Biomedical subjects

G L Bennett

Publications and source records attributed to G L Bennett.

At least 19 recordsLinked to original sources

PubMed1992

Characterization of two high affinity human interleukin-8 receptors.

Interleukin 8 (IL-8) and melanocyte growth-stimulatory activity/gro (MGSA) are structurally related proinflammatory cytokines that are chemoattractants and activators of neutrophils. Recently, cDNA clones encoding a high affinity IL-8 receptor (IL-8R-A) and a "low affinity" IL-8 receptor (IL-8R-B) have been isolated from human cDNA libraries. These two receptors have 77% amino acid identity and are members of the G protein-coupled superfamily of receptors with seven transmembrane domains. We have expressed these two receptors in mammalian cells and find that in this system both receptors bind IL-8 with high affinity (Kd approximately 2 nM). The receptor affinities differ for MGSA, however. IL-8R-A binds MGSA with low affinity (Kd approximately 450 nM); IL-8R-B binds MGSA with high affinity (Kd approximately 2 nM). The transfected cells respond to ligand binding with a transient increase in the intracellular Ca2+ concentration. A Ca2+ response is found for IL-8R-A following the binding of IL-8; no response is found for MGSA. A Ca2+ response for IL-8R-B follows the binding of both ligands. Blot hybridization with oligonucleotide probes specific for the two receptors shows that mRNA for both receptors is present in human neutrophils. Analysis of IL-8 and MGSA binding data on neutrophils as well as Ca2+ response and desensitization data shows that the presence of these two IL-8 receptors on the cell surface can account for the profile of these two ligands on neutrophils.

Amino Acid Sequence↗

PubMed1992

Structure-function analysis of hepatocyte growth factor: identification of variants that lack mitogenic activity yet retain high affinity receptor binding.

Hepatocyte growth factor (HGF) is a potent mitogen for parenchymal liver, epithelial and endothelial cells. Structurally, it has similarities to kringle-containing serine proteases, although it does not possess proteolytic activity. A structure-activity relationship study of human HGF was performed by functional analysis of HGF substitution and deletion variants. Analysis of HGF variants was accomplished by defining their ability to induce DNA synthesis on hepatocytes in primary culture and to compete with wild-type HGF for binding to a soluble form of the HGF receptor. Three groups of variants were made: (i) substitutions at the cleavage site, (ii) substitutions within the protease-like domain and (iii) deletions of the beta-chain and/or kringle domains. Our results show that: (i) single-chain HGF is a zymogen-like promitogen in that cleavage into a two-chain form is required for biological activity, however, the single chain form of HGF still retains substantial receptor binding capacity; (ii) certain mutations in the protease-like domain result in variants that are completely defective for mitogenic activity, yet exhibit apparent receptor binding affinities similar to wild-type HGF (Kd approximately 50-70 pM); and (iii) a variant containing the N-terminal 272 residues of mature HGF showed only a 4-fold increase in Kd when compared with wild-type HGF indicating that a primary receptor binding determinant is located within this sequence.

Animals↗

PubMed1992

Recovery of cholinergic phenotype in the injured rat neostriatum: roles for endogenous and exogenous nerve growth factor.

Polyclonal antibodies against recombinant human nerve growth factor (rhNGF) potently inhibited PC12 neurite outgrowth, blocked high-affinity 125I-rhNGF binding but not its receptor, and cross-reacted with rat, mouse, and human nerve growth factor (NGF) but not with brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor, insulin-like growth factor, epidermal growth factor, or activin A. Immunocytochemistry revealed many NGF-positive neurons in the rat neostriatum. The NGF-positive neurons disappeared by 3 days after mechanical injury to the neostriatum and were replaced by intensely NGF- and glial fibrillary acidic protein-positive astrocytes. Enzyme-linked immunosorbent assay measurements revealed that the NGF content of the injured striatum was elevated by eightfold 3 days postinjury and by twofold 2 weeks later. The high-affinity choline uptake (HACU) into cholinergic nerve terminals was decreased by 23% at 2 and 4 weeks postinjury, yet choline acetyltransferase (ChAT) activity in these neurons was unchanged at 2 weeks and decreased by 14% at 4 weeks. Daily infusion of 1 microgram of rhNGF into the injury area did not alter the loss of HACU. However, this treatment elevated ChAT activity by 23-29% above intact neostriatal levels and by 53-65% relative to HACU at both survival times. Thus, lesion-induced increases in NGF levels within astrocytes are associated with maintenance of striatal ChAT activity at normal levels following cholinergic injury, even with decreases in HACU. Pharmacologic doses of rhNGF can further augment ChAT activity in damaged cholinergic neurons, showing the usefulness of exogenous NGF even when endogenous NGF is elevated in response to injury.

Animals↗

PubMed1992

Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues.

Vascular endothelial growth factor (VEGF) is a secreted heparin-binding mitogen; its growth-promoting activity is limited to vascular endothelial cells in vitro and VEGF also stimulates angiogenesis in vivo. To identify target cells for VEGF and investigate the potential physiological role of this factor, iodinated recombinant human VEGF (125I-rhVEGF) was used for in vitro ligand autoradiography on tissue sections from adult rats. 125I-rhVEGF exhibited saturable, displaceable binding to a single class of sites with high affinity and low capacity in all tissues and organs examined. Colocalization of 125I-rhVEGF binding with Factor VIII-like immunoreactivity demonstrated binding sites associated with vascular endothelial cells of both fenestrated and nonfenestrated microvessels and the endothelium of large vessels, while no displaceable binding was evident on nonendothelial cells. Specific binding was associated with quiescent as well as proliferating vessels. These findings support the hypothesis that VEGF plays a specific role in both the maintenance and in the induction of growth of vascular endothelial cells.

Animals↗

PubMed1992

Thyroid follicular cells produce interleukin-8.

Interleukin-8 (IL-8) is a potent proinflammatory cytokine known to be produced by several cell types. To elucidate whether endocrine cells can also make IL-8, we have tested supernatants from eleven thyroid follicular cell primary cultures. IL-8 was readily detected under basal conditions (range 3.4-32.1 ng/ml from 1 x 10(5) cells in 3 days) and was increased 4-20 fold by stimulation with IL-1. TSH and tumor necrosis factor had an inconsistent effect, while gamma-interferon reduced basal and IL-1-stimulated IL-8 production. Since IL-8 can act as a chemoattractant for lymphocytes, these observations may explain in part the accumulation of lymphocytes within the gland in autoimmune thyroiditis.

Cells, Cultured↗

PubMed1992

Predicting lean growth while accounting for correlated traits.

Lean tissue growth rate is usually estimated from indirect measurements including growth rate. A procedure to determine prediction equations for lean tissue growth rate is proposed. The procedure restricts the regression of fat growth rate on predicted lean growth rate to be equal to the regression of fat growth rate on actual lean growth rate. The restriction can be phenotypic or genetic if suitable parameter estimates are available. When applied phenotypically, selection on predicted lean tissue growth rate will result in selection differentials for both fat and lean tissue growth rates that are proportional to those obtained by direct selection for lean tissue growth rate. This restriction is desirable because expected correlated changes in fat are used to justify selection for lean tissue growth. Conventional prediction procedures have ignored correlated changes and obscured the original intent of using lean tissue growth rate as a biological selection criterion. When using conventional procedures to predict a biological selection criterion from indirect measurements, changes in important correlated traits may depend more on the choice of indirect measurements than on the choice of selection criterion.

Adipose Tissue↗

PubMed1992

A computer model to predict the effects of level of nutrition on composition of empty body gain in beef cattle: I. Theory and development.

A computer model was developed to simulate differences in the composition of empty body gain of beef cattle resulting from differences in postweaning level of nutrition that are not associated with empty BW. Differences in rate of empty body gain of cattle of similar genotype reflect differences in the level of nutrition; hence, empty body gain was used to represent level of nutrition. The model was based on four assumptions: 1) as animals mature, the proportion of fat is greater in gain than in body weight, 2) effects on body composition caused by plane of nutrition that are not associated with empty BW are predictable from rate of empty BW gain, 3) effects resulting from changes in nutrition are not exerted immediately nor are they permanent, and 4) cattle approach an empty body composition equilibrium when empty body gain is zero. Six parameters were used to characterize mature fat-free matter, rate of change to body composition equilibrium, minimum and maximum fractional growth rate relative to fractional growth rate for empty BW, time lag of response to change in nutrition, and influence of stage of maturity and nutrition on rate of change for fat-free matter. Two parameters were specific for genotype and gender. Using results from the model, we were able to explain conflicting results obtained from compensatory gain experiments.

Adipose Tissue↗

PubMed1992

A computer model to predict the effects of level of nutrition on composition of empty body gain in beef cattle: II. Evaluation of the model.

A computer model developed to predict composition of empty body gain of beef cattle fed at different levels of nutrition was evaluated with data from one unpublished and seven published experiments. These experiments used several breeds of beef cattle growing at rates that varied from negative to fast and various combinations of these growth rates. There was close agreement between observed and simulated absolute treatment means for fatness, except when animals were fed diets low in protein. In this case, experimental animals fed inadequate protein grew at slower rates and became fatter than contemporaries fed adequate protein, whereas the model predicted the opposite. Ability of the model to predict effects of nutrition not associated with changes in BW was evaluated using the proportion of residual variation in experimental fat percentage accounted for by the model, after fitting linear and quadratic terms for empty BW. The model accounted for 13.8 to 56.2% of the residual variation in observed fatness in four experiments in which significant differences in fatness were observed among nutritional treatments after accounting for differences in empty BW. Regression of observed fatness on model-simulated fatness resulted in regression coefficients that were positive and close to 1 in these four experiments. This suggests that the model can accurately predict some of the effects of nutrition on fatness that are not associated with changes in empty BW.

Adipose Tissue↗

PubMed1992

Brain tumours and lymphomas in transgenic mice that carry HTLV-I LTR/c-myc and Ig/tax genes.

The human T-cell leukemia virus type 1 (HTLV-I) is associated with adult CD4+ T-cell leukemia (ATL) and tropical spastic paraparesis (TSP). In as much as only a small percentage of individuals infected with HTLV-I develop either disease, we set out to model a genetic partner for this virus in an effort to understand and possibly reproduce its pathophysiology. To this end we have developed a binary set of transgenic mice, one bearing the relatively inactive HTLV-I long terminal repeat (LTR) positioned to drive the c-myc oncogene and another bearing a fusion transgene consisting of the immunoglobulin promoter/enhancer driving the gene for the HTLV-I transcription activator, tax. Alone, the tax construct, though expressed in the thymus, spleen, lung and brain, has no deleterious effect. Alone, the HTLV-I LTR/c-myc construct is expressed at very low levels in lymphoid cells and occasionally induces lymphomas in older animals. When these two transgenic lines are mated, bigenic offspring harboring both transgenes exhibit dramatic tumor formation. As in the human, these animals develop CD4+ T-cell lymphomas, but they also develop central nervous system tumors by 25-90 days of age. The syndrome, which is 100% penetrant and lethal, provides an animal model for adult T-cell lymphoma and a source of cultured cells of neurogenic origin.

Animals↗

PubMed1991

Extracellular domain-IgG fusion proteins for three human natriuretic peptide receptors. Hormone pharmacology and application to solid phase screening of synthetic peptide antisera.

The natriuretic peptide receptors (NPRs) are a family of three cell surface glycoproteins, each with a single transmembrane domain. Two of these receptors, designated NPR-A and NPR-B, are membrane guanylyl cyclases that synthesize cGMP in response to hormone stimulation. The third receptor, NPR-C, has been reported to function in the metabolic clearance of ligand and in guanylyl cyclase-independent signal transduction. We engineered three chimeric proteins consisting of the natriuretic peptide receptor extracellular domains fused to the Fc portion of human IgG-gamma 1. These molecules provide material for detailed studies of the human receptor's extracellular domain structure and interaction with the three human natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and type-C natriuretic peptide (CNP). The homodimeric fusion proteins, designated A-IgG, B-IgG, and C-IgG, were secreted from Chinese hamster ovary cells and purified by protein-A affinity chromatography. We present here the primary characterization of these fusion proteins as represented by the intrinsic hormone affinities measured by saturation binding and competition assays. The dissociation constant of 125I-ANP for A-IgG was 1.6 pM and for C-IgG, 1.2 pM. The dissociation constant of 125I-Y0-CNP (CNP with addition of tyrosine at the amino terminus) for B-IgG was 23 pM. The rank order of potency in competitive binding for A-IgG was ANP greater than BNP much greater than CNP, whereas for B-IgG the ranking was CNP much greater than ANP greater than BNP. For C-IgG, we observed ANP greater than CNP greater than or equal to BNP. These data demonstrate that the receptor-IgG fusion proteins discriminate among the natriuretic peptides in the same manner as the native receptors and provide a basis for future structural studies with these molecules. The purified fusion proteins have a variety of potential applications, one of which we illustrate by a solid phase screening assay in which rabbit sera from a series of synthetic-peptide immunizations were titered for receptor reactivity and selectivity.

Amino Acid Sequence↗

PubMed1991

Increase in glia-derived nerve growth factor following destruction of hippocampal neurons.

It is currently believed that under normal conditions hippocampal neurons synthesize nerve growth factor (NGF) which may provide trophic support for cholinergic neurons projecting from the basal forebrain. The concept that glial cells are mobilized to increase the production of NGF following destruction of hippocampal neurons was examined. Excitotoxin-induced destruction of the dorsal hippocampal neurons resulted in a massive and prolonged increase in NGF-like immunoreactivity (LI). Immunostaining for NGF-LI and the glial marker, glial fibrillary acidic protein (GFAP), revealed that the source of increased NGF-LI production following the lesion were reactive astrocytes. Thus, glial cells assume the role of providing trophic support following loss of target neurons.

Animals↗

PubMed1991

Increase in nerve growth factor-like immunoreactivity and decrease in choline acetyltransferase following contusive spinal cord injury.

We have previously described a graded spinal cord injury model in the rat. Mild contusive injury results in an initially severe functional deficit that is attenuated over time to reveal the mild chronic deficits that characterize this injury. In this study, we have shown that mild contusive injury also results in a significant decrease in choline acetyltransferase (ChAT) activity during the first week after injury. At 1 week ChAT activity is maximally reduced at the site of the contusion and is also significantly lowered throughout the spinal cord. ChAT activity then rebounds during the following 3 weeks, partially at the injury site where there is considerable loss of gray and white matter, and completely in rostral and caudal cord segments. The rebound in ChAT activity is temporally associated with the partial recovery of function. Further, the changes in ChAT activity after injury are mirrored by changes in nerve growth factor-like immunoreactivity (NGF-LI) as determined by a specific two-site ELISA. NGF-LI increases significantly after injury, reaching a maximum at 7 days after contusion and at the injury site. However, levels of NGF-LI are also significantly increased throughout the spinal cord. NGF-LI then decreases at 2 and 4 weeks as ChAT activity rebounds. Further experiments will be needed to examine the possibility of a role for NGF in promoting the recovery of function after spinal cord injury.

Animals↗

PubMed1991

Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP).

The natriuretic peptides are hormones that can stimulate natriuretic, diuretic, and vasorelaxant activity in vivo, presumably through the activation of two known cell surface receptor guanylyl cyclases (ANPR-A and ANPR-B). Although atrial natriuretic peptide (ANP) and, to a lesser extent, brain natriuretic peptide (BNP) are efficient activators of the ANPR-A guanylyl cyclase, neither hormone can significantly stimulate ANPR-B. A member of this hormone family, C-type natriuretic peptide (CNP), potently and selectively activated the human ANPR-B guanylyl cyclase. CNP does not increase guanosine 3',5'-monophosphate accumulation in cells expressing human ANPR-A. The affinity of CNP for ANPR-B is 50- or 500-fold higher than ANP or BNP, respectively. This ligand-receptor pair may be involved in the regulation of fluid homeostasis by the central nervous system.

Animals↗

PubMed1991

Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific.

Complementary DNA clones encoding two distinct tumor necrosis factor receptors were isolated from a mouse macrophage cDNA library. The cDNA for murine tumor necrosis factor receptor type 1 (mTNF-R1) predicts a mature polypeptide of 425 amino acids that is 64% identical to its human counterpart, whereas the cDNA of murine tumor necrosis factor receptor type 2 (mTNF-R2) predicts a mature protein of 452 amino acids that is 62% identical to human tumor necrosis factor receptor type 2. The two murine tumor necrosis factor receptors have limited sequence homology (approximately 20% identity) in their extracellular regions but no apparent similarity in their cytoplasmic portions. Northern (RNA) analysis indicates a single 2.6-kilobase (kb) transcript for mTNF-R1; a 3.6-kb and a more predominant 4.5-kb transcript are observed for mTNF-R2. A human cell line transfected with either mTNF-R1 or mTNF-R2 expression vectors specifically bound 125I-labeled recombinant murine tumor necrosis factor alpha (TNF-alpha). Although mTNF-R1 had a similar affinity for both recombinant murine TNF-alpha and human TNF-alpha, mTNF-R2 showed strong specificity for recombinant murine TNF-alpha. This result suggests that the various activities of human tumor necrosis factor alpha reported in mice or in murine cell lines are probably mediated by mTNF-R1.

Amino Acid Sequence↗

PubMed1991

Recombinant human nerve growth factor is biologically active and labels novel high-affinity binding sites in rat brain.

Iodinated recombinant human nerve growth factor (125I-rhNGF) stimulated neurite formation in PC12 cell cultures with a half-maximal potency of 35-49 pg/ml, compared with 39-52 pg/ml for rhNGF. In quantitative ligand autoradiography, the in vitro equilibrium binding of 125I-rhNGF to brain sections showed a 10-fold regional variation in density and was saturable, reversible, and specifically displaced by up to 74% with rhNGF or murine NGF (muNGF). At equilibrium, 125I-rhNGF bound to these sites with high affinity (Kd 52-85 pM) and low capacity (Bmax less than or equal to 13.2 fmol/mg of protein). Calculation of 125I-rhNGF binding affinity by kinetic methods gave average Kd values of 24 and 31 pM. Computer-generated maps revealed binding in brain regions not identified previously with 125I-muNGF, including hippocampus; dentate gyrus; amygdala; paraventricular thalamus; frontal, parietal, occipital, and cingulate cortices; nucleus accumbens; olfactory tubercle; subiculum; pineal gland; and medial geniculate nucleus. NGF binding sites were distributed in a 2-fold increasing medial-lateral gradient in the caudate-putamen and a 2-fold lateral-medial gradient in the nucleus accumbens. 125I-rhNGF binding sites were also found in most areas labeled by 125I-muNGF, including the interpedunucular nucleus, cerebellum, forebrain cholinergic nuclei, caudoventral caudate-putamen, and trigeminal nerve nucleus. 125I-rhNGF binding sites were absent from areas replete with low-affinity NGF binding sites, including circumventricular organs, myelinated fiber bundles, and choroid plexus. The present analysis provides an anatomical differentiation of high-affinity 125I-rhNGF binding sites and greatly expands the number of brain structures that may respond to endogenous NGF or exogenously administered rhNGF.

Animals↗

PubMed1991

Triple arthrodesis in adults.

Twenty-two adult patients who underwent a triple arthrodesis because of hindfoot pain and/or deformity were reviewed retrospectively with a minimum 3-year follow-up. All procedures were performed by the same surgeon using a two-incision technique and internal fixation. Objective results were good in eight (36%) patients, fair in 13 (59%), and poor in one (5%). There were two asymptomatic nonunions of the talonavicular joint, no infections, and no neuromas. Eleven patients had radiographic evidence of tibiotalar arthritis, and eleven patients had midfoot arthritic changes that had progressed after the operation. Patient satisfaction with the procedure was high, and 21 of 22 (95%) patients felt that they were improved overall and would have the surgery again if indicated. Although the procedure is technically demanding, patient satisfaction is high and complications can be kept to a minimum if proper surgical technique is utilized.

Adult↗

PubMed1991

Genetic and environmental effects on carcass characteristics of Southdown x Romney lambs: I. Growth rate, sex, and rearing effects.

Carcass data from more than 4,400 Southdown x Romney ewe and wether lambs collected over a 16-yr period were analyzed for the effects of sex, rearing status, and growth rate. Ewe lambs grew more slowly than wethers and had .78 kg less carcass weight at the same age. The carcass weight advantage for wethers was nearly all caused by heavier fat-free weight. Based on fat depths, the fat on ewe lambs was distributed in more anterior and ventral parts of the carcass relative to wether lambs. Lambs reared as twins had 1.73 kg less carcass weight and correspondingly reduced carcass measurements compared with lambs reared as singles. Sex and rearing status interacted for some traits. However, in no case was a significant sex difference reversed in single- and twin-related lambs. Growth rate effects were determined by regressing average change in carcass measurements on average carcass weight gain over a 5-wk period. When carcass weight remained constant over a 5-wk period, fat weight increased by .12 kg, fat-free weight and muscle measurements decreased, and bone lengths increased. For each kilogram of increase in 5-wk carcass weight gain, the marginal increase in fat weight was .41 kg and that of fat-free weight was .59 kg. At the average 5-wk carcass weight gain of 1.4 kg, fat and fat-free gains were As carcass weight gain increased above 1.4 kg, fat-free gain exceeded fat gain.

Adipose Tissue↗

PubMed1991

Genetic and environmental effects on carcass characteristics of Southdown x Romney lambs: II. Genetic and phenotypic variation.

Approximately 4,400 crossbred lambs from Southdown sires and Romney ewes were slaughtered at approximately 18, 23, and 28 wk of age over a 16-yr period. Live weights, carcass measurements, and chemical percentages were analyzed to estimate genetic and phenotypic parameters. Heritabilities of postweaning weights and gains were about .20. Heritabilities of fat and water percentages were about .35 adjusted for age. Heritability of kidney fat percentage was .53. Heritabilities of fat depth and muscle measurements ranged from .21 to .37. Crutch depth (h2 = .73) and cannon bone length (h2 = .74) were the most highly heritable carcass measurements. The genetic correlation between carcass fat and fat-free weight was .47 when lambs were slaughtered at a constant age. Fat-free weight was nearly uncorrelated with percentages of fat, water, and protein when lambs were slaughtered at the same age. Carcass measurements increased accuracy of selection for fat-free weight at a constant age very little compared with using only carcass weight. However, this does not mean that additional measurements are useless. The addition of carcass measurements to the selection criteria would result in correlated responses in chemical composition that more closely resembled direct selection for fat-free weight. Carcass weight would be of little value when used by itself to reduce fat weight adjusted for carcass weight. Direct measurement of carcass composition resulted in 1.6 to 2.6 times more predicted response for reduced fat weight than any combination of carcass weight and one fat depth measurement.

Adipose Tissue↗