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Biomedical subjects

J D Johnson

Publications and source records attributed to J D Johnson.

At least 199 records · Page 11Linked to original sources
PubMed1988

Properties of the insulin receptor ectodomain.

To study the properties of the extracellular insulin-binding domain of the human insulin receptor (hIR), we have expressed portions of the parent molecule in mammalian cells. Receptor cDNAs encoding the entire hIR ectodomain, the alpha subunit of the hIR alone, or a portion of the alpha subunit containing the cysteine-rich region were placed within an expression vector and in turn used to transfect CHO cells. Only cells expressing mRNA for the entire hIR ectodomain secreted hIR-related protein, suggesting that the truncated versions of this domain are unstable. The ectodomain molecules were extensively glycosylated, properly processed heterotetramers. Further, they bound insulin with an affinity similar to that of the intact hIR. In the electron microscope the secreted ectodomains appeared as discrete globular structures. After incubation with roughly equimolar quantities of insulin, the ectodomains associated to form loops or branched and folded linear macroarrays. However, these structures were not restricted to the specific ligand, insulin, since epidermal growth factor also produced the effect. Nevertheless, it seems that the receptor ectodomains can exist in two structural states. The conversion of the singular to the aggregated state may somehow be associated with transmembrane communication and activation of the biological response.

Animals↗
PubMed1988

Protein turnover in tissues of the fetal rat after prolonged maternal malnutrition.

Protein turnover in fetal diaphragm, heart, liver, and brain was determined at 21 days gestation in fetal rats whose mothers had received a protein-restricted diet (6% protein) throughout gestation. This diet resulted in severe combined protein-calorie malnutrition. Fetal body weight was significantly decreased at days 19-22 gestation versus controls (27% protein diet) when pregnant animals were protein-restricted (e.g. 40% decrease in body weight at day 22 gestation versus controls). Protein synthesis was determined by intravenous injection of "massive" amounts of [3H]phenylalanine to pregnant animals and measuring free and protein-bound specific radioactivities in fetal tissues. Rate constants for protein degradation were calculated by subtracting fractional growth rate from protein synthesis. Fractional protein synthesis was reduced in diaphragm (0.26 versus 0.41 days-1), heart (0.41 versus 0.52 days-1), and liver (0.35 versus 0.89 days-1) in fetuses from malnourished mothers relative to controls. Similarly, fractional protein degradation was decreased in these fetal tissues-diaphragm (0.03 versus 0.06 days-1), heart (0.14 versus 0.18 days-1), and liver (0.25 versus 0.80 days-1). Reduced protein accretion during maternal malnutrition in these fetal tissues is secondary to decreased protein synthesis out of proportion to the concurrent reduction in protein degradation. Protein synthesis and degradation in fetal brain from malnourished mothers were not altered versus controls. These effects of malnutrition on protein turnover in fetal tissues throughout pregnancy in the rat correspond closely with the effect of protein restriction in young adult rats and the effect of protein-calorie malnutrition on whole body protein turnover in human infants.

Animals↗
PubMed1987

The 5'-flanking sequence of yeast tRNA(Leu3) genes enhances the rate of transcription from stable pre-initiation complexes.

A conserved sequence that enhances transcription is present at or near the 5'-end of genes which code for several abundant yeast tRNAs, one of which is tDNA(Leu3). Mutants with alterations in this region have been compared to the parent tDNA(Leu3) and a yeast tDNA(Ser) that normally lacks the conserved sequence. The apparent KM of the transcription apparatus is insensitive to 5'-flanking sequence variation. Replacement of the normal 5'-flanking sequence does result in 3-18 fold reductions in VMAX and a 7-13 fold loss of transcription in tests with competitor genes. A second template exclusion assay revealed that sequences upstream of the conserved region effect a 2 fold change in the ability of the genes to stably sequester a component(s) of the obligatory pre-initiation complex while positions -22 to +9 have little or no effect.

Base Sequence↗
PubMed1987

Suggested mechanism for the selective excretion of glucosylated albumin. The effects of diabetes mellitus and aging on this process and the origins of diabetic microalbuminuria.

In previous studies in the Sprague-Dawley rat, Williams and coworkers reported the phenomenon of selective urinary excretion of glucosylated albumin (editing, i.e., the percent glucosylation of urinary albumin is more than that of plasma albumin) by the mammalian kidney. Ghiggeri and coworkers subsequently found that the extent of editing is reduced in human diabetics. Moreover, the reduction in editing in diabetes correlates inversely with levels of microalbuminuria. We also find reduction in the extent of editing in diabetic humans. We find a striking inverse correlation not only with the magnitude of microalbuminuria but also with the extent of plasma albumin glucosylation. In contrast, we found little correlation between the reduction in editing and the duration of diabetes in human subjects. Stz induced diabetes in the Sprague-Dawley rat is associated with a striking and rapid reduction in editing which develops virtually with the same kinetics exhibited by the appearance of hyperglycemia. This loss of editing is rapidly reversed by daily administration of insulin but not by aldose reductase inhibitors. Mannitol infusion in anesthetized Wistar rats resulted in an increase in urine volume, GFR, and microalbuminuria, and was also accompanied by a marked reduction in editing. This reduction was rapidly reversed by a cessation of mannitol infusion. We propose here that glucosylated albumin (in contrast to unmodified albumin) is not reabsorbed by the proximal tubule, and thus, is preferentially excreted in the urine. We postulate that the increase in GFR which emerges as a consequence of increased plasma osmolality in diabetes mellitus delivers more albumin to the proximal tubule than can be reabsorbed. This results in a dilution of excreted glucosylated albumin molecules by excreted unmodified albumin, which appears as the early microscopic albuminuria of diabetes. Paradoxically, the fall in apparent editing is accompanied by an absolute increase in the total quantity of glucosylated albumin excreted. In contrast, we found that editing of glucosylated albumin by the normal kidney is found to gradually decline as a function of age without the appearance of microalbuminuria. This suggests that a different mechanism operates to produce the loss of editing seen with aging in man, and as clearly (but in a shorter absolute time intervals) in the Fischer-344 rat.

Adolescent↗
PubMed1987

Peroxidation of brain lipids following cyanide intoxication in mice.

Lipid peroxidation of brain lipids as determined by the conjugated diene method was observed in mice following administration of sublethal doses of potassium cyanide (KCN). Conjugated diene production was dose- and time-dependent; 10 mg/kg KCN produced detectable levels of conjugated dienes at 30 min post cyanide, whereas, 15 mg/kg produced marked levels of conjugated dienes over a 10-60-min period after KCN. Pretreatment of mice with either diltiazem (600 micrograms/kg, i.v.) or allopurinol (25 mg/kg, i.v.) blocked the generation of conjugated dienes. These results suggest lipid peroxidation of neuronal membranes play a role in cyanide intoxication and this action is related to altered regulation of neuronal calcium homeostasis and activation of xanthine oxidase.

Allopurinol↗
PubMed1987

A continuous fluorescence assay for cyclic nucleotide phosphodiesterase hydrolysis of cyclic GMP.

The fluorescent 2'-methylanthraniloyl derivative of cyclic GMP undergoes a 45% decrease in fluorescence when it is cleaved by brain phosphodiesterase in the presence of calmodulin. This fluorescence decrease is dependent upon calcium, calmodulin, and phosphodiesterase, and correlates well (r = 0.996) with the disappearance of substrate as monitored by high-performance liquid chromatography. The Kd values determined by this fluorescence method and HPLC suggest that cyclic GMP and its fluorescent derivative exhibit similar kinetic parameters in their hydrolysis.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1987

Alteration of cytosolic calcium levels in PC12 cells by potassium cyanide.

The effect of KCN on cytosolic Ca2+ levels was measured in PC12 cells using Quin II/AM, a fluorometric calcium indicator. The resting cytosolic Ca2+ concentration, 115.0 +/- 4.9 nm, increased gradually and steadily over a 30-min time period following addition of 10(-4), 10(-3), or 10(-2)M KCN to the cells. After 15 min, 10(-3) and 10(-2)M KCN produced a three- and sixfold increase in the cytosolic Ca2+ concentration, respectively. In K+-depolarized cells, KCN induced a more rapid rise of intracellular calcium than in cells treated with KCN or KCI alone. KCN and/or K+-induced accumulation of cytosolic Ca2+ was blocked when the cells were pretreated with 10(-5)M diltiazem (a calcium channel blocker). These results demonstrate in a cell model that cyanide induces an accumulation of cytosolic Ca2+ and this additional cytosolic Ca2+ load appears to originate primarily from the extracellular compartment. This study supports previous reports implicating calcium as an intracellular mediator of cyanide toxicity.

Animals↗
PubMed1987

The interaction of felodipine with calcium-binding proteins.

Felodipine is unique among the dihydropyridine calcium antagonists in that it is the most potent in relaxing porcine coronary arteries (IC50 = 1.5 X 10(-10) M); it is not as sensitive to photoinactivation as nifedipine and nisoldipine, and it is fluorescent. The fluorescence of felodipine has allowed us to study many aspects of its interaction with various calcium-binding proteins in muscle, including calmodulin, skeletal troponin C, and cardiac troponin C. Calcium binding to the calcium-specific regulatory sites on these proteins exposes allosterically related felodipine-binding sites. The binding of other calmodulin antagonists and calcium antagonists, including prenylamine, R24571, and diltiazem, to these calcium-binding proteins abolishes the cooperativity between two felodipine-binding sites, resulting in felodipine binding to the remaining site with a 20-25-fold greater affinity. In addition, the binding of high-affinity drugs to these calcium-dependent hydrophobic sites on these calcium-binding proteins produces dramatic increases (40-50-fold) in their affinity for calcium. The affinity of felodipine for these calcium-binding proteins is 100-1,000 times lower than felodipine's IC50 for relaxing tension in coronary arteries, indicating that these calcium-binding proteins are probably not the primary receptors for felodipine. Similarities between the binding of dihydropyridines to the calcium channel and to these calcium-binding proteins have led us to suggest that a "calmodulin-like" calcium-binding protein on the calcium channel is the actual pharmacological receptor for dihydropyridine calcium channel antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Effect of patent ductus arteriosus on gentamicin pharmacokinetics in very low birth weight (less than 1,500 g) babies.

The effect of patent ductus arteriosus (PDA) and its therapy on the pharmacokinetic disposition of gentamicin in very low birth weight neonates was studied. Twenty-four neonates weighing less than 1,500 g with PDA were compared to 16 patients without PDA. Patients with PDA had significantly greater apparent volumes of distribution (0.64 +/- 0.20 vs. 0.41 +/- 0.08 l/kg; p less than 0.001) and serum half-life (8.49 +/- 2.69 vs. 6.23 +/- 1.92 h; p less than 0.01). The total body clearance was not significantly different between the two groups (56 +/- 20 ml/kg/h with PDA vs. 50 +/- 24 ml/kg/h without PDA). In addition, 7 patients were studied before and after PDA closure. The apparent volume of distribution fell in every case (p = 0.02). A statistically significant effect on clearance and half-life was not found. These findings suggest that PDA increases extracellular fluid volume, but does not affect glomerular filtration rate adversely. Additionally, serum gentamicin concentrations must be reevaluated after PDA closure in premature neonates.

Ductus Arteriosus, Patent↗
PubMed1987

Effect of the topical application of some newer antibiotics on the cerebral cortex.

Five currently used antibiotics--moxalactam, cefotaxime, cefoperazone, metronidazole, and piperacillin--and bacitracin were compared for epileptogenetic effect against penicillin G as a standard when applied directly to cat cerebral cortex. Piperacillin was half as epileptogenic as penicillin. Moxalactam, cefotaxime, and cefoperazone showed epileptogenicity one-fourth to one-eighth that of penicillin on a milligram per cubic centimetre basis. Neither metronidazole nor bacitracin produced any focal seizure activity at the highest concentrations tested. Some of the newer cephalosporin antibiotics may have a significant risk of inducing seizure activity if high concentrations come in direct contact with neocortical tissue.

Animals↗
PubMed1986

Pertussis toxin inhibits retinoic acid-induced expression of tissue transglutaminase in macrophages.

Retinoic acid rapidly induces the accumulation of a specific enzyme, tissue transglutaminase (EC 2.3.2.13), in mouse macrophages. We have used the induction of tissue transglutaminase to study the regulation of gene expression by retinoic acid. In this study we report that pertussis toxin can inhibit retinoic acid-induced expression of tissue transglutaminase in mouse resident peritoneal macrophages. This inhibition is paralleled by the ADP-ribosylation of 41,000-dalton macrophage membrane protein.

Animals↗
PubMed1986

The in vitro effects of methotrexate on the phagocytosis and intracellular killing of Staphylococcus aureus by human neutrophils.

The in vitro effect of therapeutic concentrations of methotrexate on the phagocytosis and intracellular killing of Staphylococcus aureus by circulating human neutrophils was assessed. Neutrophils were isolated from whole blood of six healthy human volunteers by density centrifugation and incubated with 10(-3) M methotrexate. Staphylococcus aureus was opsonized in human serum and added to the prepared neutrophils. Phagocytosis was determined by serial dilutions and plating of unphagocytized bacteria. After treatment with lysostaphin and trypsin, intracellular killing of bacteria was determined at set intervals by serial dilutions and platings of viable bacteria released by neutrophil lysis. Methotrexate-treated neutrophils phagocytized 56% and control neutrophils 67% of bacteria in 15 minutes (P greater than 0.5), and 96% of ingested bacteria were killed in 15 minutes by both populations. In these experiments, the in vitro incubation of circulating human neutrophils with methotrexate produced no significant alterations in neutrophilic phagocytosis or intracellular killing of S. aureus.

Humans↗
PubMed1986

Localization of a felodipine (dihydropyridine) binding site on calmodulin.

The fluorescent dihydropyridine calcium antagonist drug felodipine binds to calmodulin (CaM) in a Ca2+-dependent manner. Its binding can be regulated by the interaction of CaM antagonist drugs through allosteric mechanisms [Mills, J.S., & Johnson, J.D. (1985) Biochemistry 24, 4897]. Here, we have examined the binding of a nonspecific hydrophobic fluorescent probe molecule TNS (toluidinylnaphthalenesulfonate) and of felodipine to CAM and several of its proteolytic fragments. While TNS interacts with sites on both the amino-terminal half of the protein [proteolytic fragment TR1C (1-77)] and carboxy-terminal half [proteolytic fragment TR2C (78-148)], felodipine binding shows more selectivity. It binds in a Ca2+-dependent manner to the proteolytic fragments TM1 (1-106) and TR2E (1-90) but exhibits only weak affinity for TR1C (1-77) and TR2C (78-148). Furthermore, felodipine exhibits selectivity over TNS and trifluoperazine (TFP) in blocking the tryptic cleavage between residues 77 and 78. These studies indicate a selective binding of felodipine to a hydrophobic site existing in residues 1-90 and suggest that productive binding requires amino acids in the region 78-90. Although the felodipine binding site is preserved in fragment 1-106, the allosteric interactions between the prenylamine and the felodipine binding sites that are observed with intact CaM are not observed in this fragment. Rather, prenylamine simply displaces felodipine from its binding site on this fragment. Our results are consistent with calmodulin containing not less than two allosterically related hydrophobic drug binding sites. One of these sites (felodipine) appears to be localized in region 1-90 and the other one in region 78-148.

Animals↗
PubMed1986

Stereochemistry and function of oxaloacetate keto-enol tautomerase.

Oxaloacetate keto-enol tautomerase, partially purified from porcine kidney, catalyzes the conversion of enol- to keto-oxaloacetate by a mechanism in which solvent protons end up equally distributed between the two prochiral positions at C3 of keto-oxaloacetate. This conclusion is based upon the observation that when enzyme catalyzed ketonization is conducted in 3H2O in the presence of excess malate dehydrogenase and NADH, only 50% of the 3H in the isolated (2S)-[3-3H]malate is labilized to solvent upon treatment with fumarase. From a stereochemical perspective, this enzyme is unlike phenylpyruvate keto-enol tautomerase that is known to catalyze stereospecific proton transfer between solvent and the pro-R position of keto-substrate. As a result of an attempt to clarify the physiological importance of oxaloacetate tautomerase activity, keto-oxaloacetate was demonstrated to be directly transported across the inner membrane of rat liver mitochondria on the basis of the results of kinetic and isotope-trapping experiments.

Animals↗
PubMed1986

Exaggerated jaundice in Navajo neonates. The role of bilirubin production.

We have explored further the mechanism of exaggerated neonatal hyperbilirubinemia in the Navajo by determining bilirubin production rates with measurements of endogenous carbon monoxide excretion. Navajo newborns had elevated serum bilirubin concentrations and endogenous carbon monoxide excretion measurements at 2 days of age compared with a Caucasian control population. These findings could not be explained by differences in gestational age, postnatal age, or hemoglobin concentration. We conclude that accentuated bilirubinemia in Navajo neonates is partially due to increased bilirubin production.

Bilirubin↗