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Biomedical subjects

L M King

Publications and source records attributed to L M King.

At least 37 records · Page 2Linked to original sources
PubMed1999

Turkey sperm mobility influences paternity in the context of competitive fertilization.

We have devised a novel means of investigating competitive fertilization in turkeys, using microsatellite genotyping to identify male parentage. Our results demonstrate that sperm mobility is a mechanism responsible in part for paternity efficiency in turkeys. Sperm mobility is composed of several parameters in which sperm motility is a component. Differences between ejaculates in the number of sperm penetrating into a dense, insert, nontoxic solution were measured and used to classify males into high, average, or low sperm mobility phenotypes. Microsatellite genotyping was used to determine parentage of poults after equal numbers of sperm from 10 males (either high or average phenotype, n = 5, mixed with low phenotype, n = 5) were inseminated simultaneously. In a separate study, the numbers of sperm hydrolyzing the perivitelline layer of eggs were compared between hens inseminated with sperm from high-, average-, or low-phenotype males. Overall, heterospermic inseminations resulted in consistently fewer offspring produced by low-mobility phenotype males. This correlated with physiological data in which semen from the low-mobility males had reduced numbers of sperm at the fertilization site as determined by sperm hole counts in the perivitelline layer of eggs. This is the first illustration of a measurable sperm trait predictive of paternity success in a competitive fertilization trial in turkeys, a species that is predominately reproduced by artificial insemination of multiple-sire pools.

Animals↗
PubMed1999

Differences in cadmium transport to the testis, epididymis, and brain in cadmium-sensitive and -resistant murine strains 129/J and A/J.

Although most animals with scrotal testes are susceptible to cadmium-induced testicular toxicity, strain-related differences are seen in mice. Resistant murine strains demonstrate a decreased cadmium concentration in the testis and also in the epididymis and seminal vesicle. In this study we analyzed cadmium transport into tissues with a vascular barrier, the testis, epididymis, and brain, in an attempt to characterize the mechanisms of strain resistance to cadmium-induced testicular toxicity. In the resistant murine strain A/J, 109Cd transport (administered as 109CdCl2) was significantly attenuated in the testis, epididymis, and brain, when compared to the sensitive murine strain 129/J. The unidirectional influx constant (Ki, in microliter g-1 min-1) for 109Cd was 0.01929 in the A/J testis as compared with 1.174 in the 129/J testis (P <.0001). The percentage of a 109Cd dose that reached the A/J testis by 60 min was over 10 times less than that which reached the 129/J testis. The transport system used by cadmium in the 129/J testis was saturable, with 20 microM unlabeled cadmium chloride inhibiting transport by over 60%. The transporter was competitively inhibited by zinc (P =. 00017), but not by calcium, indicating a specificity in ion transport. Studies with isolated tubules and analysis of testicular fluid compartments demonstrated no significant difference in cadmium uptake or efflux between the strains when corrected for the amount of 109Cd entering the testis. Therefore, murine strain differences in testicular sensitivity to cadmium appear to be related to the variable presence of a transport system for cadmium in the testicular vasculature.

Animals↗
PubMed1998

Physiological and pathological secretion of cartilage oligomeric matrix protein by cells in culture.

Abnormalities in cartilage oligomeric matrix protein (COMP), a pentameric structural protein of the cartilage extracellular matrix, have been identified in pseudoachondroplasia and multiple epiphyseal dysplasia, two human autosomal dominant osteochondrodysplasias. However, the function of the protein remains unknown. With the goal of establishing a model to study the mechanisms by which COMP mutations cause disease, we have analyzed synthesis and secretion of COMP in cultured chondrocytes, tendon, and ligament cells. Pentameric protein detected inside of control cells suggested that pentamerization is an intracellular process. Patient cells expressed mutant and normal RNA and secreted COMP at levels similar to controls, suggesting that abnormal pentamers are likely to be found in the extracellular matrix. Inclusions within patient cartilage stained with anti-COMP antibodies, and cultured cells presented similar inclusions, indicating that presumably abnormal COMP pentamers are less efficiently secreted than normal molecules. We conclude that the COMP disorders are likely to result from a combination of a decreased amount of COMP in the matrix and a dominant negative effect due to the presence of abnormal pentamers in cartilage.

Base Sequence↗
PubMed1998

Energy substrate metabolism, myocardial ischemia, and targets for pharmacotherapy.

Myocardial ischemia is essentially a metabolic event. In this review we will try to distill the essence of a complex series of molecular reactions triggered by the sudden reduction or cessation of blood flow to the heart. We recognize that it is difficult to describe even simple metabolic changes occurring in ischemia without a brief recap of pathways of energy transfer in the normal myocardium. We will therefore begin with a description of the energy substrate supply to a system that is best defined as the heart's remarkable ability for efficient conversion of chemical into mechanical energy. At the core of the system are rates of oxidative phosphorylation of adenosine diphosphate (ADP) that exactly match rates of adenosine triphosphate (ATP) hydrolysis. We will then describe the consequences of a sudden interruption to this balance, namely ischemia. At the same time we will explore metabolic strategies that may be employed to lessen the consequences of ischemia on contractile function, highlighting areas of future research and clinical investigation. The review is not meant to be comprehensive. Its main aim is to discuss the concept of pharmacotherapy as an intervention in altered cellular metabolism, akin to the concept of reperfusion therapy as an intervention in obstructed coronary arteries.

Adenosine Diphosphate↗
PubMed1998

F(ab')2 molecules made from Escherichia coli produced Fab' with hinge sequences conferring increased serum survival in an animal model.

Fab's with hinges based on the human gamma1 sequence containing 1, 2, or 4 cysteines have been produced by high level Escherichia coli periplasmic secretion, and coupled in vitro by reduction/oxidation to form F(ab')2. We find that the F(ab')2 made with hinges containing 2 or 4 cysteines have a high level (approximately 70%) of multiple disulphide bonds. These F(ab')2 molecules have an increased pharmacokinetic stability as measured by area under the curve compared to those made by direct coupling through a single disulphide bond. One particular molecule containing 4 hinge cysteines has a greater pharmacokinetic stability than a F(ab')2 formed by chemical cross-linking. F(ab')2 made from the Fab' with 4 hinge cysteines is also relatively resistant to chemical reduction in vitro allowing partial reduction to expose reactive hinge thiols. These hinge sequences provide a simple method for producing robust F(ab')2 in vitro, obviating the need to use chemical cross-linkers, and provide a route to hinge specific chemical modification with thiol-reactive conjugates.

Amino Acid Sequence↗
PubMed1998

Murine strain differences and the effects of zinc on cadmium concentrations in tissues after acute cadmium exposure.

The role of strain differences in cadmium tissue distribution was studied using sensitive (129/J) and resistant (A/J) mice. These murine strains have previously been shown to differ in their susceptibility to cadmium-induced testicular toxicity. Cadmium concentration was measured in testis, epididymis, seminal vesicle, liver, and kidney at 24 h after cadmium chloride exposure (4, 10, and 20 micromol/kg CdCl2). The 129/J mice exhibited a significant increase in cadmium concentration in testis, epididymis, and seminal vesicle at all cadmium doses used, compared to A/J mice. However, cadmium concentrations in liver and kidney were not different between the strains, at any dose, indicating that cadmium uptake is similar in these organs at 24 h. These murine strains demonstrate similar hepatic and renal cadmium uptake but significantly different cadmium accumulation in the reproductive organs at 24 h. The mechanism of the protective effect of zinc on cadmium toxicity was studied by assessing the impact of zinc acetate (ZnAc) treatment on cadmium concentrations in 129/J mice after 24 h. Zinc pretreatment (250 micromol/kg ZnAc), given 24 h prior to 20 micromol/kg CdCl2 administration, significantly decreased the amount of cadmium in the testis, epididymis, and seminal vesicle of 129/J mice, and significantly increased the cadmium content of the liver after 24 h. Cadmium levels in the kidney were unaffected at this time. Zinc pretreatment also prevented the cadmium-induced decrease in testicular sperm concentration and epididymal sperm motility seen in 129/J mice. These findings suggest that the differences in the two murine strains may be attributed partly to the differential accumulation of cadmium in murine gonads. This may be caused by strain differences in the specificity of cadmium transport mechanisms. The protective role of zinc in cadmium-induced testicular toxicity in the sensitive strain may be due to an interference in the cadmium uptake by susceptible reproductive organs.

Animals↗
PubMed1998

Glucose delivery is a major determinant of glucose utilisation in the ischemic myocardium with a residual coronary flow.

BACKGROUND: Experimental data from isolated rat hearts suggest that glycolysis in severe myocardial ischemia is inhibited by accumulation of glycolytic metabolites. In contrast, positron emission tomography (PET) in patients with myocardial ischemia records a 'mismatch' between the decreased coronary flow in viable ischemic tissue and an increased fluorodeoxyglucose (18FDG) signal. To resolve this contradiction, we investigated glucose uptake at very low coronary flows in the isolated rat heart model. METHODS: Rates of glucose uptake were measured in the isolated Langendorff-perfused Wistar rat heart, at control (12-15 ml/g wet wt/min) and low coronary flows (0.1, 0.2 and 0.5 ml/g wet wt/min) and at a range of glucose concentrations (2.75, 5.5, 11 and 22 mM). RESULTS: The steady-state rate of glucose uptake versus glucose concentration could be described by a double rectangular hyperbola at each coronary flow. Glucose uptake fell to levels significantly below control at low coronary flows. However, the extraction of glucose (glucose uptake as % of glucose delivered) rose sharply, from 1% at control coronary flows, to 25-30% at low coronary flows. Crossover analysis of glycolytic intermediates in freeze-clamped tissue indicated little inhibition at any specific site, although phosphofructokinase activity was restricted when glycolytic substrate availability was high. Insulin and preconditioning both increased glucose uptake with 11 mM glucose, possibly by increasing membrane transporter density and thus increasing glucose delivery to the cytosol. CONCLUSIONS: Despite the reduction in absolute glucose uptake at low coronary flow-rates, the extraction of glucose was greatly increased, possibly following GLUT4 translocation. Delivery of glucose to the glycolytic pathway appears to be a major controlling site of glycolysis in low-flow ischemia. Downstream regulation is then distributed along the pathway with no one site exerting greater inhibition than reduced glucose delivery.

Animals↗
PubMed1998

Mutations in orthologous genes in human spondyloepimetaphyseal dysplasia and the brachymorphic mouse.

The osteochondrodysplasias are a genetically heterogeneous group of disorders affecting skeletal development, linear growth and the maintenance of cartilage and bone. We have studied a large inbred Pakistani family with a distinct form of recessively inherited spondyloepimetaphyseal dysplasia (SEMD) and mapped a gene associated with this dwarfing condition to chromosome 10q23-24, a region syntenic with the locus for the brachymorphic mutation on mouse chromosome 19. We identified two orthologous genes, ATPSK2 and Atpsk2, encoding novel ATP sulfurylase/APS kinase orthologues in the respective regions of the human and mouse genomes. We characterized a nonsense mutation in ATPSK2 in the SEMD family and a missense mutation in the region of Atpsk2 encoding the APS kinase activity in the brachymorphic mouse. ATP sulfurylase/APS kinase catalyses the metabolic activation of inorganic sulfate to PAPS, the universal donor for post-translational protein sulfation in all cell types. The cartilage-specificity of the human and mouse phenotypes provides further evidence of the critical role of sulfate activation in the maturation of cartilage extracellular matrix molecules and the effect of defects in this process on the architecture of cartilage and skeletogenesis.

Amino Acid Sequence↗
PubMed1998

Diverse mutations in the gene for cartilage oligomeric matrix protein in the pseudoachondroplasia-multiple epiphyseal dysplasia disease spectrum.

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are autosomal dominant osteochondrodysplasias that result in mild to severe short-limb dwarfism and early-onset osteoarthrosis. PSACH and some forms of MED result from mutations in the gene for cartilage oligomeric matrix protein (COMP; OMIM 600310 [http://www3.ncbi.nlm. nih.gov:80/htbin-post/Omim/dispmim?600310]). We report the identification of COMP mutations in an additional 14 families with PSACH or MED phenotypes. Mutations predicted to result in single-amino acid deletions or substitutions, all in the region of the COMP gene encoding the calmodulin-like repeat elements, were identified in patients with moderate to severe PSACH. We also identified within this domain a missense mutation that produced MED Fairbank. In two families, one with mild PSACH and the second with a form of MED, we identified different substitutions for a residue in the carboxyl-terminal globular region of COMP. Both the clinical presentations of these two families and the identification of COMP-gene mutations provide evidence of phenotypic overlap between PSACH and MED. These data also reveal a role for the carboxyl-terminal domain in the structure and/or function of COMP.

Achondroplasia↗
PubMed1998

The role of gene conversion in determining sequence variation and divergence in the Est-5 gene family in Drosophila pseudoobscura.

Nucleotide sequences of eight Est-5A and Est-5C genes corresponding to previously sequenced Est-5B genes in Drosophila pseudoobscura were determined to compare patterns of polymorphism and divergence among members of this small gene family. The three esterase genes were also sequenced from D. persimilis and D. miranda for interspecific comparisons. The data provide evidence that gene conversion between loci contributes to polymorphism and to the homogenization of the Est5 genes. For Est-5B, which encodes one of the most highly polymorphic proteins in Drosophila, 12% of the segregating amino acid variants appear to have been introduced via gene conversion from other members of the gene family. Interlocus gene conversion can also explain high sequence similarity, especially at synonymous sites, between Est-5B and Est-5A. Tests of neutrality using interspecific comparisons show that levels of polymorphism conform to neutral expectations at each Est-5 locus. However, McDonald-Kreitman tests based on intraspecific gene comparisons indicate that positive selection on amino acids has accompanied Est-5 gene duplication and divergence in D. pseudoobscura.

Alleles↗
PubMed1998

Glucose and glycogen utilisation in myocardial ischemia--changes in metabolism and consequences for the myocyte.

Experimentally, enhanced glycolytic flux has been shown to confer many benefits to the ischemic heart, including maintenance of membrane activity, inhibition of contracture, reduced arrhythmias, and improved functional recovery. While at moderate low coronary flows, the benefits of glycolysis appear extensive, the controversy arises at very low flow rates, in the absence of flow; or when glycolytic substrate may be present in excess, such that high glucose concentrations with or without insulin overload the cell with deleterious metabolites. Under conditions of total global ischemia, glycogen is the only substrate for glycolytic flux. Glycogenolysis may only be protective until the accumulation of metabolites (lactate, H+, NADH, sugar phosphates and Pi ) outweighs the benefit of the ATP produced. The possible deleterious effects associated with increased glycolysis cannot be ignored, and may explain some of the controversial findings reported in the literature. However, an optimal balance between the rate of ATP production and rate of accumulation of metabolites (determined by the glycolytic flux rate and the rate of coronary washout), may ensure optimal recovery. In addition, the effects of glucose utilisation must be distinguished from those of glycogen, differences which may be explained by functional compartmentation within the cell.

Animals↗
PubMed1997

Early effects of CI-924 on hepatic peroxisome proliferation, microsomal enzyme induction, PCNA, and apoptosis in B6C3F1 mice and Wistar rats.

The lipid lowering agent 5,5'[[1,1'-biphenyl]-2,5-diylbis(oxy)]bis[2,2-dimethylpentanoic acid] (CI-924) is a peroxisome proliferator in rats and mice, but increased the incidence of hepatic tumors in mice only. Male and female B6C3F1 mice and albino Wistar rats were treated with CI-924 at doses of 0, 25 and 75 mg/kg for 1, 3, 7 and 28 days. Our aim was to identify species differences potentially related to tumorigenicity and to establish the time course of early events related to or associated with peroxisome proliferation. After 24 h of exposure to CI-924 in the diet there were increases in carnitine acyl transferase and CYP4A1 activity in mice at 25 and 75 mg/kg. In rats, carnitine acyl transferase activity was increased after 24 h and CYP4A1 activity increased after 3 days at 75 mg/kg. Acyl CoA oxidase activity was increased at both doses in male and female rats and mice by 3 days. In general the changes in enzyme activity were of greater magnitude in rats. In contrast to the rapid peroxisome proliferation, increases in the amount of PCNA were observed in CI-924 treated rats and mice at later times after administration and only at 75 mg/kg. PCNA was increased to a similar extent in both rats and mice, while apoptosis was decreased at both doses of CI-924 after 3 days in female rats, 7 days in male rats, and was largely unchanged in mice. It was concluded that the sequence of peroxisome proliferation was generally similar in rats and mice. Early changes in cell proliferation and programmed cell death were not directly correlated with subsequent CI-924-induced hepatotumorigenicity.

Animals↗
PubMed1997

Satellite DNA repeat sequence variation is low in three species of burying beetles in the genus Nicrophorus (Coleoptera: Silphidae).

Three satellite DNA families were identified in three species of burying beetles, Nicrophorus orbicollis, N. marginatus, and N. americanus. Southern hybridization and nucleotide sequence analysis of individual randomly cloned repeats shows that these satellite DNA families are highly abundant in the genome, are composed of unique repeats, and are species-specific. The repeats do not have identifiable core elements or substructures that are similar in all three families, and most interspecific sequence similarity is confined to homopolymeric runs of A and T. Satellite DNA from N. marginatus and N. americanus show single-base-pair indels among repeats, but single-nucleotide substitutions characterize most of the repeat variability. Although the repeat units are of similar lengths (342, 350, and 354 bp) and A + T composition (65%, 71%, and 71%, respectively), the average nucleotide divergence among sequenced repeats is very low (0.18%, 1.22%, and 0.71%, respectively). Transition/transversion ratios from the consensus sequence are 0.20, 0.69, and 0.70, respectively.

Animals↗
PubMed1996

Does preconditioning act by glycogen depletion in the isolated rat heart?

Preconditioning hastens the time to onset of ischaemic contracture and increases peak contracture in an isolated perfused rat heart, but improves recovery of function. The preconditioning ischaemic episode is also known to deplete glycogen stores. We tested whether a depletion in glycogen is related to the protection conferred by preconditioning. The isolated Langendorff perfused rat heart, with a left ventricular balloon to record function, was perfused with either glucose 11 mM, acetate 5 mM, or glucose 11 mM + insulin to alter pre-ischaemic glycogen levels prior to 30 min total global ischaemia. In addition, hearts were preconditioned by an episode of 5 min ischaemia and 5 min reperfusion. Time to onset of contracture (TOC-min), peak contracture and recovery of developed pressure after 20 min reperfusion with glucose-containing perfusate (both expressed as percentage pre-ischaemic developed pressure) were measured (n = 9-10). Parallel groups of hearts were clamped at various times for assessment of tissue metabolites. Acetate pre-perfusion reduced glycogen levels compared to glucose hearts, from 16.27 +/- 0.44 to 10.77 +/- 0.96 mumol/g wet wt. TOC was reduced and peak contracture increased, with poor functional recovery. Glucose + insulin pre-perfusion increased glycogen (21.39 +/- 1.08 mumol/g wet wt) with opposite effects on contracture, but functional recovery was still poor. Preconditioning hastened the time to onset of contracture, which could be partially attributed to glycogen depletion. Preconditioning significantly improved functional recovery in glucose hearts, but had little or no effect in the other groups. Thus the protective effect on functional recovery could not be linked to glycogen depletion. Pre-ischaemic glycogen appeared to play a dual role. When low, preconditioning was ineffective, presumably because of lack of production of glycolytic ATP, and severe contracture. When pre-ischaemic glycogen was increased, preconditioning was also relatively ineffective, presumably because of excess accumulation of the metabolites of glycogenolysis.

Acetates↗