Comparison of two media in a human in vitro fertilization program: lack of significant differences in pregnancy rate.
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Culture Media↗
Biomedical subjects
G Shaw
Publications and source records attributed to G Shaw.
A mutant of Escherichia coli fumarate reductase decoupled from electron transport.
The terminal electron-transfer enzyme fumarate reductase of Escherichia coli is a complex iron-sulfur flavoenzyme composed of four nonidentical subunits organized into two domains: FrdA and -B (a membrane-extrinsic catalytic domain) and FrdC and -D (a transmembrane anchor domain). We have identified a mutation within the membrane-intrinsic domain that alters the electron transfer properties of the iron-sulfur and flavin redox centers of the catalytic domain. Functional electron flow from the quinone analog 2,3-dimethyl-1,4-naphthoquinone or from the electron transport chain is impaired. However, the mutant enzyme can be reduced normally by single-electron donors such as the dye benzyl viologen. The mutant phenotype results from a single A----G transition changing His-82, within the second transmembrane alpha-helix of the FrdC anchor sequence, to an arginine. The mutation, physically located within the anchor domain, is manifested by altered catalytic properties, indicating that the intrinsic and extrinsic domains are conformationally connected. These results confirm the important role of the anchor subunits in functional electron transport and have implications for communication between intrinsic and extrinsic domains of membrane proteins.
Base Sequence↗
Effects of N-linked carbohydrate on the in vivo properties of human GM-CSF.
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Animals↗
Uterine and embryonic metabolism after diapause in the tammar wallaby, Macropus eugenii.
Pouch young were removed from lactating tammars to terminate embryonic diapause. Uterine metabolism was assessed at periods afterwards by incubating endometrial explants with [3H]leucine, and measuring the incorporation into acid-soluble material. Blastocysts were incubated with [3H]uridine to assess uptake and incorporation into acid-soluble material. Uterine reactivation, shown by an increase in the rate of leucine incorporation into secreted protein, was evident by Day 4 after removal of pouch young and was significantly more in both secreted and tissue protein by Day 6. Both continued to increase in gravid and non-gravid uteri up to Day 12. By the end of pregnancy (Day 26) uterine metabolism in the gravid uterus produced 2-3 times more secreted protein than in the non-gravid uterus, demonstrating a local feto-placental influence on the uterus. Tissue incorporation had declined in endometrium of gravid and non-gravid uteri by Day 26. Day 5 embryos were metabolically more active than in quiescence, although expansion of the embryos was not seen until Day 9. The early reactivation of the uterus and embryo from diapause suggests that it is not triggered by the previously described peaks of progesterone and oestradiol in plasma at Day 5, although there may be an earlier, increased sensitivity to these steroids which allows uterine reactivation to precede changes in peripheral plasma concentration.
Animals↗
Urogenital vasculature and local steroid concentrations in the uterine branch of the ovarian vein of the female tammar wallaby (Macropus eugenii).
The urogenital vasculature of the tammar comprises 4 major paired arteries and veins: the ovarian, the cranial urogenital, the caudal urogenital and the internal pudendal artery and vein. The ovarian artery and vein and their uterine branches which supply the ovary, oviduct and uterus, ramify extensively. Each anterior urogenital artery and vein supplies the caudal regions of the ipsilateral uterus, lateral and median vagina and cranial parts of the urogenital sinus. The caudal urogenital arteries and veins supply the urogenital sinus and caudal regions of the bladder. The internal pudendal artery and vein vascularize the cloacal region, with some anastomoses with branches of the external pudendal vessels. Anastomoses connect the uterine branch of the ovarian artery with the uterine branch of the cranial urogenital and cranial branches of the caudal urogenital arteries, and connect the caudal urogenital and the internal pudendal arteries. Anastomotic connections between the left and right arterial supply also occur across the midline of the cervical regions of the uteri and the anterior lateral vaginae. Similar connections are seen in the venous system. The uterine branch of the ovarian artery ramifies extensively very close to the ovary, giving a plexiform arrangement with the ovarian veins, and also with the uterine venous system on the lateral side of each uterus. This plexiform structure provides an anatomical arrangement which could allow a local transfer of ovarian hormones from ovarian vein into the uterine arterial supply, and thence to the ipsilateral uterus. Progesterone concentrations in plasma from the mesometrial side of the uterine branch of the ovarian vein are markedly higher than in tail vein plasma, especially during the 'Day 5 peak' early in pregnancy, and also at full term. There is also a marked decrease in progesterone concentration from all sites immediately before birth as previously reported for peripheral plasma. These results support the suggestion of a countercurrent transfer mechanism, at least for progesterone, and possibly other hormones, between the ovarian vein and uterine artery. Such a local transfer could explain the different morphological responses of the endometria of the two adjacent uteri during pregnancy in macropodid marsupial species.
Animals↗
Reactivity of a panel of neurofilament antibodies on phosphorylated and dephosphorylated neurofilaments.
The work of the Sternbergers and their colleagues has shown that monoclonal antibodies reactive with neurofilament subunit proteins may be sensitive to the state of phosphorylation of these proteins. We therefore examined the ability of our previously described panel of monoclonal and polyclonal neurofilament antibodies to bind to normal and to enzymatically dephosphorylated neurofilament subunits. All the monospecific antibodies, both mono- and polyclonal, which we had previously documented as reactive with neurofilament H protein proved to bind only to the phosphorylated form of this protein, and H antibody staining of neurofilamentous profiles in frozen sections could be abolished by appropriate pretreatment of sections with alkaline phosphatase. In contrast, all monospecific antibodies, both mono- and polyclonal, reactive with native M and L proved to bind with apparently undiminished affinity following enzymatic dephosphorylation of the appropriate antigen, either in frozen sections or on Western blots. The class of monoclonal antibodies which react with both H and M were variable in their response to dephosphorylated neurofilaments; some completely lost their reactivity whilst others were partially or wholly unaffected. We stained frozen sections of nervous tissues from various mammalian species with the panel of antibodies, and observed filamentous staining of the perikarya and dendrites of a variety of different types of neuron with all antibodies, both mono- and polyclonal, directed against L and M. Antibodies with strong reactivity for phosphorylated H always failed to stain neurofilamentous dendritic and perikaryal profiles. We further describe the isolation and characterization of a new monoclonal antibody, which recognizes both phosphorylated and enzymatically dephosphorylated forms of H.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
alpha-MSH and neurofilament M-protein share a continuous epitope but not extended sequences. An explanation for neurofibrillary staining with alpha-MSH antibodies.
The long recognized neurofibrillary immunoreactivity of the nervous system with alpha-MSH antibodies arises from an epitope on neurofilament M-protein which we have now characterized. It is situated in the amino terminal residues where M-protein and alpha-MSH exhibit similar but not identical sequences. Their divergence past residue 5 precludes a physiological significance of the crossreactivity which seems to have arisen fortuitously. Our results question previous speculations as to the existence of extrapituitary alpha-MSH-like hormones.
Acetylation↗
The human T-cell leukemia virus family, adult T cell leukemia, and AIDS.
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Acquired Immunodeficiency Syndrome↗
HTLV-III env gene products synthesized in E. coli are recognized by antibodies present in the sera of AIDS patients.
The envelope gene of HTLV-III, the retrovirus directly linked to AIDS, encodes a protein of 856 amino acids. Our sequence analysis of the cloned HTLV-III (HXB-3) env gene and its comparison with other isolates reveal significant divergence, especially in the external portion of this protein. A large segment of the env gene (1800 bp) was inserted into the expression vector pEV-vrf3, and a corresponding 68 kd protein, which encompasses both the extracellular and the membrane-associated regions of the native protein, was produced in E. coli. Several smaller polypeptides, which appear to be internal initiation products, were also produced. All 50 AIDS patient sera obtained from different locations in the United States specifically recognized the bacterially synthesized envelope proteins, as judged by Western blots. This suggests that these proteins will be useful for the diagnosis of HTLV-III infection and possibly as a vaccine against AIDS.
Acquired Immunodeficiency Syndrome↗
Cytokinin metabolism in phaseolus embryos : genetic difference and the occurrence of novel zeatin metabolites.
The metabolism of trans-[8-(14)C]zeatin was examined in embryos of Phaseolus vulgaris cv Great Northern (GN) and P. lunatus cv Kingston (K) in an attempt to detect genetic variations in organized plant tissues. Metabolites were fractionated by HPLC, and identified by chemical and enzymic tests and GC-MS analyses. Five major metabolites were recovered from P. vulgaris embryo extracts: ribosylzeatin, ribosylzeatin 5'-monophosphate, an O-glucoside of ribosylzeatin, and two novel metabolites, designated as I and II. Based on results of degradation tests and GC-MS analyses, I and II were tentatively identified as O-ribosyl derivatives of zeatin and ribosylzeatin. In embryos of P. lunatus, however, metabolites I and II were not present. The major metabolites were ribosylzeatin, ribosylzeatin 5'-monophosphate, and the O-glucosyl derivatives of zeatin and ribosylzeatin. The zeatin metabolites recovered were the same for embryos of different sizes but their quantities varied with embryo size and incubation time. The genetic differences appear to be embryo-specific and may be useful in the studies of the possible relationship between abnormal interspecific hybrid embryo growth and hormonal derangement in Phaseolus. In addition, analyses of both organized (intact) and unorganized (callus) tissues of the same genotype may provide an opportunity to address the problem of differential expression of genes regulating cytokinin metabolism during plant development.
Journal Article↗
Effect of epidermal growth factor on reproductive function of ewes.
Adult Merino ewes were infused via the jugular vein with either saline (n = 5) or epidermal growth factor (EGF) (4.2 micrograms/kg per h, n = 6) for 24 h in either the luteal phase or the follicular phase of the oestrous cycle and reproductive function was examined. Infusion of EGF during the luteal phase caused no detectable change in plasma progesterone or prolactin concentrations over a 7-day period compared with the controls. Infusion of EGF during the follicular phase suppressed the oestrous rise in plasma oestradiol. Luteinizing hormone pulse amplitude was increased and pulse frequency was decreased by the end of the infusion. All control ewes had a pro-oestrous LH surge and mated, but the LH surge and oestrus were prevented by EGF infusion. Nevertheless, plasma progesterone levels rose subsequently in the EGF-infused ewes in parallel with the control ewes, suggesting that the preovulatory follicle had luteinized. Both LH and FSH rose over the 7 days after EGF infusion to levels similar to those in ovariectomized ewes. Thus EGF appears to inhibit follicular oestradiol production, although it does not affect luteal progesterone production or follicular luteinization. We suggest that the alteration in gonadotrophin secretion patterns results from a disturbance of feedback mechanisms between the ovary and the hypothalamopituitary axis, although a direct effect in the brain or the pituitary gland cannot yet be excluded.
Animals↗
Molecular biology of human T-lymphotropic retroviruses.
The generic name for a family of human T-lymphotropic retroviruses is HTLV. Two of the three members in this family have been linked etiologically to human diseases: HTLV-I with adult T-cell leukemia and HTLV-III with the acquired immunodeficiency syndrome. In addition to their T-cell tropism and a number of other common biological and biochemical properties, the most unique common features of these viruses from a molecular biological point of view are the presence of the x-lor gene towards the 3' end of the genome and the phenomenon of a virus-induced trans-acting factor in activation of transcription initiated in the viral long terminal repeat. These features may not only be key in understanding the mechanism of transformation or cell killing by these viruses, but they also provide a basis for new classification of retroviruses. In spite of these similarities among HTLV-I, -II, -III, and bovine leukemia virus, the genome of HTLV-III is only distantly related to these other viruses. Instead, it shows greater homology to members of the Lentivirus family. Therefore, all these viruses may have a common progenitor. Two other salient features arose from the analyses of HTLV-III and acquired immunodeficiency syndrome. (a) HTLV-III frequently infects the brain of acquired immunodeficiency syndrome patients who suffer from central nervous system disorders. This not only identifies HTLV-III as the direct candidate in these central nervous system disorders but also poses the problem of crossing the blood-brain barrier in therapy strategies to eradicate the virus. (b) Different HTLV-III isolates comprise a spectrum of related viruses, with the degree of divergence varying from virtual identity to 10-15% difference. The most divergent region resides in the envelope gene. Whether this finding has implications in the development of an effective vaccine for acquired immunodeficiency syndrome remains to be determined.
Acquired Immunodeficiency Syndrome↗
Cytoskeletal proteins at the cholinergic synapse: distribution of desmin, actin, fodrin, neurofilaments, and tubulin in Torpedo electric organ.
Treatment of the electric organ of Torpedo marmorata with Triton X-100 in the presence of 2 mM MgCl2 generated a cytoskeletal fraction in which a 54 kDa polypeptide is a major constituent. This 54 kDa polypeptide accounted for about 8% of the cellular protein when total electric organ tissue was analyzed by two-dimensional gel electrophoresis. Immunoblotting experiments showed that this protein reacts with monoclonal antibodies to desmin, the major intermediate filament protein of avian and mammalian muscle tissue. Negative stain analysis revealed that filaments of about 10 nm diameter are the major structural elements of the electric organ cytoskeleton. In the presence of Ca2+ there was a rapid degradation of the desmin-like protein and intermediate filaments due to a Ca2+-activated protease. Some of the resulting fragments retained antigenic activity against the desmin antibodies. Immunoblotting of membrane fractions enriched in acetylcholine receptor revealed desmin in addition to some actin. A further cytoskeletal component was identified from biochemical and immunological properties as a homologue of the mammalian neurofilament L-polypeptide. Thus Torpedo expresses proteins homologous to the mammalian desmin and neurofilament L-protein which can be detected using immunological approaches. Immunofluorescence microscopy was used to map the location of various cytoskeletal proteins of the cholinergic synapse on paraffin sections and on en face preparations of membranes. Desmin staining was restricted to electrocytes and in en face preparations was seen associated with both the ventral receptor-containing membrane and with the non-innervated dorsal membrane. Antibodies to neurofilament L-protein stained only the axons and not the electrocytes. Staining for fodrin, a non-erythrocyte spectrin, resulted in submembraneous decoration of both the axons and the electrocytes. Axonal staining for neurofilaments and microtubules did not extend into the ends of the nerve terminal arborizations.
Actins↗
An immunofluorescence study of neurofilament protein expression by developing hippocampal neurons in tissue culture.
We have studied the development of intermediate filament proteins in the neurons found in hippocampal cell cultures using single and double label immunofluorescence with both monoclonal and polyclonal antibodies. Neurons in these cultures are known to differentiate in a manner similar to their counterparts in situ: in particular they develop axonal and dendritic processes which differ from each other in form, in ultrastructure, and in synaptic polarity. During the first days in culture, developing neurons could not be stained with antibodies against any of the neurofilament proteins, although many cells reacted with anti-vimentin. Later in the first week, antibody staining revealed clearly filamentous staining for the L (68 000 daltons) and the M (145 000 daltons) neurofilament subunits, though M reactivity was much stronger at this earlier stage of development. Some neurofilament positive profiles in many cells could also be stained with vimentin, though the vimentin immunoreactivity became progressively less pronounced during further development, and disappeared after about two weeks in culture. Also at about two weeks in vitro we noted the first appearance of neurofilament H protein (200 000 daltons) immunoreactivity, which was localized to a subset of long neurites which could be identified on morphological grounds as axons. These processes lacked staining for microtubule associated protein 2 (MAP2), a dendritic marker. They tended to be close to islands of glial cells, suggesting that H induction may require complex neuron-glial interactions. These results are consistent with the suggestion that H protein immunoreactivity is a marker for axonal outgrowth. In addition to obvious filamentous staining, we were able to localize neurofilament antigens to an interesting class of small ring-like structures, found increasingly frequently as the cultures aged. We also present evidence that tyrosinated alpha-tubulin is present both within dendrites and axons of neurons in these cultures.
Animals↗
Intermediate filaments, microtubules and microfilaments in epidermis of sea urchin tube foot.
Tube foot epidermal cells of the sea urchin Strongylocentrotus purpuratus were examined by transmission electron microscopy and fluorescence microscopy to identify the chemical nature of prominent bundles of cytoplasmic filaments. Cross sections revealed filaments of roughly 7-8 nm in diameter closely packed into dense bundles. These bundles, in turn, were each surrounded by a loose sheath of microtubules. The filament size and negative reaction with the fluorescent F-actin binding drug NBD-phallacidin indicated that they were not actin. Indirect immunofluorescence microscopy of whole tissues and frozen sections revealed a strong reaction of the filaments with a monoclonal antibody prepared against porcine stomach desmin. In SDS-polyacrylamide gels of whole tube foot protein, a band of apparent molecular weight around 50 000 daltons reacted with the anti-desmin monoclonal antibody. The combined data provide evidence that the epidermal filament bundles are related to vertebrate intermediate filaments, but further biochemical studies will be necessary to assign them to a particular class of filament proteins.
Actins↗
Association of childhood leukemia with maternal age, birth order, and paternal occupation. A case-control study.
Previous investigations have drawn conflicting conclusions concerning the association between childhood leukemia and maternal age, birth order, socioeconomic status, and paternal occupation. In this study, 255 childhood leukemia cases diagnosed in California between 1975 and 1980 were individually matched with two living controls on the basis of sex, date of birth, and county of birth. Data were derived from subjects' birth certificates. Socioeconomic status and paternal benzene exposure were determined from the paternal occupation stated on the birth certificate. Hispanics and males were overrepresented in the case group. Cases were also less likely to be of birth order one. No difference was found between case and control groups for maternal age or paternal occupation. Analyses with stratification on age, sex, and leukemia cell type are reported.
Adolescent↗
Concentrations of oestradiol-17 beta in plasma and corpora lutea throughout pregnancy in the tammar, Macropus eugenii.
Oestradiol-17 beta concentrations were measured by radioimmunoassay in peripheral blood samples from 10 tammar wallabies after their pouch young were removed to terminate embryonic diapause. Oestradiol concentrations rose from 8.3 +/- 1.2 pg/ml on Days 3 and 4 to peak of 15.8 +/- 2.9 pg/ml on Day 5, coincident with an increase in 'progesterone' concentrations, and then fell to 10.5 +/- 2.7 pg/ml on Day 7. No changes in oestradiol concentrations were associated with parturition. Five females came into oestrus and mated 9.8 +/- 6.1 h post partum; peak concentrations of plasma oestradiol (20.9 +/- 2.1 pg/ml) occurred around the time of mating. None of the females that did not mate up to the end of the experiment at Day 30 had a rise in plasma oestradiol concentrations. Corpora lutea contained 20-100 pg oestradiol during pregnancy. The highest ovarian oestradiol content (greater than 1200 pg) was measured in whole ovaries containing Graafian follicles from full-term pregnant females. The rise in oestradiol concentrations at Day 5 may be important in the termination of diapause. The post-partum increase in plasma oestradiol concentrations coincides with oestrus. The source of this oestrogen appears to be the preovulatory follicle.
Animals↗