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Biomedical subjects

L A Johnson

Publications and source records attributed to L A Johnson.

At least 127 records · Page 7Linked to original sources
PubMed1990

Sperm DNA and sex chromosome differences between two geographical populations of the creeping vole, Microtus oregoni.

The relative DNA content of the "O" and Y chromosome-bearing sperm is presented for the creeping vole, Microtus oregoni. The animals had been trapped in Oregon and in Washington State. The two populations had very similar autosomal chromosome relationships but differed greatly in the size of their X chromosome (which is not carried by vole sperm) and in their Y chromosome. The greater size and banding differences of the Y chromosome of the Washington State vole compared to the Oregon vole paralleled the greater differences in sperm DNA between the Y-bearing sperm and the sperm carrying no sex chromosome (O). The actual DNA differences between O and Y sperm was 12.5% for the sperm from the Washington State voles and 9.1% for sperm from the Oregon voles. The difference in sperm DNA content (12.5%) for Washington State voles was far greater than the difference shown for other voles or other mammals.

Animals↗
PubMed1990

Thymocytes stimulate metabolism of arachidonic acid in rat thymic epithelial cells.

Arachidonic acid metabolites play an important role in the development of T cells in the thymus. In the normal animal, prostaglandin levels in the thymus are significantly higher than in plasma. Herein, we have studied the regulation of arachidonic acid metabolism using the thymic endocrine epithelial cell line, TEA3A1, and thymocytes. We have found that TEA3A1 cells, but not thymocytes, produced prostaglandins and thromboxanes. We have also found that thymocytes could stimulate the production of arachidonic acid metabolites in TEA3A1 cells when both cells were cocultured. The strongest stimulation was observed when TEA3A1 cells were cocultured with thymocyte subpopulations either negative for OX8 or W3/25 functional surface markers (either double-negative or single-positive thymocytes). Furthermore, cell-cell contact seems to be absolutely required for the activation of arachidonic acid metabolism in TEA3A1 cells. The study presented here describes the existence of a novel regulatory mechanism of arachidonic acid metabolism which may play an important role in the development of T cells in the thymus.

Animals↗
PubMed1989

Crohn's disease uveitis. Parasitization of vitreous leukocytes by mollicute-like organisms.

Crohn's disease (CD) is an idiopathic chronic inflammatory gut disease with frequent extragut inflammatory manifestations in the eyes, orbit, lungs, joints, and skin. A bacterial cause of CD is suspected, but cultivation of a specific pathogen has not been forthcoming. Mollicute-like organisms (MLOs) were recently reported to cause human chronic ocular inflammatory disease. Inoculation of this MLO into mouse eyelids produced chronic progressive granulomatous ocular and orbital inflammatory disease. In addition, MLOs disseminated to produce similar disease in the gut, heart, and lungs. MLOs are noncultivable cell wall-deficient bacterial pathogens. Because they also pass bacteria-retaining 0.450-micron filters, they can be overlooked or confused with viruses. Because MLOs have a characteristic ultrastructural appearance, they can be identified in diseased cells with the use of a transmission electron microscope. MLOs parasitize and destroy leukocytes. They alter the nucleus, replace the cytoplasm, and destroy organelles. MLO-caused disease is treatable by certain antibiotics. This report describes MLO-parasitized vitreous lymphocytes, monocytes, and polymorphonuclear leukocytes from three patients with CD who had chronic uveitis. The results indicate that MLOs probably caused the uveitis of these patients with CD. The gut as the possible source of the MLO is suggested. Rifampin therapy of Crohn's and MLO-caused disease is discussed.

Adult↗
PubMed1989

Sex preselection in rabbits: live births from X and Y sperm separated by DNA and cell sorting.

Intact, viable X and Y chromosome-bearing sperm populations of the rabbit were separated according to DNA content with a flow cytometer/cell sorter. Reanalysis for DNA of an aliquot from each sorted population showed purities of 86% for X-bearing sperm and 81% for Y-bearing sperm populations. Sorted sperm were surgically inseminated into the uterus of rabbits. From does inseminated with sorted X-bearing sperm, 94% of the offspring born were females. From does inseminated with sorted Y-bearing sperm from the same ejaculates, 81% of the offspring were males. The probability of the phenotypic sex ratios differing from 50:50 were p less than 0.0003 for X-sorted sperm and p less than 0.004 for Y-sorted sperm. Thus, the phenotypic sex ratio at birth was accurately predicted from the flow-cytometrically measured proportion of X- and Y-bearing sperm used for insemination.

Animals↗
PubMed1989

Isolation, molecular characterization and expression of the ushB gene of Salmonella typhimurium which encodes a membrane-bound UDP-sugar hydrolase.

The UDP-sugar hydrolase of Salmonella typhimurium has previously been reported to be located in both the inner and the outer membrane. We have cloned the gene, designated ushB, encoding this enzyme and determined its nucleotide sequence. No significant sequence homology with the periplasmic UDP-sugar hydrolase of Escherichia coli was found at either the DNA or protein level. However, a sequence is detectable, in the E. coli genome, which weakly hybridizes with a specific ushB probe. Polypeptide analysis has allowed the identification of the Salmonella hydrolase which has an Mr of 28,349 as compared to an Mr of 60,767 for the E. coli hydrolase. Most of the protein (approximately 90%) is located in the inner membrane. Two independent membrane fractionation procedures indicate that the remainder may be associated with the outer membrane. The deduced primary structure indicates the presence of an N-terminal signal peptide, although certain features of the region surrounding the putative processing site indicate that processing may be inefficient, or may not occur. Experiments with several inhibitors of signal peptidase function fail to demonstrate the appearance of a precursor form.

Amino Acid Sequence↗
PubMed1988

Inactivation of chymotrypsin and human skin chymase: kinetics of time-dependent inhibition in the presence of substrate.

The reaction of chymase, a chymotryptic proteinase from human skin, and bovine pancreatic chymotrypsin with a number of time-dependent inhibitors has been studied. An integrated equation, relating product formation with time, has been derived for the reaction of enzymes with time-dependent inhibitors in the presence of substrate. This is based on a two-step model in which a rapidly reversible, non-covalent complex (EI) is formed prior to a tighter, less readily reversible complex (EI)*). The equation depends on the simplifying assumption [I] much greater than [E], but is applicable to reversible and irreversible slow-binding and tight-binding inhibitors whether or not they show saturation kinetics. The method has been applied to the reaction of chymase and chymotrypsin with the tetrapeptide aldehyde, chymostatin, basic pancreatic trypsin inhibitor and Ala-Ala-Phe-chloromethylketone (AAPCK). The irreversible inhibitor, AAPCK, showed the expected saturation kinetics for both enzymes and the apparent first-order rate constants (k2) and dissociation constants (Ki) for the non-covalent complexes were determined. Chymostatin was a much more potent inhibitor which failed to show a saturation effect. The second-order rate constant of inactivation (k2/Ki), the first-order reactivation rate constant (k-2), and the dissociation constant of the covalent complex (Ki*) were determined. Basic pancreatic trypsin inhibitor, a potent inhibitor of chymotrypsin, had similar kinetics to chymostatin but failed to inhibit chymase. The applicability of the two-step model and the integrated equation to slow- and tight-binding inhibitors is discussed in relation to a number of examples from the literature.

Amino Acid Chloromethyl Ketones↗
PubMed1988

Attempted sexing of bovine spermatozoa by fractionation on a Percoll density gradient.

Bovine spermatozoa were fractionated on Percoll density gradients into two major subpopulations of motile spermatozoa and a minor fraction containing mostly nonmotile spermatozoa with abnormal morphology. Fractionation required the addition of bovine serum albumin and a continuous Percoll gradient buffered with sodium bicarbonate. It is postulated that, under suitable ionic conditions, the binding of bovine serum albumin to spermatozoa amplifies subtle differences between subpopulations. These studies were directed toward separating Y- and X-bearing spermatozoa. However, when the subpopulations were evaluated by flow cytometry, their Y:X ratios were similar to that of an unfractionated control.

Animals↗
PubMed1988

Flow sorting of X and Y chromosome-bearing mammalian sperm: activation and pronuclear development of sorted bull, boar, and ram sperm microinjected into hamster oocytes.

Flow cytometric techniques were used to measure relative DNA content of X and Y chromosome-bearing bull, boar, and ram sperm populations and to separate the two sex-determining populations. Neat semen was prepared for flow cytometric analysis by washing, light sonication, and staining with 9 microM Hoechst 33342. Computer analysis of the bimodal histograms showed mean X-Y DNA differences of 3.9, 3.7, and 4.2% for bull, boar, and ram, respectively. Flow cytometric reanalysis of sorted bull, boar, and ram sperm showed purities greater than 90%. Bull, boar, and ram sperm nuclei were microinjected into hamster oocytes. Microinjected sperm were either unsorted, sorted, unsorted plus dithiothreitol (DTT) exposure, or sorted plus DTT exposure. Following microinjection, eggs were incubated 3 hr, fixed, and stained. A total of 579 eggs was observed for sperm activation (decondensation or formation of a male pronucleus). A lower percentage of sorted than unsorted (3 vs. 23%) boar sperm was activated (P less than .05). However, sorted and unsorted DTT-exposed boar sperm or sorted and unsorted bull or ram sperm, regardless of DTT treatment, did not differ significantly. Sorted sperm nuclei of both rams and bulls exhibited higher activation rates than sorted boar sperm (P less than .05). Treatment of sperm with DTT increased the activation rate (P less than .05) for sorted boar sperm but not for bull or ram sperm.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Fluorometric evaluation of cryopreserved bovine spermatozoa extended in egg yolk and milk.

A comparison of fluorogenically quantifiable parameters of cryopreserved, bovine spermatozoa that had been processed in homogenized milk and egg yolk citrate-based extenders was made using flow cytometry. Semen from four bulls was processed in egg yolk-citrate or homogenized milk extenders, packaged in straws and frozen at -196 degrees C. Samples were thawed at 37 degrees C, subdivided into three portions and stained after 0, 1.5 and 3 h of incubation at 37 degrees C. Spermatozoa were stained using a combination of carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) and analyzed by dual parameter flow cytometry. The sperm cells were quantified fluorometrically at each time interval for both green and red fluorescence. The proportion of spermatozoa retaining the fluorescent CFDA derivative was larger at each time interval for samples in egg yolk citrate than those in milk. Differences in the retention of spermatozoal viability were detected between identical samples of bovine spermatozoa extended in milk or egg yolk based media.

Journal Article↗
PubMed1988

Factors related to successful sperm microinjection of hamster eggs: The effect of sperm species, technical experience, needle dimensions, and incubation medium on egg viability and sperm decondensation following microinjection.

A series of experiments was designed to identify factors in a sperm microinjection system that could influence egg viability and decondensation of sperm nuclei after microinjection. Egg viability and sperm decondensation rates were not different among eggs microinjected with rodent sperm. The microinjection of ram sperm required a larger diameter needle for injection, which resulted in low egg viability and sperm decondensation in the first 3 mo of the study but improved greatly after 9 mo of technical experience. The degree of technical experience (3 vs 9 mo) also improved (P<0.05) egg viability after microinjection with rodent sperm; however, the rate of sperm decondensation remained unaffected. Altering the dimensions of the injection needle from a tapered needle barrel to a more uniform needle barrel increased egg viability from 61 to 96% and sperm decondensation from 3 to 27%. The use of medium 199 for incubating microinjected eggs further increased (P<0.05) the percentage of eggs containing decondensed sperm nuclei (52%) compared to eggs incubated in Holmes defined medium (28%). By altering the dimensions of the injection needle, by selecting an appropriate incubation medium, and by gaining technical experience in microinjection, the efficiency of a sperm microinjection system was improved for both rodents and domestic animals.

Journal Article↗
PubMed1988

The creeping vole, Microtus oregoni: karyotype and sex-chromosome differences between two geographical populations.

The G-banded karyotype of the creeping vole, Microtus oregoni, prepared from animals trapped in Oregon and Washington, is presented. The two populations had similar autosomal banding patterns but exhibited striking differences in their sex chromosomes. The X chromosome of voles captured in Oregon was 39% longer than that of voles trapped in Washington. The length difference was primarily due to an increase in size of light G-bands, which, in both populations, comprised large segments of the X chromosome. On C-banding, the X chromosome exhibited major blocks of constitutive heterochromatin corresponding to the light G-bands. In contrast, the Y chromosome of the Oregon voles was 24% shorter than that of the Washington voles and lacked the short arm and some terminal bands present in the Washington voles.

Animals↗
PubMed1988

Enhanced responsiveness of gonadotropes after protein kinase-C activation: postreceptor regulation of gonadotropin releasing hormone action.

GnRH stimulates the release of LH from pituitary gonadotropes in a Ca2+-and calmodulin-dependent manner. Although GnRH also appears to activate protein kinase-C in gonadotropes, the role of this enzyme in GnRH action remains undetermined. In the present work we have assessed the effect of pretreatment of pituitary cell cultures with a protein kinase-C-activating phorbol ester on gonadotrope responsiveness to GnRH. Pretreatment for 6 h with phorbol 12-myristate 13-acetate (PMA) reduced the EC50 for GnRH-stimulated LH release approximately 8-fold without altering the maximum proportion of total cellular LH release. This increase in the potency of GnRH occurred in the absence of any measurable change in receptor affinity. Subsequent studies revealed that PMA pretreatment did not alter the EC50 for GnRH-stimulated [3H]inositol phosphate accumulation (an indicator of phosphoinositide hydrolysis), but did cause a modest reduction (approximately 2-fold) in the EC50 for LH release in response to the Ca2+ ionophore A23187 and the Ca2+ channel-activating compound maitotoxin. These observations demonstrate that the efficiency of coupling of the GnRH receptor to LH release can be regulated at a postreceptor locus by activation of protein kinase-C and that an increased responsiveness of Ca2+-regulated effector systems to mobilized Ca2+ appears to contribute to the observed effect.

Animals↗
PubMed1988

Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws.

Experiments were conducted to study the effect of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa after cryopreservation in straws. Semen (split ejaculate) in maxi-straws (6 mm o.d.) was frozen using a programmable freezing chamber. Three methods for in vitro sperm evaluation were used: motility (MOT), acrosome integrity (NAR) and flow cytometric analysis of sperm treated with carboxyfluorescein diacetate and propidium iodide to assess sperm plasma membrane integrity (PMI). No interactions were found among the three variables evaluated. Length of prefreeze exposure to glycerol, ranging from .5 min to 75 min, had no effect on post-thaw sperm viability. Exposure of sperm to a glycerol-containing extender medium at 5 degrees C gave improved post-thaw viability over that exposed at 0 degree C (P less than .05). Glycerol at a concentration of 3 or 4% resulted in maximum post-thaw MOT. Acrosome integrity values were greatest for 2 and 3% glycerol, whereas PMI was greatest when glycerol concentration was 4 to 6%. The primary cryoprotective effect of glycerol on boar semen may be extracellular. It is concluded that 3 or 4% glycerol gives maximum viability of frozen-thawed spermatozoa when the present methods are employed.

Acrosome↗
PubMed1988

Mouse interstitial lung disease and pleuritis induction by human Mollicute-like organisms.

Mollicute-Like Organisms (MLO) are cell-wall deficient intracellular bacterial pathogens. As MLO are non-cultivable, detection is based on finding typical Mollicute bodies within the host cell using a transmission electron microscope. Extracellular Mollicutes cause disease by a variety of mechanisms. MLO cause disease by similar mechanisms, and in addition directly alter the host cell nucleus, replace the cytoplasm, and destroy the organelles. MLO parasitization of plant cells causes a well studied chronic vascular disease reversible by tetracycline antibiotics. Recently similar MLO were reported to cause human chronic ocular vasculitis. As it parasitizes, lyses, and destroys leucocytes, it has been termed Leucocytoclastic MLO. Inoculation of this MLO into mouse eyelids produced delayed onset chronic ocular and lethal cardiac vasculitis. All lesions demonstrated tissue lysis with leucocytic infiltrates and MLO parasitized leucocytes. MLO-caused human and mouse disease responds to Rifampin. This report describes the 40 interstitial lung disease lesions in 21 of 100 of those MLO inoculated mice vs 0 in 200 controls (P less than 0.05) and 27 pleuritis lesions in 17 mice vs 0 control mice (P less than 0.05). The lung and pleural disease were associated in 13 lesions and unassociated in 41 lesions. MLO parasitized leucocytes were found in both the lung and pleural lesions from six of six MLO inoculated mice versus none of six controls. As most human interstitial lung and pleural diseases are idiopathic and closely resemble this mouse disease, they may be induced by MLO and treatable by Rifampin.

Animals↗
PubMed1987

A random phase II study of mitoxantrone and cisplatin in patients with hepatocellular carcinoma. An ECOG study.

Of 86 patients entered in an Eastern Cooperative Oncology Group (ECOG) random Phase II study of mitoxantrone (DHAD) and cisplatin (DDP) in primary liver cancer, 69 were eligible. Nine of the 13 ineligible patients were excluded after a pathology review. Sixty-one percent of the patients were North American, and 39% were South African. The most common severe or the worst toxicity on DHAD was hematologic; and to DDP, hematologic and vomiting. Of the 69 eligible patients, 21 experienced severe, life-threatening or fatal toxic reactions. Two patients treated with DDP had partial responses. With a 95% confidence interval, the true response rate to DHAD was less than 8%, and to DDP, less than 17%. The median survival time was 14 weeks on both drugs. Assuming a proportional hazards model, factors that are significantly associated with survival are patient performance status, the presence of the symptoms, raised bilirubin and hepatomegaly, and clinical evidence of cirrhosis. Any differences between survival rates for South African and North American patients were largely explainable by these factors.

Antineoplastic Combined Chemotherapy Protocols↗