Biomedical subjects

R L Johnson

Publications and source records attributed to R L Johnson.

At least 163 records · Page 9Linked to original sources

PubMed1993

Bicyclic thiazolidine lactam peptidomimetics of the dopamine receptor modulating peptide Pro-Leu-Gly-NH2.

Bicyclic thiazolidine lactam peptidomimetics 3-5 have been synthesized as potential analogues of the dopamine receptor modulating peptide Pro-Leu-Gly-NH2 (PLG). Peptidomimetics 3 and 4 were designed to constrain two, psi 2 and phi 3, of the four torsion angles that define a beta-turn to values approximating those found for a type-II beta-turn, while 5 was designed as a compound that could not achieve a beta-turn conformation. Peptidomimetics 3 and 4 were found to enhance the binding of the dopamine receptor agonist ADTN to the dopamine receptor, while 5 was found to be inactive. Like PLG the dose-response curves for 3 and 4 were bell-shaped in nature with the maximum effect occurring at a concentration of 1 microM. Both 3 and 4 were more effective than PLG in enhancing the binding of ADTN to dopamine receptors. The 5,5-bicyclic thiazolidine lactam peptidomimetic 3 enhanced the binding of ADTN by almost 200%, while the 6,5-bicyclic thiazolidine lactam peptidomimetic 4 enhanced the binding of ADTN by about 75%. These results provide further evidence in support of the hypothesis that the bioactive conformation of PLG is a type-II beta-turn.

Animals↗

PubMed1993

Synthesis and dopamine receptor modulating activity of lactam conformationally constrained analogues of Pro-Leu-Gly-NH2.

A series of analogues of the potent analogue of Pro-Leu-Gly-NH2 (PLG), 2-oxo-3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-1-pyrrolidineacet amide (2) were synthesized in which the (R)-gamma-lactam residue of 2 was replaced with a (R)-beta-lactam, (R)-aminosuccinimide, (R)-cycloseryl, (R)-delta-lactam, (R)-epsilon-lactam, or (S)-epsilon-lactam residue to give analogues 3-8, respectively. These substitutions were made so as to vary the psi 2 torsion angle. The analogues were tested for their ability to enhance the binding of the dopamine receptor agonist ADTN to the dopamine receptor. Analogues 3-6 and 8 exhibited dose-response curves that were bell-shaped in nature with the maximum effect occurring at a concentration of 1 microM. Analogue 7 was inactive. Analogues 3 and 4 were found to be as effective as PLG, while analogues 5, 6, and 8 appeared to be more effective than PLG in terms of enhancing the binding of ADTN to dopamine receptors. The activity of analogues 3-6 and 8 with their psi 2 angles in the vicinity of that observed in a type II beta-turn is consistent with the hypothesis that this type of turn is the bioactive conformation of PLG.

Amino Acid Sequence↗

PubMed1993

Cloning and expression of goldfish opsin sequences.

Five opsin cDNA clones were isolated from a goldfish retina cDNA library and sequenced. On the basis of homology to previously characterized visual pigments, one clone was identified as goldfish rod opsin and a second as a goldfish red cone opsin. Two rhodopsin-like clones were found to be similar to the chicken green opsin, a pigment which shares properties with both rod and cone pigments. A fifth clone was equally homologous to human blue cone opsin and human rod opsin. In order to characterize the spectral properties of the encoded pigments, the five clones were expressed in tissue culture cells and the apoproteins reconstituted with 11-cis-retinal. The wavelength of maximal absorption for goldfish rhodopsin is 492 nm and for the fifth pigment, identified as the goldfish blue pigment, 441 nm. Pigments encoded by the two rhodopsin-like clones absorb at 505 and 511 nm and are likely to correspond to the goldfish green pigment previously characterized by microspectrophotometry. The putative red cone opsin cDNA may encode a pigment that is a polymorphic variant of goldfish red since it absorbs maximally at 525 nm.

Amino Acid Sequence↗

PubMed1993

HLA homozygosity and the risk of related-donor transfusion-associated graft-versus-host disease.

Based on the prevailing interpretation of recent data implicating donor homozygosity for an HLA haplotype shared with a heterozygous recipient, it is our conclusion that the risk of TA-GVHD for a sibling of the blood donor is about half that of a parent or a child, and the risk for a grandparent, a grandchild, or a blood-related aunt, uncle, niece, or nephew is about the same as the risk for the donor's sibling. Consequently, the current recommendation and the practice of irradiating cellular blood products for transfusion to first-degree relatives but not for transfusion to more distant relatives is insufficient. We believe that the recommendation for prophylactic irradiation of cellular blood products, based on the current understanding of its apparent safety, should be extended at least to include transfusion to the donor's grandparents, grandchildren, and blood-related aunts, uncles, nieces, and nephews.

Blood Donors↗

PubMed1993

Blood component supplementation during massive transfusion of AS-1 red cells in trauma patients.

Coagulation component transfusions in trauma patients given more than 10 units of AS-1 red cells during the first 24 hours after injury were studied. Serial coagulation tests were obtained to direct component therapy. Coagulopathy developed in more than 70% of patients. Patients with blunt or penetrating trauma had similar coagulation abnormalities during massive transfusions. More than 40% of patients with either exsanguinating hemorrhage from penetrating trauma or blunt trauma with associated brain injuries had abnormal coagulation tests on admission. In contrast, patients with blunt trauma, but without brain injury, did not have coagulopathy on admission. Patients received an average of 25 units (range, 10-77) of AS-1 red cells and were exposed to an average of 80 (range, 11-252) donors. Exsanguination and brain injuries primarily caused death in 57%. Abnormal coagulation tests will be frequent and profound during a massive transfusion with AS-1 red cells. Serial coagulation testing is recommended.

Adolescent↗

PubMed1993

Identification and targeted gene disruption of cAR3, a cAMP receptor subtype expressed during multicellular stages of Dictyostelium development.

Extracellular cAMP acts through cell-surface receptors to coordinate the developmental program of Dictyostelium. A cAMP receptor (cAR1), which is expressed during early aggregation, has been cloned and sequenced previously. We have identified a new receptor subtype, cAR3, that has approximately 56% and 69% amino acid identity with cAR1 and cAR2, respectively. cAR1, cAR2, or cAR3 expressed from plasmid in growing Dictyostelium cells can be photoaffinity labeled with 8-N3[32P]cAMP and phosphorylated when stimulated with cAMP. cAR3 RNA was not present during growth but appeared during late aggregation. Its expression peaked at 9 hr and then fell to a reduced level that was maintained until culmination. The expression of cAR3 protein followed a similar pattern, but with a 3-hr lag, and reached a maximum at the mound stage. In contrast, cAR1 protein was expressed predominantly during early aggregation and at low levels during later stages. At their respective peaks of expression, there were approximately 5 x 10(3) cAR3 sites per cell compared with approximately 7 x 10(4) cAR2 sites per cell. The cAR3 gene was disrupted by homologous recombination in several different parental cell lines. Surprisingly, the car3- cell lines display no obvious phenotype.

Amino Acid Sequence↗

PubMed1993

Nitrogen limitation and nitrogen fixation during alkane biodegradation in a sandy soil.

We investigated nutrient limitations during hydrocarbon degradation in a sandy soil and found that fixed nitrogen was initially a limiting nutrient but that N limitation could sometimes be overcome by N2 fixation. Hydrocarbon biodegradation was examined in an unsaturated sandy soil incubated aerobically at 20 degrees C with propane or butane and various added nutrients. Propane and butane degradation proceeded similarly during the first 3 months of incubation. That is, bacteria in soil amended with N oxidized these hydrocarbons more rapidly than in controls without nutrient additions or in soil with added phosphate or trace minerals. Both propane- and butane-amended soil apparently became N limited after the initial available inorganic N was utilized, as indicated by a decrease in the rates of hydrocarbon degradation. After 3 months, propane and butane degradation proceeded differently. Bacteria in propane-degrading soil apparently remained N limited because propane degradation rates stayed low unless more N was added. In contrast, bacteria in butane-degrading soil appeared to overcome their N limitation because butane degradation rates later increased regardless of whether more N was added. Analyses of total N and acetylene reduction assays supported this apparent surplus of N in butane-amended soil. Total N was significantly (P < 0.01) higher in soil incubated with butane and no N amendments than in soil incubated with propane, even when the latter was amended with N. Acetylene reduction occurred only in butane-amended soil. These results indicate that N2 fixation occurred in butane-amended soil but not in propane-amended soil.

Acetylene↗

PubMed1993

Cardiopulmonary adaptations to pneumonectomy in dogs. II. VA/Q relationships and microvascular recruitment.

Ventilation-perfusion relationships, diffusing capacity for O2, and hemodynamic response were measured at rest and during exercise while five adult dogs breathed air and 15% O2 2 mo (Pnx-A, n = 2) or 12 mo (Pnx-B, n = 3) after right pneumonectomy (removal of 58% of lung tissue). Results were compared with those in five sham-operated controls. The multiple inert gas elimination technique was employed. Maximal O2 uptake was reduced by 50% in Pnx-A and by 15% in Pnx-B. Ventilation-perfusion matching was impaired in Pnx-A but not in Pnx-B. The increase in O2 diffusing capacity during exercise was significantly restricted in Pnx-A but was partially restored in Pnx-B. Mean pulmonary arterial pressure at a given blood flow through the remaining lung was normal in Pnx-A but lower than normal in Pnx-B compared with control values for a single lung. Stroke volume and cardiac output were lower in both Pnx-A and Pnx-B than in controls at a given exercise level. All functional abnormalities were more severe at 2 mo than at 12 mo postpneumonectomy. Gas phase diffusion resistance was present in both Pnx-A and Pnx-B but not in controls. We conclude that physiological compensation postpneumonectomy is progressive. Inability to recruit pulmonary vascular bed with incremental perfusion pressure or flow appears to be the major pathophysiological process that limits early functional capacity after right pneumonectomy.

Animals↗

PubMed1993

Structural changes underlying compensatory increase of diffusing capacity after left pneumonectomy in adult dogs.

To determine if the functional compensation in diffusing capacity of the remaining lung following pneumonectomy is due to structural growth, we performed morphometric analysis of the right lung in three adult foxhounds approximately 2 yr after left pneumonectomy (removal of 42% of lung) and compared the results to those in normal adult dogs previously studied by the same techniques. Diffusing capacity was calculated by an established morphometric model and compared to physiologic estimates at peak exercise in the same dogs after pneumonectomy. The major structural changes after left pneumonectomy are hyperinflation of the right lung, alveolar enlargement, and thinning of the alveolar-capillary tissue barrier. These changes confer significant functional compensation for gas exchange by reducing the overall resistance to O2 diffusion. The magnitude of compensation in diffusing capacity estimated either morphometrically or physiologically is similar. In spite of morphometric and physiologic evidence of functional compensation, there is no evidence of significant growth of structural components. After pneumonectomy, morphometric estimates of diffusing capacity are on average 23% higher than physiologic estimates in the same dogs at peak exercise. We conclude that the previously reported large differences between morphometric and physiologic estimates of diffusing capacity reflects the presence of large physiologic reserves available for recruitment.

Animals↗

PubMed1993

Relationship between body image and percent body fat among college male varsity athletes and nonathletes.

Three groups of students from a large midwestern university (15 varsity swimmers, 15 varsity football players, and 15 nonathletes) were administered a 20-item questionnaire concerned with body image and subsequently underwent skinfold measurements to assess percent body fat. Scores obtained were correlated to estimate the relationship between body image and adiposity. For the groups combined (N = 45) the relationship was significant and inverse (r = -0.51). For the nonathletic group a significant value -0.76 was found; for the two groups of athletes there was no relationship (rs = 0.002 and -0.059 for football players and swimmers, respectively). We conclude that body image as measured in this study in inversely related to percent body fat among college men, especially among students not engaged in varsity sports.

Adipose Tissue↗

PubMed1993

Effects of high- vs moderate-intensity exercise on natural killer cell activity.

The effect of 45 min of high- (80% VO2max) vs moderate- (50% VO2max) intensity treadmill exercise on natural killer cell cytotoxic activity (NKCA) was investigated in 10 well-conditioned (66.0 +/- 1.9 ml.kg-1.min-1), young males (22.1 +/- 1.3 yr). Blood samples were taken before and immediately after exercise, with three more samples taken during 3.5 h of recovery, and analyzed for proportion of NK cells (CD3-CD16+CD56+) and NKCA. Exercise at 80% vs 50% VO2max resulted in a greater immediate postexercise increase in proportion of NK cells, followed by a 1-h and 2-h decrease below preexercise levels for both intensity conditions. NKCA rose significantly above preexercise levels following high- but not moderate-intensity exercise. For both exercise intensity conditions, NKCA tended to drop below preexercise levels by 1 h postexercise, rising back to preexercise levels by 3.5 h postexercise. When NKCA was expressed on a per-NK cell basis, however, no change relative to preexercise levels occurred following moderate-intensity exercise, while a significant increase occurred after 2-h recovery from high-intensity exercise. These data demonstrate that both high- and moderate-intensity exercise are associated with significant shifts in circulating proportions of NK cells which significantly influence interpretation of NKCA data based on assays using separated mononuclear cells.

Adolescent↗

PubMed1993

Flow cytometry. From research to clinical laboratory applications.

Flow cytometry is a technology that has made a successful transition from the research laboratory to the clinical laboratory. There are numerous clinical applications of flow cytometry. In hematology, the major applications have been in leukocyte phenotyping for the diagnosis and classification of leukemias and lymphomas and enumeration of peripheral blood lymphocytes in immune disorders. Applications in the phenotyping and functional characterization of hematopoietic and lymphoid cells such as erythroid cells, including reticulocytes, platelets and megakaryocytes, monocytes/macrophages, and granulocytes, are likely to increase and demonstrate additional diagnostic and prognostic clinical utility.

Antibodies, Monoclonal↗

PubMed1992

Quisqualic acid analogues: synthesis of beta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site.

Hippocampal CA1 pyramidal cell neurons are sensitized over 30-fold to depolarization by L-2-amino-4-phosphonobutanoic acid (L-AP4) following exposure to L-quisqualic acid. This phenomenon has been termed the QUIS effect. In the present study several novel L-quisqualic acid analogues have been synthesized and tested for their interaction with the different components of the QUIS-effect system. Replacement of the oxadiazolidinedione ring of L-quisqualic acid with several other types of heterocyclic rings yielded the following quisqualic acid analogues: maleimide 2, N-methylmaleimide 3, N-(carboxymethyl)maleimide 4, succinimides 5A and 5B, and imidazolidinedione 6. None of these analogues were able to mimic the effects of L-quisqualic acid and sensitize hippocampal CA1 neurons to depolarization by L-AP4. Also, unlike L-serine O-sulfate, L-homocysteinesulfinic acid, or L-alpha-aminoadipic acid, none of the analogues were able to preblock or reverse the QUIS effect. However, when the IC50 values for inhibition of the CA1 synaptic field potential of analogues 2-6 were determined both before and after hippocampal slices were exposed to L-quisqualic acid, the IC50 values of analogues 3 and 4 were found to decrease more than 7-fold. Thus, these two compounds behave like L-AP4 rather than L-quisqualic acid in this system in that they exhibit increased potencies in slices that have been pretreated with L-quisqualic acid even though they cannot themselves induce this sensitization. Compounds 3 and 4, therefore, represent the first non-phosphorus-containing compounds to which hippocampal neurons become sensitized following exposure to L-quisqualic acid. No change in the IC50 values was observed for 5A or 5B. Analogues 2 and 6, on the other hand, displayed a high potency for inhibition of the evoked field potential even prior to treatment of the slices with L-quisqualic acid.

Aminobutyrates↗

PubMed1992

Studies of the catalytic activities and substrate specificities of Saccharomyces cerevisiae myristoyl-coenzyme A: protein N-myristoyltransferase deletion mutants and human/yeast Nmt chimeras in Escherichia coli and S. cerevisiae.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential, 455-residue, monomeric enzyme. Amino- and carboxyl-terminal deletion mutants of Nmt1p were genetically engineered to determine the minimal domain necessary to maintain catalytic activity. Enzyme activity was assessed by (i) sequentially inducing Nmt1p or its mutant derivatives and one of two eukaryotic substrates for the wild type enzyme (S. cerevisiae Gpa1p and rat Go alpha) in Escherichia coli, a bacterium with no endogenous myristoyltransferase activity, and monitoring Nmt-dependent incorporation of exogenous [3H]myristate into the G protein alpha subunits or (ii) an in vitro enzyme assay using lysates prepared from bacteria producing wild type or mutant Nmts. The data indicate that the minimal catalytic domain of Nmt1p is located between Ile59-->Phe96 and Gly451-->Leu455. Analyses of the ability of mutant nmtps to rescue the lethal phenotype of an nmt1 null allele in a haploid strain of yeast grown on rich media, with or without blockade of cellular fatty acid synthetase, suggest that the amino-terminal 59 residues of Nmt1p may play an important noncatalytic role, functioning as a targeting signal so this cytosolic enzyme can access cellular myristoyl-CoA pools generated from activation of exogenous C14:0 by acyl-CoA synthetase(s). Moreover, there appear to be differences in the location or accessibility of myristoyl-CoA pools derived from fatty acid synthetase and acyl-CoA synthetases. The E. coli co-expression system was used to map structural elements that determine differences in the peptide substrate specificities of Nmt1p and the orthologous human Nmt. Rat Go alpha is a substrate for both enzymes, whereas human Gz alpha is a substrate only for human NMT. Studies of a series of chimeric enzymes composed of elements from the amino- or carboxyl-terminal portions of human and yeast Nmts indicate that (i) recognition/utilization of Gz alpha involves elements distributed from the amino-terminal half through the region defined by Leu352-->Lys410 of the 416 residue human enzyme and (ii) formation of a fully functional peptide binding site and a fully functional myristoyl-CoA binding site in either of these enzymes requires contributions from both their amino-terminal and carboxyl-terminal halves.

Acyltransferases↗

PubMed1992

Structure-function relationships for analogues of L-2-amino-4-phosphonobutanoic acid on the quisqualic acid-sensitive AP4 receptor of the rat hippocampus.

Hippocampal CA1 pyramidal cell neurons are sensitized to depolarization by L-2-amino-4-phosphonobutanoic acid (L-AP4) following exposure to L-quisqualic acid (QUIS). We have examined the interaction of 43 structural analogues of L-AP4 with both the 'induction' site and the QUIS-sensitive AP4 site in rat hippocampus. The synthesis of cis- and trans-4-phosphonoxy-L-proline, 3-(RS)-amino-5-phosphonopentanoic acid and 2(RS)-amino-5-phenyl-4(RS)-phosphonopentanoic acid (gamma-benzyl AP4) are described. None of the test compounds interact with the induction site; thus L-QUIS remains the only compound known to induce this effect. However, one compound (L-2-amino-3-(5-tetrazolyl)-propanoic acid (L-aspartate tetrazole) 'pre-blocked' and reversed the effects of QUIS. In addition, the potency of 16 analogues increased more than 4-fold following exposure of slices to L-QUIS. Among these, L-AP4, L-AP5, 2-amino-4-(methylphosphino)butanoic acid (AMPB), and E-1(RS)-amino-3(RS)-phosphonocyclopentanecarboxylic acid (E-cyclopentyl AP4) displayed IC50 values of less than 0.100 mM after QUIS. The results presented here suggest that the QUIS-sensitive AP4 site requires a spatial configuration of functional groups similar to that present in E-cyclopentyl AP4. The presence of a primary amino group and a phosphorus-containing group (either monoanionic or dianionic) appear to be required, however, a carboxyl group is not essential for interaction. The pharmacology of the QUIS-sensitive AP4 site suggests that it is distinct from other known binding sites for L-AP4 in the central nervous system (CNS).

Aminobutyrates↗

PubMed1992

The cyclic nucleotide specificity of three cAMP receptors in Dictyostelium.

cAMP receptors mediate signal transduction pathways during development in Dictyostelium. A cAMP receptor (cAR1) has been cloned and sequenced (Klein, P., Sun, T. J., Saxe, C. L., Kimmel, A. R., Johnson, R. L., and Devreotes, P. N. (1988) Science 241, 1467-1472) and recently several other cAR genes have been identified (Saxe, C. L., Johnson, R., Devreotes, P. N., and Kimmel, A. R. (1991a) Dev. Genet. 12, 6-13; Saxe, C. L., Johnson, R. L., Devreotes, P. N., and Kimmel, A. R. (1991b) Genes Dev. 5, 1-8). We have expressed three receptor subtypes, cAR1, cAR2, and cAR3, in growing cells and have investigated their affinity and pharmacological specificity in a series of [3H]cAMP binding studies. In phosphate buffer, there were two affinity states of about 30 and 300 nM for cAR1 and 20 and 500 nM for cAR3 but no detectable affinity for cAR2. In the presence of 3 M ammonium sulfate, there was one affinity state of 4 nM for cAR1 and 11 nM for cAR2 and two affinity states of approximately 4 and 200 nM for cAR3. The relative affinities of 14 cyclic nucleotide derivatives were tested for each cAR in ammonium sulfate. These studies suggest a model (Van Haastert, P. J. M., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) in which cAMP binds to all three receptor subtypes by maintaining hydrogen bond interactions at the N6 and O3' positions. Interactions at the exocyclic oxygens of cAMP varied between the receptors; cAR2 and cAR3 lacked a stereoselective interaction at the axial oxygen which was present in cAR1. The cleft, which binds the adenine ring of cAMP, was hydrophobic in cAR1 and cAR3 but relatively polar in cAR2. The analog specificity of cAR1 and cAR3 in phosphate buffer was similar to that measured in ammonium sulfate though the derivatives' relative affinity to cAMP was reduced. We conclude that these cAMP receptor subtypes can be distinguished by distinct pharmacological properties which will allow selective activation of each cAR during development.

Amino Acid Sequence↗

PubMed1992

Activity of the conformationally rigid 2-amino-4-phosphonobutanoic acid (AP4) analogue (RS)-1-amino-3-(phosphonomethylene)cyclobutane-1-carboxylic acid (cyclobutylene AP5) on evoked responses in the perforant pathway of rat hippocampus.

The highly rigid and conformationally extended 2-amino-4-phosphonobutanoic acid (AP4) analogue (RS)-1-amino-3-(phosphonomethylene)-cyclobutane-1-carboxylic acid (cyclobutylene AP5) was synthesized and found to inhibit evoked responses in the rat lateral perforant path (LPP) with an IC50 of 41 (+/- 1.5 S.E.M.) microM and the medial perforant pathway with an IC50 of 218 (+/- 3.7 S.E.M.) microM. Furthermore, paired pulse potentiation experiments suggest that cyclobutylene AP5 acts, in part, at a presynaptic site in the LPP. Thus, cyclobutylene AP5 appears to act in a similar manner to L-AP4 in the perforant pathway. These data support the hypothesis that L-AP4 assumes an extended conformation at the L-AP4 receptor of the LPP.

Animals↗

PubMed1992

Capillaries measured in canine diaphragm by two methods.

We have measured capillary distribution in costal and crural canine diaphragm using two methods: histochemical processing and perfusion fixation. Each of 18 dogs was deeply anesthetized, the abdomen opened, and the left inferior phrenic artery cannulated. The animal was heparinized and overdosed with pentobarbital. The right hemidiaphragm was frozen, either postexcision (Protocol 1) or intact with no preload (Protocol 2), for histochemical processing. The left hemidiaphragm was fixed by perfusion in situ using 2% glutaraldehyde, either with preload (Protocol 1) or without (Protocol 2). Costal and crural regions of each hemidiaphragm were sampled for analysis. Frozen samples were sectioned and processed for acid-stable (pH 4.0) ATPase activity; perfusion-fixed samples were postfixed, stained, embedded in Epon, and sectioned. Measurements were made using a digital imaging system. We found that muscle fibers had smaller cross-sectional areas in costal than in crural diaphragm; capillary-to-fiber ratio (C:F) did not differ by region and regional differences in capillary density could be attributed to differences in fiber size. Results depended critically on methodology. In perfusion-fixed muscle, fiber area was less, C:F was greater, and capillary density was greater than in histochemically-processed tissue. We conclude that capillary distribution is similar in costal vs. crural diaphragm and that perfusion fixation identifies capillaries more effectively than histochemistry.

Animals↗