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Biomedical subjects

J L Johnson

Publications and source records attributed to J L Johnson.

At least 19 recordsLinked to original sources
PubMed1992

High-level expression of a heterologous protein in the milk of transgenic swine using the cDNA encoding human protein C.

Transgenic pigs were generated that produced human protein C in their milk at up to 1 g/liter. The gene construct was a fusion gene consisting of the cDNA for human protein C inserted into the first exon of the mouse whey acidic protein gene. These results demonstrate that the mouse whey acidic protein gene contains regulatory elements that can direct cDNA expression at high levels in the pig mammary gland. Recombinant human protein C that was produced at about 380 micrograms/ml per hr in transgenic pig milk possessed anticoagulant activity that was equivalent to that of protein C derived from human plasma. These studies provide evidence that gamma-carboxylation can occur at high levels in the mammary gland of a pig.

Animals↗
PubMed1992

Kinetics of Bacillus cereus phosphatidylinositol-specific phospholipase C with thiophosphate and fluorescent analogs of phosphatidylinositol.

Thiophosphate analogs (C-S-P bond) of phosphatidylinositol (Cn-thio-PI: racemic hexadecyl-, dodecyl-, and octylthiophosphoryl-1-myo-inositol) and a fluorescent analog (pyrene-PI: rac-4-(1-pyreno)-butylphosphoryl-1-myo-inositol) were all substrates for phosphatidylinositol-specific phospholipase C from Bacillus cereus. Hydrolysis of thio-PI was followed by coupling the production of alkylthiol to a disulfide interchange reaction with dithiobispyridine. Hydrolysis of pyrene-PI was followed using a HPLC-based assay with fluorescence detection. The activity of PI-PLC with thio-PI analogs showed an interfacial effect. C16-Thio-PI, which had a critical micelle concentration (CMC) of 7 microM, gave a hyperbolic activity versus concentration curve between 0 and 2 mM, while C8-thio-PI, which had a CMC above 10 mM, showed very low activity which increased greatly upon introduction of an interface in mixed micelles with hexadecylphosphocholine (HDPC). Pyrene-PI, which aggregates above 0.3 mM, gave a sigmoidal activity curve with much higher activity above the CMC. All three thio-PI homologs as mixed micelles with HDPC gave hyperbolic activity curves with PI-PLC that were a function of bulk concentration of substrate at constant surface concentration and surface concentration of substrate at constant bulk concentration. The maximal activity of PI-PLC with pure C16-thio-PI micelles was 6.25 mumol min-1 mg-1, while that with pyrene-PI was estimated to be 68 mumol min-1 mg-1. With pure C16-thio-PI micelles, 0.022 mM substrate gave half Vmax, similar to that in mixed micelles with HDPC.

Bacillus cereus↗
PubMed1992

MgATP and fructose 6-phosphate interactions with phosphofructokinase from Escherichia coli.

A thermodynamic linked-function analysis is presented of the interactions of MgATP and fructose 6-phosphate (Fru-6-P) with phosphofructokinase (PFK) from Escherichia coli in the absence of allosteric effectors. MgATP and Fru-6-P are shown to bind in random fashion by product inhibition of the back-reaction as well as by the kinetically competent binding of each ligand individually as monitored by the consequent changes in the intrinsic fluorescence of E. coli PFK. When Fru-6-P is saturating, the dissociation of MgATP is sufficiently slow that it cannot achieve a binding equilibrium in the steady state, causing the observed Km (49 microM) to significantly exceed the Kd (1.7 microM) deduced from a thermodynamic linkage analysis. The following features distinguish the interactions of MgATP and Fru-6-P with E. coli PFK: MgATP and Fru-6-P antagonize each other's binding to the enzyme in a saturable manner with an overall apparent coupling free energy equal to +2.5 kcal/mol at 25 degrees C; MgATP induces positive cooperativity in the Fru-6-P binding profile, with the Hill coefficient calculated from the Fru-6-P binding curves reaching a maximum of 3.6 when MgATP is saturating; and MgATP exhibits substrate inhibition at low concentrations of Fru-6-P. Simulations based upon the rate equation pertaining to a two-active-site, two-substrate dimer indicate that these features can all result from two independent couplings: an antagonistic MgATP-Fru-6-P coupling extending at least in part between active sites and a MgATP-induced Fru-6-P-Fru-6-P coupling.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

Sertraline, a serotonin-uptake inhibitor, reduces food intake and body weight in lean rats and genetically obese mice.

Sertraline was found to inhibit weight gain and decrease food intake without affecting locomotion in rats and genetically obese (ob/ob) mice. Doses of 10, 17.8, and 32 mg/kg, administered intraperitoneally, (bid) significantly reduced the time rats spent in contact with their feeders and body weight in a dose-related manner. During a 5-d bid treatment regimen, vehicle-treated rats gained 37 +/- 3 g (mean +/- SEM), whereas animals treated with 32 mg sertraline/kg lost 34 +/- 4 g. The effects of sertraline on feeding and body weight in rats appeared to be specific because locomotor activity was not altered. In ob/ob mice, sertraline (44 mg/kg, ip, bid) lowered body weight relative to vehicle-treated controls for the duration of a 12-d study. There was no evidence for tolerance to the hypophagic and weight-loss effects of sertraline during either of the chronic dosing studies. These results suggest a potential role for sertraline in the treatment of human obesity.

1-Naphthylamine↗
PubMed1992

Hydralazine does not restore uterine blood flow during cocaine-induced hypertension in the pregnant ewe.

Cocaine abuse is widespread, and its use by the parturient has potential significant adverse effects in both the mother and the newborn. This study was undertaken in gravid ewes to determine the effects of treatment of cocaine-induced hypertension with hydralazine (Apresoline) on the maternal and fetal cardiovascular systems, catecholamine response, blood gas and acid-base status, and uterine blood flow (UBF). Twenty-one experiments were performed in 15 chronically instrumented ewes near term gestation. After a 30-min control period, cocaine was given intravenously to all ewes for 55 min to induce and maintain increased maternal mean arterial pressure (MMAP) and reduced UBF. The sheep were randomly assigned to receive either cocaine alone (n = 11, control group) or hydralazine (n = 10, treatment group), starting 15 min after the cocaine administration. Both drugs were discontinued 55 min after the start of the cocaine administration, followed by a 35-min recovery period. In the control group, cocaine administration resulted in a 31 +/- 13% (SD) increase in MMAP (P less than 0.05) and a 26 +/- 21% reduction in UBF (P less than 0.05). In the treatment group, the initial cocaine administration resulted in a similar increase in MMAP and decrease in UBF. Hydralazine therapy restored MMAP toward baseline after 20 min of administration, but UBF remained reduced (37 +/- 17%) throughout therapy (P less than 0.05) and recovery (18 +/- 13%) (P less than 0.05). The maternal heart rate increased maximally by 121 +/- 33% (P less than 0.05) after the administration of hydralazine, compared with a 14 +/- 21% increase (P less than 0.05) in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Emended descriptions of Prevotella denticola, Prevotella loescheii, Prevotella veroralis, and Prevotella melaninogenica.

During studies of human periodontal disease, a number of bacterial strains were encountered that, on the basis of results of standard biochemical tests, appeared to be Prevotella buccalis, Prevotella denticola, Prevotella melaninogenica, or Prevotella loescheii. However, use of the standard biochemical tests, cellular fatty acid analyses, and the polyacrylamide gel electrophoresis patterns of soluble proteins resulted in conflicting identifications of these strains. The results of tests for cellobiose fermentation, inulin fermentation, and pigment production were responsible for most of the discordant results. Cellular fatty acid analyses in which the Microbial Identification System was used did not differentiate these strains from validly described species, even though separate library entries were created for them. DNA reassociation determinations in which the S1 nuclease procedure was used showed that cellobiose fermentation and pigment production are variable among strains of P. melaninogenica and P. denticola and that fermentation of xylan is not a reliable characteristic for differentiating P. buccalis from Prevotella veroralis. In contrast to previous indications, most strains of P. veroralis do not ferment xylan. These species can be differentiated by DNA-DNA reassociation and by cellular fatty acid analysis, using the Microbial Identification System, but differentiation by currently described phenotypic characteristics is not reliable. Similarly, P. loescheii and the genetically distinct (but closely related) D1C-20 group cannot be distinguished reliably from each other or from P. veroralis, P. denticola, and P. melaninogenica on the basis of currently described phenotypic tests other than cellular fatty acid composition or, for some species, electrophoretic patterns of soluble whole-cell proteins.

Bacterial Typing Techniques↗
PubMed1992

Isolation and characterization of a newly identified Haemophilus species from cats: "Haemophilus felis".

A gram-negative coccobacillus was isolated from the lower respiratory tract of a cat with chronic obstructive pulmonary disease. The isolate required CO2 and V factor for growth and was initially identified as Haemophilus paraphrophilus on the basis of its nutritional requirements, colony morphology, and some biochemical tests. Because of the host specificity of Haemophilus species and discrepancies in catalase, oxidase, and hemolytic activities, additional testing was done. Extensive biochemical testing, G+C content, and DNA reassociation studies indicated that the organism was distinct from other Haemophilus species. Therefore, the organism was identified as a previously unrecognized Haemophilus species and was tentatively named "Haemophilus felis." Bacteria identical to the original isolate were isolated from the nasopharynxes of 6 of 28 apparently normal cats, indicating that H. felis or H. felis-like organisms may be common members of the feline upper respiratory tract flora.

Animals↗
PubMed1992

Glucoregulation during rest and exercise in depancreatized dogs: role of the acute presence of insulin.

To determine the effects of the presence of insulin in poorly controlled diabetes, depancreatized (PX) dogs (n = 5) were studied during rest and 150 min of exercise in paired experiments in which saline alone was infused (IDEF) and in which insulin was replaced intraportally (200 microU.kg-1.min-1) with glucose clamped at the levels in IDEF (IR+G). PX dogs (n = 4) were also studied with insulin, but glucose was allowed to fall (IR). Insulin was not detectable, 6 +/- 1 and 6 +/- 2 microU/ml in IDEF, IR+G, and IR. Plasma glucose was 470 +/- 47, 480 +/- 48, and 372 +/- 35 mg/dl at rest in IDEF, IR+G, and IR, respectively. Levels were unchanged with exercise in IDEF and IR+G, but fell by 139 +/- 13 mg/dl in IR. Basal glucose rate of appearance (Ra) was 7.0 +/- 0.9, 1.3 +/- 1.1, and 6.0 +/- 0.7 mg.kg-1.min-1 in IDEF, IR+G, and IR, respectively. Exercise elicited a rise in Ra in only IDEF. The rises in Rd and metabolic clearance rate in IDEF were reduced (delta 2.6 +/- 0.7 and delta 0.8 +/- 0.3 ml.kg-1.min-1 at 150 min) compared with IR+G (delta 5.3 +/- 1.9 and delta 1.7 +/- 0.2 ml.kg-1.min-1 at 150 min) and IR (delta 3.7 +/- 1.2 and delta 2.4 +/- 0.8 ml.kg-1.min-1). The insulin sensitivity of glucose utilization (Rd) was elevated by approximately 75% at 150 min. Basal glycerol was similar in IDEF and IR but was reduced by approximately 70% in IR+G. Glycerol rose similarly with exercise in IDEF and IR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Regional cerebral glucose utilization in chronic organic mental disorders associated with alcoholism.

Localized cerebral utilization rates for glucose (CMRglu) were determined in 10 detoxified patients with alcoholic organic mental disorders and in 7 age-equivalent normal volunteers using [18F]2-fluoro-2-deoxyglucose positron emission tomography. Although gray and white matter CMRglu were not significantly different, normalized CMRglu was increased in the left cerebellar and parietal cortical regions and decreased in the right posterior white matter and anterior temporal regions of alcoholic patients, and the pattern of regional CMRglu differed between the two groups. The results suggest functional disruption of right-sided and frontal brain regions and hyperactivity of cerebellar-cortical connections in alcoholic chronic organic mental disorders.

Aged↗
PubMed1992

Classification and characteristics of Army stress casualties during Operation Desert Storm.

During the Persian Gulf War, the U.S. Army provided mental health care to soldiers using a model that emphasized the identification and management of factors associated with combat stress. Nonpathological labels, such as combat stress reaction, a non-hospital-based care delivery system, and simple interventions were used to promote return to duty. The authors describe such services provided to the U.S. Army 7th Corps. A total of 158 patients were treated; 72 percent were men. Seventy percent were from the junior ranks. Thirteen soldiers were treated for axis I disorders, four for axis II disorders, and 118 for stress reactions. Ninety-nine percent of the soldiers treated were returned to effective duty after a brief intervention.

Adult↗
PubMed1992

In vitro development of zygotes from prepubertal gilts after microinjection of DNA.

The effect of pronuclear microinjection of DNA and culture in excised mouse oviducts on the development of porcine zygotes was assessed in this study. Precocious ovulation was induced in prepubertal gilts with pregnant mare's serum gonadotrophin and hCG. Zygotes received either pronuclear microinjection of buffer alone, buffer containing a DNA construct, or no microinjection. Zygotes were cultured in vitro in either modified Krebs-Ringer bicarbonate medium (KRB) for 144 h or in mouse oviduct (MO) explant culture with KRB for 48, 72, 96, or 120 h. Pronuclear microinjection of DNA resulted in a lower (P less than .05) cleavage index (CI) than did buffer or no microinjection (CI 2.16 +/- .10 vs 2.80 +/- .13 and 2.93 +/- .10). The CI loss was greatest for DNA-injected zygotes at the two-cell stage of development. Coculture of zygotes in MO resulted in a higher CI (P less than .01) than did culture in KRB. Culture in MO for 72 h was the most beneficial system compared with MO for 48, 96, or 120 h (P less than .05; CI 3.25 +/- .12 vs 2.66 +/- .18, 2.79 +/- .14, and 2.40 +/- .14, respectively). Microinjection of DNA, not merely the mechanical procedure, was detrimental to early zygote development and may be the cause of low pregnancy rates.

Animals↗
PubMed1992

31P-NMR of free and protein-bound molybdopterin guanine dinucleotide.

Molybdopterin guanine dinucleotide was studied by 31P-NMR in the free, iodoacetamide derivatized form [di(carboxamidomethyl)molybdopterin] and in the native state in the dimethyl sulfoxide reductase from Rhodobacter sphaeroides. The spectra confirm the presence of a pyrophosphate moiety in the cofactor molecule. Comparison of the spectrum of the free pterin with that of the protein-bound cofactor reveals a substantial upfield shift of the 31P resonances in the enzyme-bound form with respect to the free form. This shift is attributed to differences in the bond and torsional angles of the phosphates. The spectrum of the protein suggests significant coupling between the two phosphorus nuclei with coupling constants of approximately 200 Hz. Comparison of the 31P-NMR spectra of molybdopterin guanine dinucleotide and flavin adenine dinucleotide suggests that the two cofactors have similar conformations in both their free and protein-bound forms.

Animals↗
PubMed1991

Molybdenum cofactor biosynthesis in Escherichia coli. Requirement of the chlB gene product for the formation of molybdopterin guanine dinucleotide.

The chlorate-resistant mutants of Escherichia coli are affected in the biosynthesis of the molybdenum cofactor and show pleiotropic loss of the activities of those enzymes which require the cofactor. The molybdenum cofactor in all molybdoenzymes other than nitrogenase is a complex of the metal with a unique pterin termed molybdopterin. The molybdenum cofactor in a number of E. coli enzymes has been shown to contain GMP in addition to the metal-molybdopterin complex, with the GMP appended in pyrophosphate linkage to the terminal phosphate ester on the molybdopterin side chain. In this paper, we have examined the biochemistry of the chlB mutant and show that the gene product of the chlB locus is essential for the addition of the GMP moiety to form molybdopterin guanine dinucleotide, a step which occurs late in the cofactor biosynthetic pathway in E. coli. Sensitive techniques were developed for the identification of fluorescent derivatives of molybdopterin and of molybdopterin guanine dinucleotide in extracts of E. coli cells. Wild type cells were shown to contain both molybdopterin and molybdopterin guanine dinucleotide, while cells of chlB mutants were found to contain elevated levels of molybdopterin but no detectable molybdopterin guanine dinucleotide.

Chromatography, High Pressure Liquid↗
PubMed1991

Bidirectional effects of cytokines on the growth of Mycobacterium avium within human monocytes.

Certain cytokines including IFN-gamma possess macrophage-activating factor activity that enhances the ability of these effector cells to destroy intracellular pathogens. A panel of recombinant and highly purified human cytokines was screened to detect this effect on the activation of human monocytes to kill Mycobacterium avium in an in vitro model. Peripheral blood monocytes obtained from 15 healthy donors were precultured for 2 days before infection. Monocytes were infected with two strains of M. avium, one AIDS-associated and relatively avirulent strain (86m2096), and the other a non-AIDS-associated isolate that demonstrated consistent and rapid growth in cultured human monocytes (LR114F). The effects of recombinant and purified human cytokines on M. avium infection were assayed by determining CFU of M. avium in lysates of infected monocytes after 0, 4, and 7 days of culture. After infection, monocytes were cultured in medium alone or continuously in the presence of the following cytokines: IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6, IFN-gamma, granulocyte-macrophage-CSF, or macrophage-CSF. In some experiments, cultures were performed in the presence of indomethacin (IM) in addition to cytokines. Culture in the presence of rIFN-gamma was associated with a decrease in mycobacterial growth within human monocytes. The combination of 300 U/ml of IFN-gamma plus 1 micrograms/ml of IM was associated with a 10-fold decrease (p less than 0.01) in intracellular growth of the virulent strain (LR114F) compared with unstimulated cultures. No other cytokine or combination of a cytokine with IM inhibited the intracellular growth of either strain of M. avium in human monocytes. Rather, several cytokines enhanced the intracellular growth of M. avium. IL-3, IL-6, and macrophage-CSF increased the growth of one, and IL-1 alpha of both strains of M. avium tested. IL-1 alpha and IL-6 also induced M. avium growth in tissue culture medium without monocytes. These studies indicate bidirectional effects of cytokines on intracellular parasitism that may influence the outcome of M. avium infection.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Inhibition of human neutrophil superoxide generation by alpha 1-antichymotrypsin.

Reactive oxygen intermediates and serine proteases are important components of host defense systems but can produce host injury if not tightly regulated. To determine whether these components can be coordinately controlled, we investigated regulation of superoxide generation by physiologically relevant concentrations of a) highly purified serum-derived antichymotrypsin (ACT), b) recombinant, wild-type ACT, c) rACT in which amino acid substitutions were engineered into the reactive center, and d) chymotrypsin/ACT complexes. These proteins and protein complexes inhibited superoxide anion production in neutrophils stimulated by f-Met-Leu-Phe, Con A, or PMA. In contrast, ligand-stimulated degranulation was not inhibited. In addition, using the recombinants and complexes, the region of ACT involved in inhibiting superoxide anion production was shown to be structurally distinct from the reactive center of the protein. The results indicate that functional domains of ACT corresponding to different biological activities can be decoupled and suggest that three species of ACT (intact ACT, a complexed protease/ACT form, and a partially denatured or proteolyzed form of ACT) that can exist in the microenvironment of an activated neutrophil may play an important role in regulating neutrophil function.

Cell Degranulation↗