Biomedical subjects
G Bennett
Publications and source records attributed to G Bennett.
Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin.
We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable enterotoxin receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously expresses STa binding sites. Scatchard analysis of displacement binding studies revealed a single STa binding site with an affinity (Ki) of 97 pM in 293-STaR compared with 55 pM in T84 cells. Saturation isotherms of STa binding gave a Kd of 94 pM for the cloned receptor expressed in 293 cells and 166 pM for the receptor present in T84 cells. Kinetic measurements of STa binding to 293-STaR gave an association rate constant, K1, of 2.4 x 10(8) M-1 min-1 and a dissociation rate constant, K2, of 0.016 min-1. The half-time of dissociation was 43 min, and the Kd calculated from the ratio of the kinetic constants was 67 pM. The pH profile of STa binding showed that the number of STa binding sites is increased 3-fold at pH 4.0 compared with pH 7.0, with no effect on binding affinity. A polyclonal antibody directed against the extracellular domain of STaR immunoprecipitated two proteins of approximately 140 and 160 kDa from both 293-STaR and T84 cells. Cross-linking of 125I-STa to 293-STaR cells resulted in the labeling of proteins with a molecular mass of approximately 153, 133, 81, 68, 56, and 49 kDa, the two smallest being the more abundant. Similar results have been reported for the STaR present on rat brush border membranes. These data suggest that the STaR-guanylyl cyclase identified by molecular cloning is the only receptor for STa present in T84 cells.
Björk-Shiley valves.
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National working conference on smoking and body weight. Task Force 3: Implications with respect to intervention and prevention.
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Medical-cause accidents in commercial aviation.
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Rectal aminophylline gel in treatment of apnoea in premature newborn babies.
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Traffic through the Golgi apparatus as studied by radioautography.
The ability to radiolabel biological molecules, in conjunction with radioautographic or cell fractionation techniques, has brought about a revolution in our knowledge of dynamic cellular processes. This has been particularly true since the 1940's, when isotopes such as 35S and 14C became available, since these isotopes could be incorporated into a great variety of biologically important compounds. The first dynamic evidence for Golgi apparatus involvement in biosynthesis came from light microscope radioautographic studies by Jennings and Florey in the 1950's, in which label was localized to the supranuclear Golgi region of goblet cells soon after injection of 35S-sulfate. When the low energy isotope tritium became available, and when radioautography could be extended to the electron microscope level, a great improvement in spatial resolution was achieved. Studies using 3H-amino acids revealed that proteins were synthesized in the rough endoplasmic reticulum, migrated to the Golgi apparatus, and thence to secretion granules, lysosomes, or the plasma membrane. The work of Neutra and Leblond in the 1960's using 3H-glucose provided dramatic evidence that the Golgi apparatus was involved in glycosylation. Work with 3H-mannose (a core sugar in N-linked side chains), showed that this sugar was incorporated into glycoproteins in the rough endoplasmic reticulum, providing the first radioautographic evidence that glycosylation of proteins did not occur solely in the Golgi apparatus. Studies with the tritiated precursors of fucose, galactose, and sialic acid, on the other hand, showed that these terminal sugars are mainly added in the Golgi apparatus. With its limited spatial resolution, radioautography cannot discriminate between label in adjacent Golgi saccules. Nonetheless, in some cell types, radioautographic evidence (along with cytochemical and cell fractionation data) has indicated that the Golgi is subcompartmentalized in terms of glycosylation, with galactose and sialic acid being added to glycoproteins only within the trans-Golgi compartment. In the last ten years, radioautographic tracing of radioiodinated plasma membrane molecules has indicated a substantial recycling of such molecules to the Golgi apparatus.
Psychological change during residence in a rehabilitation centre for female drug misusers. Part I. Drug misusers.
Fifty consecutive entrants to a year-long Christian residential rehabilitation programme for female drug misusers received a comprehensive psychological assessment on intake, 3 and 6 months later, and at completion of the programme. Significant changes were seen in emotional state, self-esteem, personality and attitudes to Christianity. Most of the changes occurred during the first 3 months, but changes in personality continued throughout the period of residence. The 11 women who completed the programme differed significantly from the others on their characteristics at intake; the 26 who dropped out during the first 3 months differed significantly from those who dropped out after that time. In both cases, the differences were at a clinically significant level. The importance of matching drug users to rehabilitation centres is discussed.
Psychological change during residence in a rehabilitation centre for female drug misusers. Part II. Children of drug misusers.
This study examined psychological changes in children of female drug misusers who were living with their mothers in a residential rehabilitation centre. The results suggest that the childrens' behaviour and cognitive skills were not adversely affected, and that there were many benefits for drug addicted mothers retaining custody of their children whilst undergoing a rehabilitation programme. The implications of the possible detrimental effects of a mother failing to complete her rehabilitation programme on her children are discussed.
Molecular nature of in vivo mutations in human cells at the autosomal HLA-A locus.
The mutations present in vivo in normal human cells were studied at the HLA-A locus by isolating mutant lymphocytes using antibody-complement immunoselection and cloning at limiting dilution. The molecular basis for mutation in 127 mutant lymphocytes from 10 individuals was determined by studying a variety of polymorphic gene loci on both arms of chromosome 6. No change was detected in 78 mutants (61.4%), gene deletion was detected in 11 (8.7%), and mitotic recombination was detected in 38 (29.9%). Neither gene conversion nor chromosome loss was detected. These observations document the mechanisms responsible for gene loss in normal human cells in vivo, emphasize the importance of mitotic recombination, and indicate the similarity between mutational mechanisms in normal cells and in cancer cells.
Developing an integrated quality improvement program.
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Versatile serum-free media formulations.
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Sexual practices and "beats": AIDS-related sexual practices in a sample of homosexual and bisexual men in the western area of Sydney.
One hundred and seventy-six men who had engaged in sexual activity with other men in the past six months were interviewed, in the western area of Sydney, about their sexual activities and knowledge as related to the acquired immunodeficiency syndrome (AIDS). Comparisons were made between men who had frequented "beats" (public toilets, parks or isolated roads where men meet for homosexual encounters) in the past six months, and those who did not use beats. One-quarter of those men who frequented beats reported beats as their only venue for meeting men for sexual contact, while other men reported them as one of many venues that they used to meet men. Although all interviewed men were engaging in sexual activities with men, 12 men described themselves as heterosexual, and 12% of those men who used beats, and 7% of the beat non-users, were married or in de-facto relationships. In comparing those who used beats with the beat non-users, 64% of beat users had engaged in sexual practices with between six and 50 different partners in the past six months compared with 8% of beat non-users. Beat users also were more likely to engage in unprotected anal intercourse with causal partners. Nineteen men claimed to be HIV-seropositive for the human immunodeficiency virus, with 11 of these men still engaging in high-risk sexual activities. The implications for AIDS education are discussed.
A potential source for the transmission of the human immunodeficiency virus into the heterosexual population: bisexual men who frequent "beats".
Fifty-four actively bisexual men with recent sexual experiences with both men and women were interviewed as part of a wider study of 176 homosexual men, many of whom frequented "beats" (public toilets, parks and isolated roads where men meet for homosexual encounters). Forty-six per cent of the bisexual men were engaging in unsafe sexual practices with at least one man and one woman. Three of these men knew that they were seropositive for the human immunodeficiency virus, with two of these men engaging in unsafe sexual practices with some of their partners. Fewer than one-quarter of the bisexual men identified with or participated actively in aspects of organized, overt "gay" culture. So-called "closeted" bisexual men are suggested to be a major, but neglected, target for educational efforts against the acquired immunodeficiency syndrome.
Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta.
Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.
Morphological and functional characteristics of myocytes isolated from human left ventricular aneurysms.
Isolated single myocytes were prepared from myocardium of developing ventricular aneurysms and from myocardium within the scar of chronic ventricular aneurysms. The morphology and function of the individual cells were compared. The cells from developing aneurysms were rod-shaped, with a distinct sarcomeric structure, but did not contract even in the presence of high calcium concentrations. The sarcomere length was significantly higher than that of cells from chronic aneurysms and approached the theoretical point at which no contraction can occur. Cells from chronic aneurysms were either rod-shaped and contractile, or rounded due to hypercontracture of the myofilaments. Electron microscopy of cells from developing aneurysms confirmed the presence of elongated sarcomeres, a loss of the actin-myosin interdigitation, and damage to the contractile proteins which was particularly evident in the thin filaments. Cells with similar characteristics have also been isolated from a ruptured, ischaemic papillary muscle. These changes, which are due either to ischaemia or to overstretching of cells, may account for the weakness of the wall of developing aneurysms and be a cause of rupture or enlargement.
Ultrastructural localization of CMPase, TPPase, and NADPase activity in neurons, satellite cells, and Schwann cells in frog dorsal root ganglia.
Sections of bullfrog dorsal root ganglia were analyzed for cytidine monophosphatase (CMPase), thiamine pyrophosphatase (TPPase), and nicotinamide adenine dinucleotide phosphatase (NADPase) activity, and the distributions of these enzymatic activities were compared with those traditionally found in other cell types (e.g., CMPase: Golgi trans-sacculotubular network; TPPase: trans-Golgi saccule(s); NADPase: intermediate Golgi saccules). In the present study, CMPase activity in neurons was localized mainly to the Golgi trans-sacculotubular network and lysosomes, but sometimes also occurred at the ends of the trans and most distal intermediate Golgi saccules. A similar distribution was found in satellite and Schwann cells. TPPase activity in neurons occurred not only in the trans-Golgi saccule but also in the trans-sacculotubular network, lysosomes, and scattered tubular elements. In satellite and Schwann cells, activity was found in both the trans saccule and trans-sacculotubular network, and substantial activity often appeared in the more distal of the intermediate saccules. NADPase activity in neurons was usually absent from the intermediate Golgi saccules and was confined to the trans-sacculotubular network and lysosomes; however, activity was sometimes also found in the intermediate and/or trans-Golgi saccules. In satellite and Schwann cells, activity appeared consistently in both the trans-sacculotubular network and intermediate saccules, as well as in lysosomes. These distributions, especially in the case of TPPase and NADPase, differ substantially from the most frequently reported localizations of the above enzymes, indicating that the Golgi complex may exhibit considerable plasticity of structure and function in different cell types.
An enzyme-linked immunosorbent assay to study human relaxin in human pregnancy and in pregnant rhesus monkeys.
A sensitive and specific double-antibody enzyme-linked immunoassay, using a synthetic analogue of human relaxin for standard and immunogen, was developed for the measurement of human relaxin (hRLX) in serum and plasma. No cross-reactivity was observed for human insulin, human insulin-like growth factor-I, hGH, human chorionic gonadotropin, hFSH, hLH or human prolactin. The assay was used to monitor RLX concentrations in samples from men, non-pregnant and pregnant women, and in pregnant rhesus monkeys infused with hRLX. RLX was not detected in serum from men nor from non-pregnant women, while a concentration of 600 ng/l was measured in pooled sera from two pregnant women (pregnancies achieved by in-vitro fertilization). Immunoreactive RLX (1.1 micrograms/g) was found in human corpora lutea taken from ectopic pregnancies at 7 weeks. In an experiment with a pregnant rhesus monkey infused with human RLX analogue, less than 1.5% of the maternal concentration was measured in the fetal circulation. Even though preliminary, these data suggest a low level of transfer of human analogue relaxin across the placenta in a rhesus monkey. Further studies of the physiology of RLX in human pregnancy will be facilitated by the availability of this immunoassay.