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Biomedical subjects

G Hull

Publications and source records attributed to G Hull.

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Sperm acrosome status and sperm antibodies in infertility.

PURPOSE: We studied whether the spermatozoa from sperm autoimmune infertile men undergo premature acrosomal loss and whether this relates to the presence of sperm antibodies in wives. MATERIALS AND METHODS: We evaluated acrosome status of live washed native and overnight capacitated spermatozoa from 17 sperm nonautoimmune fertile and 23 sperm autoimmune infertile men using an immunofluorescent peanut lectin binding assay. We used cytotoxic and immunobead binding assays to prescreen the serum and seminal plasma of these men, and serum and cervical mucus of the wives for immunological infertility. We performed immunofluorescent sperm antibody assays on all study samples to ascertain sperm antibody isotype levels in each sample. Levels of acrosomal loss in husband native and capacitated spermatozoa were correlated with levels of IgG, IgA and IgM sperm antibodies in the study samples. RESULTS: Sperm autoimmune infertile men had a significantly larger percentage of sperm (p <0.0001) that had lost the acrosome and a lower percentage of sperm with intact acrosome (p <0.0001) in native and capacitated preparations in contrast to those of fertile controls. Levels of cytotoxic and IgA antibodies, especially in seminal plasma and cervical mucus, correlated significantly with percentages of sperm with a total loss of acrosome in native and capacitated sperm preparations (p < or = 0.01). CONCLUSION: Infertile men with sperm antibodies in serum and seminal plasma undergo premature acrosome loss. This loss may expose the reproductive tract immune system, especially that involving IgA, in autoimmune infertile men and the wives to high immunogenic levels of sperm acrosome membrane antigens, thereby rendering them immunologically infertile.

Acrosome

Molecular weights and isoelectric points of sperm antigens relevant to autoimmune infertility in men.

PURPOSE: We determined the molecular weights and isoelectric points of antigens in the uncapacitated and capacitated spermatozoa of fertile men binding to the serum immunoglobulin G (IgG) from 8 autoimmune infertile men and 8 fertile nonautoimmune men. MATERIALS AND METHODS: We used double fluorochrome cytotoxicity and immunobead binding assays to determine the sperm antibody status of the study subjects. 2-Dimensional gel electrophoresis and Western blot analysis were used to determine the molecular weights and isoelectric points of sperm antigens binding to serum IgG from these men. Amino acid sequencing of the digested peptides of chosen proteins was accomplished. Immune reactivity to the proteins in autoimmune infertile men was further verified. RESULTS: Serum IgG from fertile men failed to react significantly. Serum IgG from all autoimmune men (100%) showed significant binding to proteins with a molecular weight of 92 kDa. and isoelectric points of 3.5 to 4.0 in the capacitated spermatozoa. Six of 8 infertile men (75%) had serum IgG binding to capacitated sperm antigens with a molecular weight of 18 kDa. and isoelectric points of 4.5 to 5.2. Amino acid sequencing of peptides of the 92 kDa. protein matched complement component 1 (C1) inhibitor, with noted differences in the amino acid sequencing from the latter. The 18 kDa. protein matched calmodulin. We verified that serum IgG from autoimmune infertile men bound with C1 inhibitor and ascertained that the 92 kDa. protein in the spermatozoa was C1 inhibitor-like protein. CONCLUSIONS: Significant antibody responses to C1 inhibitor-like protein and calmodulin were noted in autoimmune men. Both of these proteins may be of testicular origin and these autoimmune responses may be highly relevant to infertility.

Amino Acid Sequence

Richard Wiseman 1622-1676.

Wiseman, like Paré, learnt his surgery on the battlefield. On his return to civilian practice in London he became the first consultant surgeon, only treating patients referred to him. Increasing ill-health allowed him time to write and the resulting Severall Chirurgicall Treatises describes over 600 of his own cases. It is a more detailed and personal account than any written by his contemporaries, and his sections on the King's Evil and cancer show a remarkable understanding of disease for his time. Wiseman raised the status of British surgery very considerably, his work having a great influence well into the 18th century.

General Surgery

Robert Chessher.

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Clubfoot

Extended-term cultures of human T-lymphocytes: a practical alternative to primary human lymphocytes for use in genotoxicity testing.

A simplified method, using recombinant interleukin-2, foetal bovine serum and freeze-killed feeder cells, has been developed for the mass culture of T-lymphocytes derived from human peripheral blood. In this protocol, bulk cultures can be cryopreserved approximately 8 days after initiation, and subsequent mass cultures generated a further week after recovery. At the end of this period, the lymphocytes have maintained a normal karyotype and cultures from different donors are very similar in terms of rate of cell division and expression of key antigenic markers. Background micronucleus frequencies and dose-responses for micronucleus induction by a reference clastogen, hycanthone, were also very similar in all the cultures examined. Such extended-term T-lymphocyte cultures are potentially valuable in genotoxicity testing, providing cells with the normal human karyotype which can be characterised and handled with the practical convenience of established rodent cell lines.

Animals

Demonstration of differential effects of cytokines on mast cells derived from murine bone marrow and peripheral blood mononuclear cells.

Mouse bone marrow (BM) was cultured in the presence of recombinant mouse (rm) interleukin-3 (IL-3), rmIL-4, rmIL-5, rmIL-7, purified mouse (m) IL-9, rmIL-10, recombinant human (rh) macrophage-colony-stimulating factors (M-CSF), rm granulocyte-macrophage colony-stimulating factors (GM-CSF) rm stem cell factor (SCF), rh interferon-alpha (IFN-alpha), rmIFN-gamma, and mNGF to determine which cytokine would give rise to mast cells in murine BM cultures. From a starting population of 1 x 10(7) cells, 1.55 x 10(7) mast cells developed within 14 days in cultures supplemented by rmIL-3. No mast cells were seen at day 14 when any of the other cytokines were present alone, except for rmSCF, which supported the growth of < 0.01% of mast cells observed in IL-3-dependent BM cultures. When rmIL-4, -5, -7, -10, mIL-9, rhM-CSF, rmGM-CSF, rmSCF, rhIFN-alpha, -gamma, or mNGF were added to BM cultures in the presence of rmIL-3, mast cell growth increased 200% with the addition of rmSCF, and 10% when rmIL-4 or IL-9 was added. However, the addition of rhM-CSF, rmGM-CSF, rmIFN-gamma, and mNGF decreased the number of mast cells. Mast cell number, as determined by metachromatic stains, generally approximated the number of Fc epsilon RI+ cells as assessed by FACS analysis. Among the cytokines, only rmIL-4 and rmSCF were able to support the survival of mast cell progenitors in the absence of obvious mast cell proliferation, similarly to rmIL-3. Only rmSCF alone, or in combination with rmIL-3 or -4, supported the growth of mast cells from mouse peripheral blood mononuclear cells (PBMC) where the number of mast cell precursors was about 90 per 10(6) PBMC. With time, mouse BM cells cultured in rmIL-3 became more responsive to rmSCF. Taken together, these data demonstrate that IL-3 is a major early mast cell growth factor, that mast cells become more dependent on SCF with time, and that the effects of IL-3 and SCF are upregulated (IL-4) or downregulated (M-CSF, GM-CSF, IFN-gamma) by both growth factors and proinflammatory cytokines.

Animals

Two different Em-like genes are expressed in Arabidopsis thaliana seeds during maturation.

Using a radish cDNA probe, we have isolated and characterized two genomic clones from Arabidopsis thaliana (GEA1 and GEA6) encoding two different proteins that are homologous to the "Early methionine-labelled" (Em) protein of wheat. GEA1 differs from GEA6 and Em clones of wheat in that a sequence coding for 20 amino acid residues is tandemly repeated 4 times. These two genomic clones correspond to two genes named AtEm1 and AtEm6. Sequencing of several cDNA clones showed that both genes are expressed. The transcription start site was determined for both genes by RNase mapping. The site of polyadenylation is variable and there is no obvious consensus sequence for polyadenylation at the 3' ends of the genes. mRNA corresponding to GEA6 is present only in nearly dry and dry seeds, whereas the corresponding to GEA1 appears in immature seeds and is maximum in dry seeds. No expression of either gene could be detected in leaf, stem, or floral buds. Expression of both genes could be detected in immature seeds when the siliques were incubated with abscisic acid (ABA), demonstrating that both genes are ABA responsive. However, examination of the 5' upstream region does not reveal any extensive homology, suggesting that regulation of the two genes differs. In situ hybridization with a GEA1 probe demonstrated that the expression of this gene is essentially located in the provascular tissues of the cotyledons and axis of the dry seed as well as in the epiderm and outer layers of the cortex in the embryo axis.

Abscisic Acid

Spatiotemporal expression of two cell-cell adhesion molecule 105 isoforms during liver development.

The rat liver adhesion molecule cell CAM 105 has been postulated to be involved in liver histogenesis. Recently, it was shown to exist in two isoforms that differ in the length of their cytoplasmic domains (O. Culic, Q-H. Huang, D. Flanagan, D. C. Hixson, and S. H. Lin, Biochem. J., 285: 47-53, 1992). Isoform-specific differences in phosphorylation and aggregation function have been observed. To study the possible roles of these isoforms during liver development, we have used both complementary DNA probes and isoform-specific antibodies to examine their temporal and spatial expression. Northern blot analysis revealed low steady-state levels of a 4.0-kilobase RNA at 15-20 days gestation, which increased dramatically at birth and remained at least 12-fold higher than fetal levels in neonatal and adult liver. Additional polyadenylated RNA species of 6.0 and 2.9 kilobases were detected after birth. Steady-state levels of cell CAM 105 RNA in cultured adult and fetal hepatocytes were comparable to in vivo levels, respectively, and were not influenced by treatment with transforming growth factor beta or by culture density. Increases in cell CAM 105 protein demonstrated by immunoblot analysis correlated with the RNA increases, suggesting that regulation of cell CAM 105 expression is largely transcriptional during development. Ratios of the long and short isoforms remained relatively constant after birth. Isoform-specific antipeptide antibodies localized both isoforms primarily to maturing bile canalicular domains of hepatocytes during liver development. The long isoform could not be detected in fetal liver in situ prior to 20 days, however, suggesting that specific roles may exist for these molecules during development.

Adenosine Triphosphatases

Restriction fragment analysis of the secalin loci of rye.

Analyses of wheat/rye addition lines by Southern blotting confirmed the presence of sequences related to the Sec 1, Sec 2, and Sec 3 loci on chromosomes 1R and 2R. Comparison of the 1R and 2R addition lines allowed the identification of gamma-secalin genes at Sec 1 and Sec 2, respectively, while omega-secalin and gamma-secalin genes at Sec 1 were discriminated by comparative hybridization with three probes: omega-secalin, total gamma-secalin, and 3' gamma-secalin. The high molecular weight (HMW) secalin genes at Sec 3 were identified using a homologous HMW subunit probe from wheat. Gene copy numbers were estimated as about 40-60 for omega-scalins, 5-10 for gamma-secalins, and 2 for HMW secalins. Comparison of individual plants of cv. Gazelle showed a high degree of polymorphism, particularly for sequences related to omega-secalins and HMW secalins.

Amino Acid Sequence