Biomedical subjects

K H Scheit

Publications and source records attributed to K H Scheit.

At least 55 records · Page 3Linked to original sources

PubMed1990

Characterization of three abundant mRNAs from human ovarian granulosa cells.

Three cDNA clones, pHGR122, pHGR11, and pHGR74 containing the coding information for abundant mRNAs were identified from a human ovarian granulosa cell cDNA library. Characterization by nucleotide sequencing revealed that pHGR122 was specific for a collagenase inhibitor and pHGR11 for melanoma-associated antigen ME491. Relative quantification by Northern analysis indicated that collagenase inhibitor mRNA is a major species in granulosa cells. This finding provides evidence for the origin of this protein in follicular fluid as a secretory product of granulosa cells. pHGR11 identified melanoma-associated antigen ME491 as the unexpected product of normal, noncarcinogenic, granulosa cells. pHGR74 has the complete coding information for an unknown protein. Three independent experiments: (i) cell-free translation of pHGR74 RNA; (ii) transcription of suitable restriction fragments followed by cell-free translation; (iii) hydrolysis of the cell-free translation product of pHGR74 RNA by endoproteinase Lys-C, identified one open reading frame coding for an acidic, highly hydrophilic protein of 111 amino acid residues. pHGR74 mRNA is expressed in human testis, prostate, seminal vesicle, and ovarian granulosa cells. A comparative Southern analysis indicates pHGR74 mRNA is species specific and encoded by a single-copy gene.

Amino Acid Sequence↗

PubMed1990

Gene expression in bovine seminal vesicles.

Seminal vesicle secretion contributes significantly to the proteins of bovine seminal plasma. The following proteins from bull seminal vesicle were isolated and characterized: major protein (PDC 109), the basic proteins BUSI II, RNAse BS1, protein P6 and seminal antimicrobial protein (SAP). Using antibodies against the proteins BUSI II, RNAse BS1, SAP and major protein, the seminal vesicle epithelium was identified as the source of the respective antigens. The biosynthesis of bovine seminal vesicle secretory proteins was studied by cell free translation of poly (A)-RNA from seminal vesicles and the respective mRNAs were characterized by cDNA cloning. Recombinant clones (103) of a cDNA library of bull seminal vesicle poly (A) + RNA were screened by colony hybridisation using radioactively labelled synthetic probes. The respective clone containing the longest cDNA insert was sequenced. In case of major protein the Mr of the 134 amino acid residue precursor polypeptide was 15,480 as deduced from direct mRNA sequencing. The precursor sequence of 25 amino acid residues has a hydrophobic character and very likely constitutes a signal peptide, directing the protein towards the secretory pathway. The deduced amino acid sequence contained no consensus sequence indicative of N-glycosylation.

Amino Acid Sequence↗

PubMed1990

Synthesis of bioactive seminalplasmin by expression of a newly constructed fusion gene.

A synthetic DNA, carrying the coding sequence for seminalplasmin (SAP), the major basic protein of bull semen, was cloned into the C-terminal part of a shortened, mutated fragment of the lacZ gene (lacZ-MF) of vector pLZPWB1. As a result of the mutation, all methionine as well as cysteine residues are replaced by other amino-acid residues. In the fusion gene lacZ-MF-SAP of the resulting construct pSAP4 the two proteins are linked through a methionine residue. Expression of pSAP4 in E. coli W3110 in the presence of the inducer isopropylthiogalactoside (IPTG) led to production of fusion protein with a yield of approximately 50% of the total proteins synthesized. All SAP-immunoreactive fusion protein was found within the insoluble protein fraction and represented 40% of total proteins produced during expression. The fusion protein was subjected to cyanogen bromide cleavage. The overall yield of crude SAP with a purity of 80% was 10 mg/l of culture. The crude SAP was further purified by calmodulin-Sepharose affinity absorption. Characterisation by protein chemical analysis indicated the identity of recombinant SAP with authentic SAP purified from bull semen.

Animals↗

PubMed1990

[A quantitative ELISA for the measurement of class-specific anti-spermatozoa antibodies].

A class-specific ELISA for the quantitative determination of anti-spermatozoal antibodies is described. The intra-assay variation at the lowest standard concentration for anti-spermatozoal-IgG- and anti-spermatozoal-IgA-specific measurements was 6%. The test possessed an inter-assay variation at the lowest standard concentration in case of the anti-spermatozoal-IgG-determination of 9% and for anti-spermatozoal-IgA-determinations of 8.5%. With this ELISA 5.7% anti-spermatozoal-IgAs and 11.3% anti-spermatozoal-IgGs were identified in sera of a group of infertile females (N = 53). A collective of anti-spermatozoal antibody positive sera, identified by an anti-spermatozoal antibody ELISA measuring total immunoglobulins was subjected to an investigation with the class-specific ELISA. The proportion of anti-spermatozoal-IgAs (54%) was significantly lower than that of anti-spermatozoal-IgAs (78%). Interestingly, no correlation was observed between the parameters IgA and IgG in antispermatozoal positive sera (r = 0.139).

Antibodies↗

PubMed1990

Functional properties of peptides derived from seminalplasmin: binding to monospecific anti-seminalplasmin immunoglobulins G and calmodulin.

Seminalplasmin was specifically hydrolysed employing the proteinases Lys-C and Glu-C. A set of peptides of seminalplasmin were obtained which were used to study their interaction with monospecific anti-seminalplasmin IgGs as well as calmodulin. Two peptides P4 (position 38-47) and P9 (position 4-32) strongly interacted with the polyclonal anti-seminalplasmin IgGs, indicating that a C-terminal (P4) as well as a N-terminal region of seminalplasmin represent major antigenic sites of the polypeptide. From the panel of peptides only peptide P9 was found to bind to calmodulin with high affinity. Thus, the structural requirements for the strong and specific interaction of calmodulin with seminalplasmin apparently reside in the N-terminal sequence 3-32 of the latter.

Amino Acid Sequence↗

PubMed1989

[Immunologically active glycoproteins of Baptisia tinctoria].

Chromatographically purified fractions of aqueous-ethanolic extracts from Baptisia tinctoria roots contained a strong lymphocyte DNA synthesis-stimulating activity. Electrophoretic analysis of these fractions revealed four distinct protein bands with molecular masses of P1 = 58 kD; P4 = 31 kD; P5 = 26 kD; and P6 = 14 kD. They contained carbohydrate as determined by periodic acid Schiff staining. An estimation of the approximate amount of sugar was done by using human transferrin as a reference, this method revealed the following values: P1 = 27%; P4 = 12%; P5 = 14%; and P6 = 8%. The mixture of proteins and every single band were immunoreactive with a polyclonal antiserum against Baptisia proteins determined in immune and dot blots, respectively. Electrophoretically purified proteins were characterized by tryptic cleavage and determination of their amino acid content. They contained several common amino acids, predominantly aspartic acid, glutamic acid, threonine, and alanine. The content of glucosamine and/or galactosamine was less than 0.2 Mol-per cent. The four proteins revealed pI values between 5.3 and 4.7. Protein P 4 was immunochemically related to phytohemagglutinin but, in contrast to PHA-P, it exhibited no hemagglutinating activity and no leucagglutinating activity like PHA-L.

Adjuvants, Immunologic↗

PubMed1989

Complete sequence of the coding region of human elongation factor 2 (EF-2) by enzymatic amplification of cDNA from human ovarian granulosa cells.

The use of two primers allowed the specific enzymatic amplification of elongation factor 2 starting with total double-stranded cDNA from human ovarian granulosa cells. The amplified DNA fragment with a length of 1765 bp was restricted and sequenced by the shot gun approach. From the sequences obtained from the amplified fragment and the cDNA insert of pHGR81 [Rapp et al. (1988) Biol. Chem. Hoppe-Seyler 369, 247-250] respectively, the DNA sequence containing the complete coding as well as the 3'-untranslated region was assembled.

Animals↗

PubMed1989

Inhibition of cell proliferation by basic proteins from bull seminal plasma.

The fraction of basic proteins has been shown to suppress the concanavalin A-induced proliferation of bovine lymphocytes effectively in a dose-dependent manner. The inhibitory effect was observed within a concentration range where the fraction of basic proteins was not cytotoxic. By separating the basic proteins, only two proteins, seminal ribonuclease and seminal antimicrobial protein were found to be active in inhibition of lymphocyte proliferation. Both proteins appear to function differently. At a concentration of 56 micrograms/ml, where both proteins inhibited DNA synthesis, seminal ribonuclease also inhibited RNA synthesis by greater than 50% whereas seminal antimicrobial protein had no effect on RNA synthesis. It was furthermore observed that the suppressive effect of the basic proteins was independent of the mitogenic stimulus and proliferation of both, T- as well as B-lymphocytes was inhibited by those proteins.

Animals↗

PubMed1989

Immunological relationships between specific sperm proteins and secretory polypeptides from seminal vesicles.

Rat and bovine testicular and posttesticular spermatozoa, in situ or removed from the seminal pathways were studied for the presence and the binding of seminal vesicle secretory proteins. A protein cross-reactive with an antibody against rat seminal secretory sulfhydryloxidase was found in mitochondria of pachytene primary spermatocytes which are sequestered during spermiogenesis and collected in the residual bodies of late spermatids. This indicates the replacement of one generation of mitochondria by another during spermiogenesis. Using an antibody against the SVS II polypeptide from rat seminal vesicles, an intrinsic immunoreactive protein was detected in the principal piece of the sperm tail as well as an extrinsic protein (presumably secreted by the epididymis) on the sperm head. The actin-capping capacity of SVS II indicates the functional relation of this immunoreactive membrane protein with actin or an actinrelated protein. Ampullary bovine spermatozoa, contrary to their epididymal precursors contain immunoreactive major protein, secreted from bull seminal vesicles, firmly incorporated into the (sub) plasmalemmal protein in the middle piece. In this case, a secretory protein from seminal vesicles is incorporated into the sperm proteins. This is presumably an important process, related with the initiation of hypermotility of spermatozoa.

Animals↗

PubMed1989

Localization of bovine seminal plasma RNA-A BS1, on the surface of bovine spermatozoa.

Using IgG antibodies raised against RNA-A BS1, the presence of this seminal RNA-A on the surface of bovine spermatozoa has been demonstrated. Indirect immunofluorescence and immunoferritin methods showed that this protein coats the surface of ejaculated bovine spermatozoa, but the pattern of binding of the label varied from cell to cell. More than 50% of the spermatozoa showed labelling all over, except the anterior head region; about 30% showed labelling all over except the region below the equatorial plate region; and the remaining were either completely labelled or showed labelling only in the head or the tail region. The head-tail junction (the neck region) was not labelled in any case.

Animals↗

PubMed1988

Binding of a major secretory protein from bull seminal vesicles to bovine spermatozoa.

The seminal vesicles synthesize in an androgen-dependent manner a neutral protein of 13.5 kDa molecular weight that makes up about 40% of their secretion ("major protein"). An antiserum against this protein raised in rabbits was used to localize the antigen within the seminal vesicles. In addition to intraluminal secretion of the seminal vesicles and the ampulla of the vas deferens, ejaculated and ampullary spermatozoa revealed an intense immunoreaction, which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Comparison of spermatozoa taken from the tail of the epididymis with ampullary spermatozoa showed that about 90% of the latter, but only 10-20% of the former presented this distributional pattern of immunoreactive sites. Epididymal epithelium as well as calf seminal vesicle epithelium showed no immunoreactivity with major protein antiserum. Using a pre-embedding staining technique with gold-labeled primary or secondary antibodies, respectively, no immunostaining could be achieved at the ultrastructural level. Incubation experiments of epididymal spermatozoa in EGTA-containing solutions in the absence of calcium resulted in a gradual labilization and eventual loss of the plasma membrane of the sperm middle piece. After removal of (at least part of) the plasma membrane, bound major protein could be visualized immunohistochemically close to the mitochondria of the middle piece using a gold-labeled primary or secondary antibody. The acceptor site for major protein therefore seems to reside inside the plasma membrane of the sperm middle piece.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

The major protein of bull seminal plasma: biosynthesis and biological function.

We isolated the major protein of apparent Mr of 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid respectively. Cell-free translation of poly(A)RNA from seminal vesicle tissue and immunoprecipitation yielded one major species with apparent Mr of 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. Cloning and sequencing of a major protein-specific cDNA led to the identification of clone pMP17, encoding a precursor of the major protein of 128 amino acid residues. We proved that the major protein is identical to protein PDC 109 (Esch et al., Biochem. Biophys. Res. Comm. 113:861-867, 1983). The seminal vesicles synthesize major protein in an androgen-dependent fashion. In addition to intraluminal secretion of the vas deferens, ampullary spermatozoa revealed an intense immunoreaction which was restricted to the neck region of the sperm head and the middle piece, while the principal piece of the tail as well as the sperm head were devoid of immunoreactive material. Epididymal epithelium (as well as calf seminal vesicle epithelium) showed no immunoreactivity with major protein antiserum. Immunoelectron microscopy demonstrated that only spermatozoa devoid of a plasma membrane around the middle piece were able to bind the antiserum against major protein. After removal of the plasma membrane from epididymal spermatozoa, binding of major protein to subplasmalemmal binding sites was visualised using gold-labeled MP. Transblotting with gold-labeled MP demonstrated a protein of about 66 kDa which appears to represent the major protein-receptor. Binding of major protein to the receptor (after loss of the plasma membrane in the mid-piece region of the spermatozoa after contact with secretions from seminal vesicles) is interpreted as a physiological process presumably related to the onset of sperm motility.

Amino Acid Sequence↗

PubMed1988

Cloning and sequence analysis of a cDNA from seminal vesicle tissue encoding the precursor of the major protein of bull semen.

A cDNA library derived from poly(A)+RNA of bull seminal vesicle tissue was screened with a synthetic DNA probe specific for the major protein of bull semen. A positive clone pMP17, containing a 680-bp insert, was sequenced. In combination with primer-extension sequencing of poly(A)+RNA, a DNA sequence of 700 bp was determined. This DNA encodes a reading frame for 134 amino acids, starting with an ATG and terminated by a TAG codon. The first 25 amino acids constitute a signal peptide segment followed by 109 amino acids with the known sequence of the major protein. The initiation methionine occurs within the sequence CTACCATGG, which is highly homologous to a putative control signal for translational efficiency of mammalian mRNAs. The DNA sequence comprises a 3' untranslated region of 276 bp and the polyadenylation signal AATAAA, 13 bp upstream from a tract of A residues. Northern analysis indicated the presence of a 750-bp mRNA species in poly(A)+RNA of seminal vesicle tissue. According to Southern analysis, one gene appears to specify the major protein of bull semen.

Animals↗

PubMed1988

Cloning and sequence analysis of a cDNA from human ovarian granulosa cells encoding the C-terminal part of human elongation factor 2.

A cDNA clone, pHGR81, encoding 358 amino-acid residues of the C-terminal region of human elongation factor 2 (EF-2), was isolated from a human ovarian granulosa cell cDNA library. The deduced amino-acid sequence of pHGR81, when compared with the known identical amino-acid sequences of hamster as well as rat EF-2 revealed a substitution of a glutamine by an alanine residue in the partially determined human sequence. The 15 amino-acid-residue sequence comprising the histidine-715, supposed to be of importance for the biological function of EF-2, is preserved in human EF-2. The coding region of the cDNA insert of pHGR81 displays a homology of 87% to hamster and of 88% to rat EF-2 cDNA. In Northern-transfer analysis, pHGR81 specifically hybridizes with an mRNA species of 3.1 kb.

Amino Acid Sequence↗

PubMed1987

Cloning and sequence analysis of a cDNA encoding poly-ubiquitin in human ovarian granulosa cells.

A cDNA clone, pHGR21 encoding poly-ubiquitin, was isolated from a human ovarian granulosa cDNA library. This clone contained three complete, and part of a fourth, ubiquitin coding sequence joined head to tail with no spacer sequences. Northern analysis employing a restriction fragment comprising a complete ubiquitin coding unit indicated the existence of two mRNA species of 1.1kb and 2.8kb. Sequence comparison of pHGR21 with the known two human ubiquitin genes revealed differences to the human ubiquitin-3 repeat gene but significant homology to the human ubiquitin-9 repeat gene. The untranslated 3'-region and the adjacent ubiquitin coding repeat were found to be identical to that of the human ubiquitin-9 repeat gene. The other 3 ubiquitin coding repeats were of close homology to the fourth ubiquitin coding repeat of the human ubiquitin-9 repeat gene. These findings suggest the existence of yet another human poly-ubiquitin gene.

Base Sequence↗

PubMed1987

An mRNA encoding poly-ubiquitin in porcine corpus luteum: identification by cDNA cloning and sequencing.

A cDNA library derived from a particular fraction of mRNA of porcine corpus luteum was screened by hybrid-release. cDNA clone pCL208 selected an mRNA which, in cell-free translation, directed the synthesis of a major polypeptide of 28 kD. Sequence analysis of clone pCL208 revealed that the entire coding sequence contained three complete repeats and a fourth incomplete repeat of porcine ubiquitin, joined head-to-tail without any spacer peptide sequences. The ultimate ubiquitin molecule contained a Phe residue as a carboxy-terminal extension. The coding sequence terminated with the stop codon TAA followed by a short untranslated region, the polyadenylation signal AATAAA, and a tract of A residues. Northern analysis indicated the presence of two types of ubiquitin-specific mRNAs, i.e., 1100 and 2500 bp, in porcine corpus luteum.

Amino Acid Sequence↗

PubMed1987

Molecular cloning and expression of a synthetic DNA coding for the antimicrobial protein of bull seminal plasma.

A DNA carrying the coding sequence for the antimicrobial protein from bull seminal plasma (SAP) was obtained by enzymic ligation of six synthetic oligonucleotides. The 162 bp synthetic DNA fragment was cloned into the C-terminal part of the lacZ-gene employing the vector pUR289. Expression in E. coli in the presence of the inducer isopropylthiogalactoside (IPTG) led to the formation of a fusion protein, which was shown by immuno-blotting to contain immuno-reactive antimicrobial protein. Approximately 90 min after induction, the cells stopped growing and the culture was found to contain no viable cells 3 h after induction. We conclude from this observation that the beta-galactosidase-antimicrobial protein fusion product was toxic for the E. coli cell and that the SAP-residue attached to beta-galactosidase was responsible for the cytotoxicity.

Animals↗

PubMed1987

Immunohistochemistry of secretory proteins in the bull seminal vesicle.

Antibodies against several proteins isolated from bovine seminal fluid (ribonucleases, bull seminal proteinase inhibitor BUSI II, seminal antimicrobial protein SAP) were used to identify the secretion sites of the respective proteins within the genital tract of the bull. Consistent positive immunoreactions were achieved with most of the antisera in the seminal vesicle epithelium, while only weak or dispersed immunoreactions were found in the epididymis, ductus deferens or the prostate. Comparison of serial sections of bull seminal vesicle stained with different antisera gave incongruous distribution patterns of positively reacting cells. This was interpreted as a sign of either a differential secretion cycle for different proteins in these cells or, more likely, a fixation or processing artifact. There was no clear cut evidence for secretion of seminal antimicrobial protein by seminal vesicle epithelium, but rather a resorption of intraluminally concentrated SAP. The secretion site of this important protein remains obscure, unless cRNA probes for in situ hybridisation studies are available.

Animals↗