenolpyruvate synthetase from Escherichia coli

A gene from the hyperthermophilic archaeon Pyrococcus furiosus, strain Vc1 (DSM 3638), contains an 817-amino-acid open reading frame which shows 42% identity to the phosphoenolpyruvate (PEP) synthetase of Escherichia coli. This putative P. furiosus PEP synthetase is slightly larger than the E. coli enzyme, the region between residues 58 and 89 being absent from the latter.     

Isolation and characterization of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3'',5''-cyclic nucleotide phosphodiesterase

An attempt has been made to suppress the ethanol-induced formation of megamitochondria (MG) in the rat liver by 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-OH-TEMPO), a free radical scavenger, and by allopurinol (AP), a xanthine oxidase inhibitor. Changes observed in the liver of animals given ethanol (EtOH) for 1 month were remarkable decreases both in the body weight gains during the course of the experiment and in the liver weight at the time of sacrifice compared to those of the control; remarkable increases in the level of thiobarbituric acid reactive substances and lipid soluble fluorophores both in microsomes and mitochondria; decreases in the content of cytochrome a+a3 and b and lowered phosphorylating ability of mitochondria; and formation of MG in the liver. A combined treatment of animals with EtOH plus 4-OH-TEMPO completely suppressed the formation of MG in the liver induced by EtOH and distinctly improved the changes caused by EtOH, as specified above, while AP partly suppressed the MG formation. Results described herein provide additional insight into chronic hepatotoxicity of EtOH besides that previously reported. A novelty of the present work is that we were able for the first time to demonstrate reversibility of EtOH-mediated ultrastructural changes of the liver by a simple administration of aminoxyl-type free radical scavenger, 4-OH-TEMPO. Our results suggest that free radicals may be involved in the mechanism of the formation of MG induced by EtOH.     

Cloning, characterization, and transcriptional analysis of a gene encoding an alpha-crystallin-related, small heat shock protein from the thermophilic cyanobacterium Synechococcus vulcanus

hspA, a gene encoding a 16-kDa heat-induced protein from the thermophilic cyanobacterium Synechococcus vulcanus, has been cloned and sequenced. The deduced amino acid sequence of the gene product showed significant homology to sequences of the family of alpha-crystallin-related, small heat shock proteins. A monocistronic mRNA of hspA increased transiently in response to heat shock. The heat shock induction occurred at a vegetative promoter but without the CIRCE (controlling inverted repeat of chaperone expression) element.     

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed lambdaSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.     

Cloning and functional expression of a swelling-induced chloride conductance regulatory protein, plCln, from rabbit ocular ciliary epithelium

The cDNA encoding a swelling-induced chloride conductance regulatory protein, plcln, was cloned from rabbit ciliary epithelium by using a polymerase chain reaction (PCR)-based approach. The open reading frame encoding 236 amino acids possesses high amino acid identity (93/%) with the previously cloned plcln from human ciliary epithelium. Outwardly rectifying currents were recorded in Xenopus oocytes injected with plcln cRNA, a result consistent with plcln expression in ciliary epithelium. A widespread distribution and marked expression of plcln mRNA in both nonpigmented ciliary epithelial (NPE) cells and pigmented ciliary epithelial (PE) cells was found for the first time. In situ hybridization analysis showed that plcln expression is more abundant in NPE than PE. These findings are consistent with the idea that plcln may be an important regulatory element in these secretory cells.     

Cloning and characterization of four types of cDNA encoding myeloperoxidase from human monocytic leukemia cell line, SKM-1

Four cDNA clones encoding myeloperoxidase were isolated from a cDNA library of monocytic leukemia SKM-1 cells. The sequences of two of them were identical to those of cDNA clones previously isolated from a HL-60 cell cDNA library. The sequences of the other two cDNA clones, MP-S34 and MP-S29, differed from those previously described. There was a deletion of 57 bp in the MP-S34 sequence, which was generated by partially skipping exon 9. MP-S29 had a 171 bp deletion, which was generated by completely skipping exon 10. Thus MP-S34 and MP-S29 encoded polypeptides lacking 19 and 57 amino acids, respectively. Both deletions were located on the sequence encoding the heavy subunit. These results indicate that the heterogeneity of the heavy subunit of MPO observed in leukocytes or leukemia could be in part produced by partial or complete skipping of an exon.     

Cloning of aas, a gene encoding a Staphylococcus saprophyticus surface protein with adhesive and autolytic properties

A gene encoding a novel cell wall-associated protein of Staphylococcus saprophyticus that binds fibronectin and to sheep erythrocytes has been cloned and sequenced. The 4392 bp open reading frame codes for an amino acid sequence that is quite similar to the Atl, an autolysin, of Staphylococcus aureus and to the AtlE of S. epidermidis. The two regions of most pronounced homology code for an N-acetyl-muramyl-L-alanine amidase and for an endo-beta-N-acetyl-D-glucosaminidase. The cloned protein lysed cells of S. saprophyticus and Micrococcus luteus exogenously. Subcloning localized the enzymatic activities to the regions of high homology and demonstrated that the interposed sequence is responsible for the adhesive activities. Two allelic replacement mutants were constructed that lacked autolytic activity and adhesive properties. The N-terminal portion of the protein contains seven highly conserved, contiguous repeats with no similarity to published sequences. It lacks the motifs typical of Gram-positive surface proteins and shows a different overall organization. This autolysin/adhesin of S. saprophyticus (Aas) appears to represent a new class of staphylococcal adhesins.     

Cloning, sequence and characterization of a sunflower (Helianthus annuus L.) pathogen-induced gene showing sequence homology with auxin-induced genes from plants

To determine whether osteoarthritic changes of the perilunate articular cartilage improve following radial osteotomy for Kienb?ck's disease and correlate with clinical results, arthroscopic examination was performed in 18 patients concurrently with radial osteotomy and at the time of removal of implants after an average of 21 months. Clinical results were satisfactory. All patients improved the preoperative level of pain. Wrist function was improved in range of motion and grip strength. Radiographic findings also demonstrated prevention of further collapse of the lunate. However, follow-up arthroscopic examination revealed progression of osteoarthritis in the area of the lunate in two thirds of the cases. There was no correlation between arthroscopic observations and clinical results. A long-term follow-up period is therefore advocated for evaluation of radial osteotomy because of the possibility of additional progression of osteoarthritic changes.     

Cloning of a cDNA encoding SjIrV1, a Schistosoma japonicum calcium-binding protein similar to calnexin, and expression of the recombinant protein in Escherichia coli

Membrane-associated proteins were isolated from adult Philippine strain Schistosoma japonicum by partitioning into the detergent phase of Triton X-114. A rabbit polyclonal antiserum raised against these proteins was used to screen an S. japonicum expression cDNA library. Positive clones were identified which encoded the species orthologue of SmIrV1, a Schistosoma mansoni protein which was initially identified by screening with sera from mice protectively vaccinated with irradiated cercariae [Hawn et al., J. Biol. Chem. 268 (1993) 7692-7698]. The S. japonicum molecule, which we term SjIrV1, is 83% identical to SmIrV1 at the predicted amino acid level and is a member of the calreticulin family of non-EF-hand, calcium-binding proteins. The Chinese strain S. japonicum orthologue of SjIrV1 was obtained by screening with the radiolabelled insert of the Philippine strain clone. Northern blot analysis revealed a single message of around 2.4 kb and gave no indication of alternative splicing. Southern blot analysis gave a simple pattern, indicating a single-copy gene, and showed a single restriction fragment length polymorphism between the genomes of Chinese and Philippine strains of S. japonicum. Recombinant, full-length SjIrV1 was expressed with a hexahistidine tag in Escherichia coli and the recombinant protein isolated by nickel-chelate chromatography. Recombinant SjIrV1 was shown to exhibit calcium-dependent, differential electrophoretic migration and to bind ruthenium red in the absence but not in the presence of calcium ions. The presence of conserved Ca(2+)-binding motifs predicted from the primary sequence, together with the Ca(2+)-dependent electrophoretic mobility of recombinant SjIrV1, confirmed that SjIrV1 was a functional calcium-binding protein.     

Identification and sequence of human PKY, a putative kinase with increased expression in multidrug-resistant cells, with homology to yeast protein kinase Yak1

The authors report a clinical case of endometriosis the abdomen rectum muscle, in woman 28 years old, after a cesarean section delivery. On the basis of literature on the topic, the following are taken into consideration, the incidence, the pathogenesis, the clinical characteristics of this kind of pathology and the aspects which might facilitate the diagnostic approach and correct therapeutic to be given or follow. Parietal endometriosis is an extremely rare disease with incidence in feminine population of 0.03-1%. The pathogenesis is still ill-known. Lack of the classical symptoms and the unusual site can make diagnosis difficult. Pathognomonics but not always present are the presence of tumescence palpable of the abdominal wall near or proximity of preceding surgical scar, the cyclic character of painful symptomatology, the augmentation of volume and the bleeding in period menstrual or premenstrual. The ultrasonography, the computerized axial tomography, the nuclear magnetic resonance can facilitate the preoperative diagnosis but they do not always furnish reports of certainty. The aspirate-needle in ultrasonography control can furnish one of orientation diagnosis. The diagnosis of certainty is founded on the histologic examination after biopsy or excision. The treatment of the abdominal wall endometriosis is surgically essential. The excision of tumescence, easy usually, it is the only means to obtain the definitive recovery. The medical therapy postoperative is adjuvant in the treatment of unrecognized pelvic centres of endometriosis.     

Cloning and chromosome assignment to 1q32 of a human cDNA (RAB7L1) encoding a small GTP-binding protein, a member of the RAS superfamily

A full-length cDNA homologous to RAB7, a member of the RAB-related GTP-binding protein subfamily, was isolated from a human placenta cDNA library. This cDNA, designated RAB7L1, has an open reading frame of 609 nucleotides encoding 203 amino acids. Northern analysis showed that the mRNA is ubiquitously expressed in human tissues, although signal intensities were different among the various organs examined. This gene was located on chromosome band 1q32 by fluorescence in situ hybridization.