Biomedical subjects
Publications and source records attributed to J H Johnson.
A new monobactam SQ 28,332 has been isolated from fermentations of a Flexibacter sp. and the structure 3 was deduced from its spectroscopic properties. SQ 28,332 exhibits weak antibacterial activity.
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A chloromethyl ketone derivative of lactic acid is a potent inhibitor of glycolysis of Ehrlich ascites tumor cells. It inhibited glycolysis of intact cells by about 50% at 200 microM (100 nmol/mg of protein) while cell-free extracts were inhibited 50% at 50 microM (50 nmol/mg of protein). N alpha-(p-Tosyl)-L-lysine chloromethyl ketone and N alpha-(p-tosyl)-L-phenylalanine chloromethyl ketone inhibited only slightly or not at all at this concentration. The inhibition was localized at the hexokinase and phosphofructokinase steps since these two enzymes added to an inactivated extract restored the glycolytic activity, whereas none of the other glycolytic enzymes did. In fact, addition of pyruvate kinase or lactate dehydrogenase, which stimulated glycolysis, resulted in a more pronounced inhibition. Glycolysis and hexokinase activities in extracts of Rous sarcoma virus transformed cells were considerably more sensitive to the inhibitor than the activities from normal chick embryo fibroblasts. Hexokinase from mouse brain required 50 times higher concentrations for inhibition than the enzyme from mouse Ehrlich ascites tumor cells. Yeast hexokinase was unaffected at all concentrations tested. Since 5,5'-dithiobis(2-nitrobenzoate) protected against the inhibition, the chloromethyl ketone appeared to inhibit by interaction with an essential SH group. A pronounced inhibition of protein kinase activity of plasma membranes of Ehrlich ascites tumor cells was observed in the presence of the chloromethyl ketone. As in the case of glycolysis, the chloromethyl ketone of lactic acid was a more potent inhibitor of protein kinase activity than several other chloromethyl ketones that were tested.
Blood samples were collected via jugular catheters from ovariectomized rats at 10-minute intervals for one hour before and two hours after microinjection of -.5 microliter of either saline vehicle or morphine sulfate (10 micrograms) into the dorsal raphe nucleus (DRN) or adjacent periaqueductal gray by means of chronically-implanted guide cannulae. LH was measured by radioimmunoassay and mean pre-injection and mean post-injection values were compared for each rat (t test) as well as for each treatment group (paired t test). Neither saline in DRN nor morphine at other sites significantly altered circulating LH. A significant decrease in LH was observed following injection of morphine into DRN. This effect of morphine was prevented by pre treatment of the animals with the narcotic antagonist naltrexone (10 mg/kg i.v.), indicating the involvement of opiate receptors. These results indicate that DRN is one site at which systemically-administered morphine might act, and suggest the possibility of participation of this mechanism in modulation of LH release by endogenous opioids.
A new cholesterol-lowering agent, surfomer (AOMA), has been developed that blocks cholesterol absorption and lowers plasma cholesterol in animals. To evaluate AOMA in man, we studied its effects on plasma cholesterol, cholesterol absorption, fecal excretion of cholesterol and its bacterial degradation products, coprostanol and coprostanone, and percent saturation of gallbladder bile with cholesterol in 20 individuals chosen for hyperlipidemia. These patients had low density lipoprotein cholesterol (LDL-C) of 215 +/- 29 mg/dl. Two dose levels of AOMA were compared (10.8 and 5.4 grams daily), each for 1 mo in a study that combined features of inpatient and outpatient investigation. AOMA was tolerated well by all volunteers. There was a statistically significant correlation between percent absorption and LDL-C in both the control and AOMA treated states. AOMA lowered mean plasma cholesterol and LDL-C by 9.1% and 12.9% at the high dose and by 6.4% and 8.3% at the low dose, respectively. Triglyceride (control = 223 +/- 58 mg/dl, treatment = 232 +/- 85 mg/dl), high density lipoprotein cholesterol (HDL-C: control = 50 +/- 11 mg/dl, treatment = 50 +/- 13 mg/dl), and other lipoprotein lipids were not affected. AOMA lowered cholesterol absorption by 25% on the high dose. For 18/20 patients there was a statistically significant (p less than 0.001) correlation (r = 0.74) between percent LDL-C reduction and percent absorption inhibition. For these patients, presumably, variable effectiveness of the agent in inhibiting absorption was the most important predictor of individual responsiveness although individual variation in other cholesterol regulatory mechanisms also played a role. Two other patients showed marked LDL-C reduction at unusually low levels of absorption inhibition. We also had the opportunity to compare the effects of AOMA with neomycin in 8 volunteers. Neomycin was 50% more effective in lowering LDL-C than AOMA; however, it was twice as effective in inhibition absorption as well. AOMA dramatically reduced fecal excretion of cholesterol bacterial conversion products; whereas cholesterol per se accounted for only 50% of total neutral steroid excretion in the control state, it accounted for 93% of steroid excretion when patients were administered 10.8 grams of AOMA daily. In four patients studied there was no adverse effect of AOMA on gallbladder saturation with cholesterol; in fact, the percent saturation tended to decrease with AOMA in these four patients.
A new polyelectrolyte was synthesized and evaluated for antitumor activity. The product is a derivative of ethylene/maleic anhydride copolymer of low molecular weight (Mn approximately equal to 1100). The anhydride groups were first converted to the half-amide, half-ammonium salt by reaction with ammonia. A percentage (14-25 wt %) of these groups was further converted to the imide by heating. The product, carboxyimamidate (Carbethimer, N-137) inhibited the growth of a number of solid tumors in vivo. Sensitive tumor models included Lewis lung carcinoma, Madison 109 lung carcinoma, M5076 ovarian tumor, colon carcinoma 26, B16 melanoma, and P815 mastocytoma. Activity was dose related between nontoxic dose levels of 300 and 2000 mg/kg ip.
Blood samples were collected via jugular catheters from ovariectomized rats at 10-min intervals for 1 h before and 2 h after microinjection of 0.5 microliter of either saline vehicle or morphine sulfate (10 micrograms) into the dorsal raphe nucleus (DRN) or adjacent periaqueductal gray by means of chronically-implanted guide cannulae. Prolactin was measured by radioimmunoassay, and mean preinjection and mean postinjection values were compared for each rat (t test) as well as for each treatment group (paired t test). Neither saline in DRN nor morphine in the surrounding periaqueductal gray significantly altered circulating prolactin. A significant rise in prolactin was observed following injection of morphine into DRN. This effect of morphine was prevented by pretreatment of the animals with the narcotic antagonist naltrexone (10 mg/kg i.v.), indicating the involvement of opiate receptors. These results indicate that DRN is one site at which systemically-administered morphine might act, and suggest the possibility of participation of this mechanism in modulation of prolactin release by endogenous opioids.
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Naloxone blockade of stimulation-produced analgesia in the rat is partial and variable. In the present study the effectiveness of the long-acting narcotic antagonist naltrexone is examined. Bipolar stainless steel electrodes were implanted in the dorsal raphe nucleus or ventral periaqueductal gray matter of male rats. Analgesia produced by electrical stimulation was tested by the tail flick method before and twenty min following the administration of saline or naltrexone. Saline administered IP failed to alter the analgesic response. Following naltrexone the degree of analgesia was reduced by a mean of 79% for IV injection (3.7 mg/kg) and by means of 26%, 52%, 81% and 83% for IP administration of 0.3, 1.0, 3.0 and 10 mg/kg, respectively. These results confirm the participation of opiate mechanisms in stimulation-produced analgesia, and indicate that, under certain circumstances, only opiate mechanisms are involved.
The capacity of haloperidol (Haldol) to block luteinizing hormone (LH) release and ovulation was investigated with particular reference to its effectiveness during the "critical period" on the afternoon of proestrus in normal, cycling female rats. Haldol was injected subcutaneously at 13.30 h in one of five dosage levels (0.125, 0.25, 0.5, 1.0, or 4.0 mg/kg), or at 09.30 h in a single dose of 1.0 mg/kg. Control animals received the corn oil vehicle at 13.30 or 09.30 h. Blood samples were taken by means of intra-atrial catheters which had been implanted on the day prior to Haldol treatment. Oviducts were examined for the presence of ova at 10.00 h on the day following the experiment (estrus). Haldol was found to be ineffective at 13.30 h when given at dosage levels which were shown by previous authors, and in the present experiment, to be capable of blocking LH release and ovulation after injection at 09.30 h. Although LH levels and the numbers of ova released were inversely related to the dose of Haldol administered at 13.30 h, well-defined LH surges were still present and the occurrence of ovulation was not significantly inhibited. Haldol was found to block LH release and ovulation, however, when injected at 13.30 h at the highest dosage level (4.0 mg/kg). The fact that low doses of Haldol blocked in the morning but not in the afternoon indicates that the morning blockade must be mediated by a secondary action of the drug which required a certain period of time to be effective. The blockade by the high dose of Haldol in the afternoon, however, was probably due to a direct action on LH releasing mechanisms.
Plasma immunoreactive cationic trypsin(ogen) levels were determined in 32 control subjects and 43 patients with varying degrees of pancreatic insufficiency including 35 with cystic fibrosis (CF) and eight with Shwachman's syndrome. In six CF infants less than 2 years of age, plasma trypsin(ogen) levels were significantly elevated (97.3 +/- 62.2 ng/ml) above the normal range for nine controls (7.0 +/- 5.9 ng/ml; P less than 0.025). Four of these infants had steatorrhea, three of whom had undetectable duodenal trypsin activity after stimulation with secretin-cholecystokinin. In two CF infants, molecular size fractionation by gel filtration of plasma followed by radioimmunoassay of the column fractions demonstrated that trypsinogen was the only immunoreactive species in the circulation. In contrast, in older CF patients with steatorrhea (mean age, 15.3 +/- 4.6 years), plasma cationic trypsin(ogen) levels were undetectable or low (1.1 +/- 1.7 ng/ml). This finding clearly distinguished them from older CF patients without steatorrhea (mean age, 14.3 +/- 3.9 years) in whom cationic trypsin(ogen) levels were significantly higher (23.3 +/- 17.6 ng/ml; P less than 0.01). The mean trypsin(ogen) concentration in the older CF patients without steatorrhea did not differ from the mean value for 23 normal subjects of similar age. Plasma cationic trypsin(ogen) levels in two Schwachman's patients with steatorrhea (0.19 and 0.86 ng/ml) were significantly lower than the values found in six Shwachman's patients without steatorrhea (5.9 +/- 2.3 ng/ml; P less than 0.025). Furthermore, in nine older CF patients and eight Schwachman's patients, circulating trypsin(ogen) levels were highly correlated with duodenal trypsin output after secretin-cholecystokinin stimulation (r = 0.946, P less than 0.01; r = 0.899, P less than 0.01, respectively). These results suggest that in CF infants high levels of circulating trypsin(ogen) persist even in those with Shwachman's syndrome, however, circulating trypsin(ogen) accurately reflects residual pancreatic function.
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Growth of Microccoccus lysodeikticus in the presence of pantoyl lactone brings about both qualitative and quantitative changes in cell membrane lipids. Significant amounts of the two major phospholipids (phosphatidylglycerol and diphosphatidylglycerol) are converted to lyso forms; the largest conversion occurs in the phosphatidylglycerol. In addition, amounts of several phospholipid fatty acids are changed. Physical alteration of the call membrane can be demonstrated using differential scanning calorimetry. Although growth and transport are significantly inhibited when pantoyl lactone is present, cells possessing altered call membrane phospholipds and phospholipid fatty acids, brought about by growth in the presence of pantoyl lactone, transport D-alanine, L-glutamic and L-aspartic acid normally when washed free of the pantoyl lactone.
The synthesis and some of the physical and biological characteristics of a new inhibitor of lactate transport are described. The inhibitor is isobutylcarbonyl lactayl anhydride (iBCLA). It is formed by the condensation of lactic acid and isobutylchloroformate. It inhibits lactate transport 50% at 0.5 microgram/mg of protein in both Ehrlich ascites tumor cells and human erythrocytes. In contrast, 15 microgram of iBCLA/mg of protein is required for 50% inhibition of phosphate transport in erythrocytes, and phosphate transport in Ehrlich ascites tumor cells is unaffected at levels as high as 50 microgram of iBCLA/mg of protein. A time-dependent and concentration-dependent reversal of lactate transport inhibition took place on exposure of iBCLA-treated Ehrlich ascites cells to hydroxylamine or dithiothreitol. These data, along with the observed sensitivity of the lactate transporter to sulfhydryl reagents [Spencer, T. L., & Lehninger, A. L. (1976) Biochem. J. 154, 405-414], suggest that iBCLA acylates an essential sulfhydryl group on the transporter. When glycolyzing Ehrlich ascites tumor cells were treated with concentrations of iBCLA sufficient for complete inhibition of lactate transport, intracellular lactate levels increased, intracellular pH and extra-cellular lactate levels decreased, and overall lactate production was inhibited.