Alpha Sequences Research Articles

The exon structure of the mouse alpha 2(IX) collagen gene shows unexpected divergence from the chick gene.

One cosmid and two overlapping phage clones covering the entire mouse alpha 2(IX) collagen gene including 12 kilobase pairs (kb) of 5'- and 8 kb of 3'-flanking sequences were isolated from two genomic libraries. The overall gene structure was determined by restriction mapping and nucleotide sequencing. The gene spans 16 kb from the start of transcription to the polyadenylation site and contains 32 exons. It codes for a mRNA of 3 kb that translates into a polypeptide of 688 amino acids. The intron-exon junctions and mRNA structure were confirmed by amplification of cDNA made for mouse cartilage RNA. The coding sequence of the mouse alpha 2(IX) collagen gene shows marked similarities to those for other type IX collagen chains. Although the overall exon-intron organization of the mouse gene is very similar to the chick alpha 2(IX) gene, some unexpected differences were observed at the splice junctions. Split codons characteristic for the central triple helical domain of the chick were not found in the mouse gene that thus exhibited a long stretch of exons with sizes that are multiples of 9 base pairs in this domain. The promoter of the mouse alpha 2(IX) collagen gene contains some G + C-rich elements including three Sp1 consensus recognition sites and a far upstream CCAAT box but no TATAA box. Both primer extension and RNase protection assays revealed several transcription start sites within 418 base pairs of the promoter. The present study reports the first complete nucleotide sequence of any type IX collagen gene and forms the basis for comparative structural studies on this collagen type and for experiments involving transgenic mice.

The human alpha 1(XV) collagen chain contains a large amino-terminal non-triple helical domain with a tandem repeat structure and homology to alpha 1(XVIII) collagen.

We have cloned and characterized cDNAs encoding the alpha 1 chain of type XV collagen from a human placenta library. Using primer extension cloning we extended the cDNAs to the 5' end of the mRNA and determined the complete deduced primary structure of the human alpha 1(XV) chain. The polypeptide chain contains nine triple helical domains separated by eight non-triple helical regions and flanked by large amino-terminal (555 amino acid residues) and carboxyl-terminal (256 amino acid residues) non-triple helical domains. Comparison of amino acid sequences of the human alpha 1(XV) chain with those of mouse alpha 1(XVIII) collagen showed remarkable similarity within both amino- and carboxyl-terminal non-triple helical domains. Within the carboxyl third of the amino-terminal domain a tandem repeat structure is found with an about 45-amino acid residue sequence repeated four times. This amino acid sequence has a strikingly high similarity to rat cartilage proteoglycan core protein. Northern blot analysis of human embryonic RNA revealed that alpha 1(XV) mRNA is expressed predominantly in internal organs such as the adrenal gland, kidney, and pancreas.

2-O-methyl-D-mannose residues are immunodominant in extracellular polysaccharides of Mucor racemosus and related molds.

In this study, the structure of the immunodominant carbohydrate epitope of the extracellular polysaccharides from mold species belonging to the order Mucorales reactive with rabbit IgG antibodies was elucidated. An exo-alpha-D-mannanase which was able to abolish the antigenicity of these polysaccharides completely was purified and characterized, and the activity was compared with that of an alpha-D-mannosidase. Analysis of the monomeric reaction products after enzymatic treatment revealed the presence of 2-O-methyl-D-mannose residues. This compound is a constituent of the polysaccharides from the mold genera Mucor, Rhizopus, Rhizomucor, Absidia, Syncephalastrum, and Thamnidium, and its occurrence in fungi has not been reported until now. Two mannan fractions which are highly reactive with rabbit IgG were isolated from the extracellular polysaccharides of Mucor racemosus and characterized with ethylation analysis. The role of the newly found 2-O-methyl-D-mannose residues in the immunoreactivity was assessed by specific degradation of these mannans with the exo-alpha-D-mannanase and subsequent ethylation analysis. It was concluded that the immunodominant carbohydrates reactive with rabbit IgG are chains composed of a single terminal non-reducing 2-O-methyl-D-mannose residue, alpha (1-2)-linked to a short sequence of alpha(1-2)-linked D-mannose residues.

Tyrosine-containing activation motif-dependent phagocytosis in mast cells.

FcR capable of triggering cell activation share with BCR and TCR a conserved intracytoplasmic tyrosine-containing activation motif (TAM). Besides cell activation, these receptors trigger other biologic responses, such as endocytosis of soluble ligands. Murine mast cells express two types of FcR that, when aggregated by antibodies and multivalent Ag, trigger the release of inflammatory mediators and cytokines. These are high affinity receptors for IgE (Fc epsilon RI) and low affinity receptors for IgG (Fc gamma RIII). They comprise each an IgE- or IgG-binding alpha-subunit and two TAM-containing subunits that associate with both receptors: a beta-subunit and a homodimeric gamma-subunit that can associate also with the other subunits of the TCR. Herein, we focused on biologic activities triggered in mast cells via the TAM of the gamma-subunits. Using rat basophilic leukemia (RBL) cells stably transfected with cDNA-encoding murine Fc gamma RIII alpha, we found that murine Fc gamma RIII trigger the phagocytosis of antibody-coated erythrocytes. Using RBL transfectants expressing Fc gamma RIII with a deletion of the intracytoplasmic domain of Fc gamma RIII alpha or chimeric receptors having the extracellular and transmembrane domains of Fc gamma RII and the intracytoplasmic domain of Fc gamma RIII alpha, we showed that intracytoplasmic sequences of Fc gamma RIII alpha are neither necessary nor sufficient for Fc gamma RIII to trigger phagocytosis. Using RBL transfectants expressing chimeric receptors having the extracellular and transmembrane domains of Fc gamma RII and the TAM-containing intracytoplasmic domain of murine Fc gamma RIII gamma, we demonstrated that intracytoplasmic sequences of Fc gamma RIII gamma are sufficient to trigger phagocytosis. Using RBL transfectants expressing the same Fc gamma RII-III gamma chimeras, in the TAM of which one, the other, or both tyrosine residues were mutated, we established that tyrosines in the TAM sequence are required for phagocytosis. Our results endow TAM gamma with previously unknown triggering capacities and Fc gamma RIII with new biologic properties.

Requirement for double-strand breaks but not for specific DNA sequences in herpes simplex virus type 1 genome isomerization events.

Herpes simplex virus type 1 (HSV-1) genome isomerization occurs as a result of DNA replication-mediated homologous recombination between several sets of inverted repeat sequences present in the viral DNA. The frequency with which this recombination occurs has been demonstrated to be dependent upon DNA homology length rather than specific sequences. However, the smallest of the viral inverted repeats, the alpha sequence, has been shown to function as a recombinational hot spot, leading to speculation that this sequence may represent a specific element through which genome isomerization is mediated. To investigate this apparent paradox, a quantitative transient recombination assay system was developed and used to examine the recombinogenic properties of a panel of alpha sequence mutants. This analysis revealed that the presence of both the pac1 and pac2 elements was both necessary and sufficient for the induction of high-frequency recombination events by the alpha sequence. However, it was the double-strand break promoted by pac1 and pac2 during cleavage and packaging at the alpha sequence, and not the DNA sequences of the elements themselves, which appeared to be critical for recombination. This was illustrated (i) by the inability of the same pac1 and pac2 sequences to mediate inversion events in cells infected with an HSV-1 mutant which was competent for DNA replication-dependent recombination but defective for the cleavage and packaging process and (ii) by the ability of double-strand breaks generated in non-HSV-1 DNA by an in vivo-expressed restriction endonuclease to significantly stimulate the initiation of recombination events in virus-infected cells. Thus, the alpha sequence appears to act as a hot spot for homologous recombination simply because it happens to coincide with the site of the double-strand break which is generated during the cleavage and packaging process, not because it contains discrete sequences which are required for this activity. However, it was found that this enhanced recombinogenicity disappeared when the element was flanked by regions of extensive sequence homology, particularly that of the large inverted repeats which flank the alpha sequence at its natural site in the HSV-1 genome. These findings are consistent with a model for HSV-1 genome isomerization in which recombination is initiated primarily by multiple random double-strand breaks which arise during DNA replication across the inverted repeats of the genome, rather than by a single specific break which occurs at the alpha sequence during the cleavage and packaging process.

Identification of a binding site on retinal transducin α for the phosphodiesterase inhibitory γ subunit

Transducin alpha (T alpha) activates retinal rod cyclic GMP phosphodiesterase (PDE) by interacting with and removing the inhibitory PDE gamma subunit. A T alpha-PDE gamma complex can be isolated in vitro, and our previous work [Morrison, Rider and Takemoto (1987) FEBS Lett. 222, 266-270; Morrison, Cunnick, Oppert and Takemoto (1989) J. Biol. Chem. 264, 11671-11681] has identified a region of PDE gamma, residues 24-45, that binds to T alpha. The C-terminal region of PDE gamma is the site that interacts with PDE alpha/beta and inhibits catalytic function. The site on T alpha that binds to the PDE gamma 24-45 region has not been identified. Synthetic peptides (15-mers) which span the bovine T alpha sequence were tested for binding to purified recombinant PDE gamma using a solid-phase assay. The peptides were also tested for ability to activate a PDE complex. We have identified a region, residues 250-275 of T alpha, which shows a high affinity of PDE gamma and for the PDE gamma (24-45) binding peptide. The peptide did not bind to the C-terminal residues 50-87 of PDE gamma. Likewise, a region of T alpha, 1-25 did not exhibit high-affinity binding to PDE gamma or to the 24-45 PDE gamma peptide. Specific binding of the 250-275 peptide to PDE gamma was confirmed by its ability to compete with T alpha binding to PDE gamma, although a higher concentration was required (10x). The T alpha-(250-275) peptide activated a fully inhibited PDE alpha beta gamma 2 complex in a dose-dependent manner. These results suggest that a region on T alpha that recognizes the PDE gamma-binding site is found within residues 250-275 of T alpha.

Molecular cloning of alpha antigen like protein gene of Mycobacterium leprae and its over production in Escherichia coli

I have constructed the genomic library of M. leprae Thai 53 strain, and cloned the alpha antigen like protein gene by plaque hybridization method by using M. leprae alpha antigen DNA fragment as probe which was characterized in the previous study, I have termed it as alpha 2 antigen gene. The alpha 2 antigen gene has been characterized by sequencing. By comparing the deduced amino acid sequence of alpha and alpha 2 antigen with 85 complex antigen of other mycobacteria. I have found the higher homology between alpha 2 antigen and 85A antigen and between alpha antigen and 85B antigen. We have constructed the over expression system of M. leprae alpha and alpha 2 antigen gene in E. coli using vector pMALc-RI. Recombinant alpha and alpha 2 antigen has been purified by amylose column chromatography at the purity of more than 95%. More than 6 mg and more than 10 mg of recombinant alpha and alpha 2 antigen has been obtained from 200 ml of liquid culture, respectively. ELISA tests have been performed with the sera of leprosy patient and healthy control against the recombinant alpha and alpha 2 antigens. The antibody titers in sera of leprosy patient against the two kinds of antigens were all much higher than healthy controls. The antibody titer against the alpha 2 antigen was higher than that against alpha antigen. Recombinant alpha and alpha 2 antigens in this study could be used as a new specific antigen for serodiagnosis of leprosy.

The fidelity of the human leading and lagging strand DNA replication apparatus with 8-oxodeoxyguanosine triphosphate.

A product of oxidative metabolism, 8-oxodeoxyguanosine triphosphate (8-O-dGTP), readily pairs with adenine during DNA replication, ultimately causing A.T-->C.G transversions. This study utilized 8-O-dGTP as a probe to examine the fidelity of the leading and lagging strand replication apparatus in extracts of HeLa cells. Simian virus (SV) 40 T antigen-dependent DNA replication reactions were performed with two M13mp2 vectors with the SV40 origin located on opposite sides of the lacZ alpha sequence used to score replication errors. The presence of 8-O-dGTP at equimolar concentration with each of the 4 normal dNTPs resulted in a > 46-fold increase in error rate for A.T-->C.G transversion over that observed in the absence of 8-O-dGTP. A similar average error rate was observed on the (+) and (-) strands in both vectors, suggesting that the fidelity of replication by leading and lagging strand replication proteins is similar for the dA.8-O-dGMP mispair. Replication fidelity in the presence of 8-O-dGTP was reduced on both strands when an inhibitor of exonucleolytic proofreading (dGMP) was added to the reaction. These data suggest that the majority of dA.8-O-dGMP mispairs are proofread by both leading and lagging strand replication proteins.

Sequence and tissue distribution of the integrin alpha 9 subunit, a novel partner of beta 1 that is widely distributed in epithelia and muscle [published erratum appears in J Cell Biol 1994 Feb;124(3):395

The integrin family of adhesion receptors consists of several heterodimeric glycoproteins, each composed of one alpha and one beta subunit. A novel integrin alpha subunit partial cDNA isolated from TGF-beta stimulated guinea pig airway epithelial cells has previously been reported (Erle, D.J., D. Sheppard, J. Bruess, C. Rüegg, and R. Pytela. 1991. Am. J. Respir. Cell Mol. Biol. 5:170-177). We have now determined cDNA and amino acid sequence for the human homolog of this subunit, named alpha 9, from a human lung cDNA library, a human small intestine cDNA library, and cDNA from the cell lines U937, HL-60 and Tera-2. This sequence is predicted to encode a 1006-amino acid mature protein that shares 39% identity with the previously identified integrin subunit alpha 4. By Northern blot analysis, alpha 9 mRNA was detected in the human carcinoma cell lines Tera-2 and Caco-2. Anti-peptide antibodies against the predicted COOH-terminal sequence of alpha 9 immunoprecipitated a heterodimer (140 kD/115 kD nonreduced; 150 kD/130 kD reduced) from Tera-2 lysates. Immunodepletion of beta 1-containing integrins with Tera-2 lysates removed alpha 9 immunoreactivity, suggesting that beta 1 is the principal beta subunit partner for alpha 9 in these cells. alpha 9 was detected by immunohistochemistry in airway epithelium, in the basal layer of squamous epithelium, and in smooth muscle, skeletal muscle, and hepatocytes.

Identification of conserved T cell receptor CDR3 residues contacting known exposed peptide side chains from a major histocompatibility complex class I‐bound determinant

We have analyzed the T cell receptor (TCR) repertoire found in the major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response to the protein ovalbumin (OVA). Despite skewing towards the expression of V beta 5.2+TCR by OVA-specific CTL from C57BL/6 mice, we found a relatively high degree of diversity in V(D)J usage in both TCR alpha- and beta-chains. Closer examination showed that the majority of these sequences encoded negatively and positively charged residues at their respective TCR alpha- and beta-chain VJ or VDJ junctions. These junctions form the third complementarity-determining regions (CDR3) of the TCR polypeptides involved in the direct interaction with the class I-bound peptide. Crystallographic analyses of Kb-peptide complexes predict that the major determinant from OVA, peptide OVA257-264 (SIINFEKL), contains two exposed charged side chains which can contact the TCR. These are the negatively charged glutamic acid at determinant position 6 (P6) and the positively charged lysine at P7. To examine whether the TCR alpha-chain makes contact with P7 lysine, we established a single chain TCR transgenic C57BL/6 mouse line where all T cells express a TCR beta-chain derived from the V beta 5.2+ clone B3. OVA-specific T cells derived from in vivo primed transgenic mice preferentially expressed TCR alpha-chains that also contained negatively charged junctional residues despite some further variation in V alpha and J alpha sequences. Stimulation of naive TCR beta-chain transgenic T cells with a P7 substitution peptide analogue induced a T cell response that was no longer cross-reactive with the wild-type OVA257-264 determinant, suggesting that the TCR alpha-chain from the T cell clone B3 can determine the specificity for this residue. Consequently, these results reveal the existence of conserved residues in the CDR3 of TCR alpha- and beta-chains specific for OVA257-264 and identify their possible orientation over the peptide-class I complex.

Impaired growth hormone-releasing hormone signal transduction in the dwarf (dw) rat is independent of a generalized defect in the stimulatory G-protein, Gs alpha.

The homozygous dwarf (dw) rat pituitary is characterized by a 95% reduction in GH content as well as a 75-80% reduction in the number of somatotrophs. The nature of the mutation responsible for this phenotype is unknown. Previous investigations from our laboratory indicate that dw somatotrophs exhibit decreased sensitivity and a reduced GH secretory response to GH-releasing hormone in vitro, accompanied by a decreased generation of cAMP. We hypothesized that dw rats have a defect in the pathway linking the GH-releasing hormone receptor to adenylyl cyclase and focused on the expression and function of the stimulatory G-protein, Gs alpha. When corrected for differences in pituitary size and cell number, GH mRNA content was reduced by 78% compared to that in controls. However, there was no difference in Gs alpha mRNA content or size in dw pituitaries. Similarly, there was no difference in the content or size of mRNA for the pituitary transcription factor pit-1 in dw pituitaries. Immunoblot analysis of pituitary membrane proteins using a Gs alpha-specific antibody revealed no differences in size, quantity, or relative distribution of Gs alpha peptides between control and dw pituitaries. In addition, cholera toxin effectively ribosylated dw Gs alpha, and there were no differences in the size, quantity, or relative distribution of ribosylated membrane proteins between dw and control pituitaries. Finally, to examine for mutations in other regions of the Gs alpha-coding sequence, we cloned the full-length Gs alpha cDNA from dw rat pituitaries by polymerase chain reaction. The sequence of this clone was identical to that of normal rat Gs alpha cDNA. These results indicate that 1) 52-kilodalton Gs alpha appears to predominate in both normal and dw pituitary; 2) the content, function, and sequence of Gs alpha in adult dw rats are normal; and 3) a generalized Gs alpha regulatory or structural mutation as the cause of the observed phenotype can be excluded. The results also demonstrate that there is no decrease in pit-1 expression in the adult dw pituitary.

Alternative extracellular and cytoplasmic domains of the integrin alpha 7 subunit are differentially expressed during development.

Examination of cDNAs for the laminin-binding alpha 7 integrin subunit identified two different sequences (designated X1 and X2) coding for the variable region between the III and IV homology repeat domains near the putative ligand-binding site. Sequencing of a mouse alpha 7 genomic clone established that the X1 and X2 regions are derived by mutually exclusive alternative mRNA splicing. Reverse transcriptase-polymerase chain reaction analysis of alpha 7 mRNA indicated that the X1 and X2 isoforms were present in equal amounts in mouse skeletal myoblasts and adult heart. However, in adult skeletal muscle, the X2 variant was exclusively expressed. Amino acid sequence homologies in the III/IV segment suggest that alpha 3 and alpha 6 are also alternatively spliced at this site. We identified alternatively spliced exons in a human alpha 6 genomic clone that encode X1- and X2-like segments. Analysis of the alpha 7 cytoplasmic domain indicated that this region was also alternatively spliced and like alpha 3 and alpha 6 could exist as the A or B form. In mouse skeletal and cardiac muscle the B form of alpha 7 was strongly expressed. However, we identified alpha 7A in neonate and adult skeletal muscle but not in cardiac tissue. High levels of alpha 7A were detected in differentiating myotubes, but in proliferating myoblasts only the alpha 7B isoform was present. These results indicate that alternative splicing of alpha 7 mRNA is differentially regulated during development and generates variant integrin chains with structurally and presumably functionally unique ligand-binding and cytoplasmic domains.

Characterization of the gene for the human high affinity IgE receptor (Fc epsilon RI) alpha-chain.

The Fc epsilon RI couples the mast cell-surface binding of IgE and Ag to a complex series of intracellular events culminating in cell activation and degranulation. The alpha-chain of Fc epsilon RI constitutes the Ig-binding subunit of this heterotetrameric receptor, and is itself a member of the Ig gene superfamily. We have isolated a human genomic DNA clone containing the entire Fc epsilon RI alpha gene, and completely sequenced a region from 1257 bp 5' of the transcription start site, to 513 bp 3' of the last exon of the gene. As with the previously characterized rat and mouse genes, human Fc epsilon RI alpha consists of five exons and four introns, and spans 5889 bp of genomic DNA. The splice donor and acceptor sites deduced by comparison with the cDNA sequence corresponded exactly to the locations found in analogous rodent genes. By mapping the 5' end of Fc epsilon RI alpha transcripts we found three major transcription initiation sites 24, 27, and 29 bp upstream of the ATG translation initiation codon. As well, several longer minor transcripts were seen, with a maximum of 60 nt of 5'-untranslated sequence. About 650 bp of DNA upstream of the ATG translation initiation codon were compared among human, rat, and mouse Fc epsilon RI alpha sequences in search of common motifs that might mediate conserved regulatory interactions with DNA binding proteins. A 172-bp region of the human Fc epsilon RI alpha 5'-flanking sequence was highly conserved in both rodent species. Further studies will be required to determine whether these or other sequences are involved in Fc epsilon RI alpha gene regulation.

The precursor region of a protein active in sperm-egg fusion contains a metalloprotease and a disintegrin domain: structural, functional, and evolutionary implications.

PH-30, a sperm surface protein involved in sperm-egg fusion, is composed of two subunits, alpha and beta, which are synthesized as precursors and processed, during sperm development, to yield the mature forms. The mature PH-30 alpha/beta complex resembles certain viral fusion proteins in membrane topology and predicted binding and fusion functions. Furthermore, the mature subunits are similar in sequence to each other and to a family of disintegrin domain-containing snake venom proteins. We report here the sequences of the PH-30 alpha and beta precursor regions. Their domain organizations are similar to each other and to precursors of snake venom metalloproteases and disintegrins. The alpha precursor region contains, from amino to carboxyl terminus, pro, metalloprotease, and disintegrin domains. The beta precursor region contains pro and metalloprotease domains. Residues diagnostic of a catalytically active metalloprotease are present in the alpha, but not the beta, precursor region. We propose that the active sites of the PH-30 alpha and snake venom metalloproteases are structurally similar to that of astacin. PH-30, acting through its metalloprotease and/or disintegrin domains, could be involved in sperm development as well as sperm-egg binding and fusion. Phylogenetic analysis indicates that PH-30 stems from a multidomain ancestral protein.

Expression of topoisomerase II alpha and beta in an adenocarcinoma cell line carrying amplified topoisomerase II alpha and retinoic acid receptor alpha genes.

Human topoisomerase II enzymes are targets for a number of widely used anticancer agents. We have analysed a lung adenocarcinoma cell line CALU3, which has co-amplified topoisomerase II alpha and ERBB2 sequences, for the structure of the amplicon and for expression of both topoisomerase II alpha and beta. The region of chromosome 17q amplified in CALU3 also includes the retinoic acid receptor alpha locus and is therefore similar to the amplicon observed in breast cancers carrying amplified topoisomerase II alpha and retinoic acid receptor sequences. The use of fluorescence in situ hybridisation localises the amplified topoisomerase II alpha sequences to a cluster on one chromosome with single copies localised to others. CALU3 express high levels of topoisomerase II alpha is determined by Western blot, immunofluorescence and enzyme activity. The enzyme activity extracted from CALU3 is sensitive to inhibition by the topoisomerase II poison etoposide. Topoisomerase II beta expression was observed in three lung cancer cell lines including CALU3 and was confined to the nucleoli. Thus, the CALU3 cell line is an ideal model to study the amplification and expression of topoisomerase II alpha in adenocarcinomas.

Regulation of Na,K-adenosine triphosphatase gene expression by sodium ions in cultured neonatal rat cardiocytes.

Na,K-ATPase (Na,K-pump) plays an important role in the regulation of intracellular ion composition. The purpose of this study is to determine whether Na+ regulates the levels of mRNA coding for Na,K-ATPase alpha and beta subunits in cultured neonatal rat cardiocytes. We measured intracellular Na+ levels ([Na+]i) in cardiocytes using a Na(+)-sensitive fluorescence dye (SBFI). 1 mM ouabain caused a significant increase in [Na+]i in cardiocytes; from 12.8 +/- 0.3 to 28.8 +/- 1.8 mM. Exposure of cardiocytes to 1 mM ouabain resulted in a three- to fourfold increase in alpha 1, alpha 2, and alpha 3 mRNA accumulation, and an approximate two-fold increase in beta 1 mRNA accumulation. A maximum elevation was reached at 60 min in both cases. The ouabain-induced alpha 1 mRNA accumulation was still observed in the Ca(2+)-free culture medium. Exposure of cardiocytes to 10 microM monensin in the absence of extracellular Ca2+ also resulted in a threefold increase in alpha 1 mRNA accumulation. The increased alpha 1 mRNA expression by 1 mM ouabain was associated with a fourfold increase in alpha 1 subunit protein accumulation. Transfection experiments with chimeric plasmids containing 5'-flanking sequences of alpha 1, alpha 2, and alpha 3 isoform genes and a luciferase reporter gene revealed that 1 mM ouabain caused a twofold increase in luciferase activity in each alpha system. These results suggest that Na+ directly regulates Na,K-ATPase gene expression in cardiocytes. The transfection study further supports the premise that Na(+)-responsive elements are located within the 5'-flanking sequences of each alpha isoform gene.

Purification and characterization of plantaricin A, a Lactobacillus plantarum bacteriocin whose activity depends on the action of two peptides

A Lactobacillus plantarum bacteriocin, plantaricin A, has been purified to homogeneity by ammonium sulphate precipitation, binding to cation exchanger and Octyl-Sepharose, and reverse-phase chromatography. The bacteriocin activity was associated with two peptides, termed alpha and beta, which were separated upon reverse-phase chromatography. Bacteriocin activity required the complementary action of both the alpha and beta peptides. From the N-terminal end, 21 and 22 amino acid residues of alpha and beta, respectively, were sequenced. Further attempts at sequencing revealed no additional amino acid residues, suggesting that either the C terminus had been reached or that modifications in the next amino acid residue blocked the sequencing reaction. Judging from their amino acid sequence, alpha and beta may be encoded by the same gene, since alpha appeared to be a truncated form of beta. Alanine, the first amino acid residue at the N-terminal end of beta was not present at this position in alpha. Otherwise the sequences of alpha and beta appeared to be identical. The calculated molecular masses of the sequenced part of alpha and beta were 2426 and 2497 Da, respectively. The molecular masses of alpha and beta as determined by mass spectroscopy were 2687 +/- 30 and 2758 +/- 30 Da, respectively, indicating that (i) the only difference between alpha and beta was the presence of the N-terminal alanine residue in beta, and that (ii) in addition to the sequenced residues, two to three unidentified amino acid residues are present at the C-terminal ends of the alpha and beta peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Transforming growth factor-alpha and its receptor are expressed in the epithelium of the rat prostate gland.

Transforming growth factor-alpha (TGF alpha) is a growth regulatory polypeptide factor that is expressed by several prostatic tumor cell lines. In this investigation, we examined whether TGF alpha is expressed during morphogenesis of the rat prostate gland. We first examined whether the prostate gland expressed TGF alpha by immunohistochemistry. Using a sheep antiserum directed against the mature sequence of TGF alpha, immunoreactivity was identified in the ventral and lateral, but not the dorsal, lobes of the prostate gland from 9-week-old rats. TGF alpha immunoreactivity was localized to a supranuclear site of the epithelial cells, with no immunoreactivity in the stromal cells or elsewhere in the prostate gland. A mouse monoclonal antibody directed against a TGF alpha epitope near cysteine-16 also identified the polypeptide. Furthermore, we confirmed that TGF alpha was synthesized in the epithelial cells of the ventral prostate gland, using an antiserum raised against the intracellular sequences of the transmembrane precursor. To assess the primary target for TGF alpha, we examined expression of receptors for TGF alpha (epidermal growth factor receptor) and found receptors to be localized to the apical surface of the epithelial cells of the ventral prostate gland. We next investigated a potential developmental role for TGF alpha in the rat prostate by examining TGF alpha expression in ventral rat prostate glands ranging from 8 days postpartum to 9 weeks of age. We found that TGF alpha expression was directly proportional to the developmental stage of the gland. Significantly, the fourth week of prostate development marked the appearance of TGF alpha expression in the supranuclear region of the epithelial cells. Immunohistochemical staining of progressively mature prostates of 5 and 6 weeks that contain more complex acinar formations exhibited more intense and highly localized TGF alpha staining. In conclusion, TGF alpha and its receptor have been identified in the apical region of epithelial cells of the rat prostate, and there is reason to believe that TGF alpha may function as an autocrine factor that promotes differentiated functions in the prostate gland.

Transforming growth factor-alpha and its receptor are expressed in the epithelium of the rat prostate gland

Transforming growth factor-alpha (TGF alpha) is a growth regulatory polypeptide factor that is expressed by several prostatic tumor cell lines. In this investigation, we examined whether TGF alpha is expressed during morphogenesis of the rat prostate gland. We first examined whether the prostate gland expressed TGF alpha by immunohistochemistry. Using a sheep antiserum directed against the mature sequence of TGF alpha, immunoreactivity was identified in the ventral and lateral, but not the dorsal, lobes of the prostate gland from 9-week-old rats. TGF alpha immunoreactivity was localized to a supranuclear site of the epithelial cells, with no immunoreactivity in the stromal cells or elsewhere in the prostate gland. A mouse monoclonal antibody directed against a TGF alpha epitope near cysteine-16 also identified the polypeptide. Furthermore, we confirmed that TGF alpha was synthesized in the epithelial cells of the ventral prostate gland, using an antiserum raised against the intracellular sequences of the transmembrane precursor. To assess the primary target for TGF alpha, we examined expression of receptors for TGF alpha (epidermal growth factor receptor) and found receptors to be localized to the apical surface of the epithelial cells of the ventral prostate gland. We next investigated a potential developmental role for TGF alpha in the rat prostate by examining TGF alpha expression in ventral rat prostate glands ranging from 8 days postpartum to 9 weeks of age. We found that TGF alpha expression was directly proportional to the developmental stage of the gland. Significantly, the fourth week of prostate development marked the appearance of TGF alpha expression in the supranuclear region of the epithelial cells. Immunohistochemical staining of progressively mature prostates of 5 and 6 weeks that contain more complex acinar formations exhibited more intense and highly localized TGF alpha staining. In conclusion, TGF alpha and its receptor have been identified in the apical region of epithelial cells of the rat prostate, and there is reason to believe that TGF alpha may function as an autocrine factor that promotes differentiated functions in the prostate gland.

Expression and characterization of functionally active fragments of the platelet glycoprotein (GP) Ib-IX complex in mammalian cells. Incorporation of GP Ib alpha into the cell surface membrane.

The platelet glycoprotein Ib-IX complex (GP Ib-IX) is essential for the initial attachment of platelets to the wall of damaged arteries. In this study, an N-terminal fragment of human GP Ib alpha (residues 1-318), containing the ligand binding sites for von Willebrand factor (vWF) and thrombin, as well as the entire human GP Ib alpha were expressed in Chinese hamster ovary cells. The transfected cells secreted a 48- and a 110-kDa protein, respectively, into the supernatant. Both recombinant proteins were purified by immunoaffinity chromatography. The purified proteins bound soluble vWF in the presence of botrocetin as demonstrated in solid-phase binding assays. The dissociation constant (Kd) for 125I-vWF binding to the recombinant 110-kDa protein was 1.2 +/- 0.2 nM as compared with 1.0 +/- 0.3 nM for vWF binding to purified platelet GP Ib-IX. Both recombinant proteins were also retained on thrombin-Sepharose 4B. The 48-kDa protein contained two N-linked oligosaccharide chains. A 125-kDa protein was identified in the lysate of cells transfected with the coding sequence for the entire GP Ib alpha. Trypsin treatment of this protein generated a 110-kDa fragment, whereas the secreted 110-kDa protein remained unchanged. Post-translational removal of the C-terminal transmembrane domain of recombinant GP Ib alpha might have facilitated the secretion of the soluble glycocalicin-like 110-kDa fragment. In addition, flow cytometry and immunofluorescence microscopy demonstrated that the expression of GP Ib alpha alone is sufficient for its incorporation into the cell surface membrane. These data indicate that two soluble fragments of human GP Ib alpha with binding activity for vWF and thrombin can be expressed in mammalian cells and that the incorporation of GP Ib alpha into the surface membrane does not depend on co-expression with GP Ib beta and/or GP IX.