Methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A and its repressibility by serine

The methylenetetrahydrofolate dehydrogenase of the amethopterin-resistant strain Streptococcus faecium var. durans A(k) was purified 100-fold. Because it is extremely labile, this enzyme required protection by 1 mm nicotinamide adenine dinucleotide phosphate (NADP(+)) during purification; 0.01 mm EADP(+) with 0.1% bovine plasma albumin stabilized the purified enzyme during storage at -20 C. Although the enzyme has properties of sulfhydryl enzymes, thiol compounds were not stabilizers. Oxidation of methylenetetrahydrofolate, catalyzed by the purified enzyme preparation, is NADP(+)-specific and yields methenyltetrahydrofolate and the reduced pyridine nucleotide. K(m) values for NADP(+) and for 5,10-methylenetetrahydrofolate (prepared as the formaldehyde adduct of biologically synthesized l,l-tetrahydrofolate) were calculated to be 0.021 and 0.026 mm, respectively. Neither purine bases and their derivatives nor serine inhibited the reaction. In growing cultures, the differential rate of synthesis of the methylenetetrahydrofolate dehydrogenase was dependent upon the composition of the medium. A medium which contained acid-hydrolyzed casein, and thus an exogenous source of serine, was repressive for this enzyme. In a serine-free, completely defined medium, the amount of folate added (for serine synthesis de novo) affected the duration of the initial exponential growth phase. At the termination of this phase, which primarily reflected the onset of a decreased rate of serine biosynthesis, synthesis of the methylenetetrahydrofolate dehydrogenase was derepressed. Exogenous serine in the completely defined medium prevented the derepression. Furthermore, physiological concentrations of l-serine were repressive not only for the dehydrogenase but also for the methenyltetrahydrofolate cyclohydrolase and the serine hydroxymethyl-transferase. Concomitantly, the differential rate of synthesis of the formyltetrahydrofolate synthetase of S. faecium var. durans A(k) was increased. Apparently, serine regulates the differential rates of syntheses of these enzymes.     

Behavioral treatment of isolated systolic hypertension in the elderly

Fifteen hypertensive patients were recruited from a geriatric medicine clinic for a research project designed to evaluate a Behavioral Stepped-Care treatment program of high blood pressure (HBP). All patients met the selection criteria of the Isolated Systolic Hypertension (ISH) in the Elderly (SHEP) clinical trial. During baseline, subjects recorded BP at home 9 times/day (3 times each, shortly after awakening, during the middle of the day, and within an hour of retiring) for 1 month and mailed that data to us daily. In addition, they came to the clinic weekly and had their BP recorded by a nurse. During treatment 1, systolic (SBP) feedback, they were trained to lower SBP at home using their sphygmomanometers. They also continued to monitor BP and to obtain weekly professional BP readings. During treatment 2 (relaxation), they were trained to relax; they followed the self-administration and data-collection protocol as in treatment 1. Each treatment phase lasted 3 months. Average monthly self-determined BP fell significantly from 166.4/85.8 (SBP/DBP) mm Hg during baseline to 153.3/81.2 by the end of the relaxation phase; average monthly professionally measured BP fell significantly, from 164.7/87.1 to 156.9/81.5. These findings show that a nurse-supervised, patient-administered behavioral treatment program of ISH can yield sustained, significant falls in BP.Ms. Pearce and Dr. Burton were supported in part by the Johns Hopkins Academic Nursing Home Award, PO, AG04402, from the National Institute on Aging. This material was presented in part at the annual meeting of the Gerontological Society of America, November 1988, San Francisco.     

ATP-Evoked Ca2+ Mobilisation and Prostanoid Release from Astrocytes: P2-Purinergic Receptors Linked to Phosphoinositide Hydrolysis

Astrocyte cultures prelabelled with either [3H]inositol or 45Ca2+ were exposed to ATP and its hydrolysis products. ATP and ADP, but not AMP and adenosine, produced increases in the accumulation of intracellular 3H-labelled inositol phosphates (IP), efflux of 45Ca2+, and release of thromboxane A2 (TXA2). Whereas ATP-stimulated 3H-IP accumulation was unaffected, its ability to promote TXA2 release was markedly reduced by mepacrine, an inhibitor of phospholipase A2 (PLA2). ATP-evoked 3H-IP production was also spared following treatment with the cyclooxygenase inhibitor, indomethacin. We conclude that ATP-induced phosphoinositide (PPI) breakdown and 45 Ca2+ mobilisation occurred in parallel with, if not preceded, the release of TXA2. Following depletion of intracellular Ca2+ with a brief preexposure to ATP in the absence of extracellular Ca2+, the release of TXA2 in response to a subsequent ATP challenge was greatly reduced when compared with control. These results suggest that mobilisation of cytosolic Ca2+ may be the stimulus for PLA2 activation and, thus, TXA2 release. Stimulation of alpha 1-adrenoceptors also caused PPI breakdown and 45 Ca2+ efflux but not TXA2 release. The effects of ATP and noradrenaline (NA) on 3H-IP accumulation were additive, but their combined ability to increase 45Ca2+ efflux was not. Interestingly, in the presence of NA, ATP-stimulated TXA2 release was reduced. Our data provide evidence that functional P2-purinergic receptors are present on astrocytes and that ATP is the first physiologically relevant stimulus found to initiate prostanoid release from these cells.     

A method of remote physiological monitoring of a fully mobile primate in a single animal cage

A system was designed to allow the physiological monitoring of a fully mobile, unstressed baboon (Papio anubis) in a single animal cage for the purpose of measuring the changes occurring in a hyperbaric environment. It was required to operate for at least three months, both inside a pressure chamber and outside, and to measure the following parameters: electroencephalogram (EEG, three channels), electrooculogram (EOG), electromyelogram (EMG, two channels), electrocardiogram (ECG), arterial blood pressure, respiration and body temperature. Also in the system were catheters through which blood samples could be taken and intravenous drugs given. The overall system consisted of a harness and jacket, an umbilical and back pack, a combined electrical and fluid transmission swivel and a monitoring implant and catheters.     

Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation.

Antibody against purified CYP2A1 recognizes two rat liver microsomal P450 enzymes, CYP2A1 and CYP2A2, that catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone, respectively. In human liver microsomes, this antibody recognizes a single protein, namely CYP2A6, which catalyzes the 7-hydroxylation of coumarin. To examine species differences in CYP2A function, liver microsomes from nine mammalian species (rat, mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and human) were tested for their ability to catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone and the 7-hydroxylation of coumarin. Antibody against rat CYP2A1 recognized one or more proteins in liver microsomes from all mammalian species examined. However, liver microsomes from cat, dog, cynomolgus monkey, and human catalyzed negligible rates of testosterone 7 alpha- and/or 15 alpha-hydroxylation, whereas rat and cat liver microsomes catalyzed negligible rates of coumarin 7-hydroxylation. Formation of 7-hydroxycoumarin accounted for a different proportion of the coumarin metabolites formed by liver microsomes from each of the various species examined. 7-Hydroxycoumarin was the major metabolite (greater than 70%) in human and monkey, but only a minor metabolite (less than 1%) in rat. The 7-hydroxylation of coumarin by human liver microsomes was catalyzed by a single, high-affinity enzyme (Km 0.2-0.6 microM), which was markedly inhibited (greater than 95%) by antibody against rat CYP2A1. The rate of coumarin 7-hydroxylation varied approximately 17-fold among liver microsomes from 22 human subjects. This variation was highly correlated (r2 = 0.956) with interindividual differences in the levels of CYP2A6, as determined by immunoblotting. These results indicate that CYP2A6 is largely or entirely responsible for catalyzing the 7-hydroxylation of coumarin in human liver microsomes. Treatment of monkeys with phenobarbital or dexamethasone increased coumarin 7-hydroxylase activity, whereas treatment with beta-naphthoflavone caused a slight decrease. These results suggest that environmental factors can increase or decrease CYP2A expression in cynomolgus monkeys, which implies that environmental factors may be responsible for the large variation in CYP2A6 levels in humans, although genetic factors may also be important. In contrast to rats and mice, the expression of CYP2A enzymes in cynomolgus monkeys and humans was not sexually differentiated. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied approximately 5-fold among the human liver microsomal samples, but this variation correlated poorly (r2 = 0.126) with the variation observed in CYP2A6 levels and coumarin 7-hydroxylase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Metal pollution recorded in extinctDreissena polymorpha communities, Lake Breitling, Havel Lakes system, Germany: a laser ablation inductively coupled plasma mass spectrometry study

Shells of probable former living communities ofDreissena polymorpha were found within sediments of the shallow polytrophic to hypertrophic hard water Lake Breitling (Havel-Lake system, Germany). Corresponding sediments have been deposited between approximately 1940 and 1970 and reflect increasing eutrophication and heavy metal pollution of the Lake during this period (Schettler, 1992). Single shells from various sediment depths were analysed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) along a line on the outer part of the shell. The response of these freshwater mussels to increasing heavy metal pollution is clearly reflected in the distribution of Pb, Cu, Cd and Zn within their valves. In general, Cd, Cu, Pb and Zn contents are lower, and the distribution more even, in the outer parts of the deepest (oldest) shells compared to shells from higher in the cored sediments. Notably higher contents of Cu, Pb and Zn were recorded from the central (umbonal) part of the more recent shells, but this behaviour is not recorded for Cd. Metabolic changes brought on by worsening environmental conditions are proposed to explain this phenomena. Acidity produced during anaerobic metabolism can be neutralised by dissolution of the carbonate part of the shell. Copper, Zn and Pb, which show an affinity for the organic component of the shell, may thus accumulate by repeated dissolution and reprecipitation of the shell during the lifetime of an individual organism. Cadmium, which is bound mainly in the aragonite of the shells, is released during the dissolution of carbonate and is not concentrated in the umbonal area of the shell.     

Enhanced stability in vivo of a thermodynamically stable mutant form of yeast iso-1-cytochrome c

Previous work has established that the N57I amino acid replacement in iso-1-cytochrome c from the yeast Saccharomyces cerevisiae causes an unprecedented increase in thermodynamic stability of the protein in vitro, whereas the N57G replacement diminishes stability. Spectrophotometric measurements of intact cells revealed that the N57I iso-l-cytochrome c is present at higher than normal levels in vivo. Although iso-1-cytochrome c turnover is negligible during aerobic growth, transfer of fully derepressed, aerobically grown cells to anaerobic growth conditions leads to reduction in the levels of all of the cytochromes. Pulsechase experiments carried out under these anaerobic conditions demonstrated that the N57I iso-l-cytochrome c has a longer half-life than the normal protein. This is the first report of enhanced stability in vivo of a mutant form of a protein that has an enhanced thermodynamic stability in vitro. Although the N57I protein concentration is higher than the normal level, reduced growth in lactate medium indicated that the specific activity of this iso-l-cytochrome c in vivo is diminished relative to wild-type. On the other hand, the level of the thermodynamically labile N57G iso-1-cytochrome c was below normal. The in vivo levels of the N57I and N57G iso-l-cytochrome c suggest that proteins in the mitochondrial intermembrane space can be subjected to degradation, and that this degradation may play a role in controlling their normal levels.     

Seven loci on human Chromosome 4 map onto sheep Chromosome 6: a proposal to restore the original nomenclature of this sheep chromosome

Seven new loci, casein alpha-S1 (CSN1S1), casein alpha-S2 (CSN1S2), casein beta (CSN2), the Hardy-Zuckerman 4 feline sarcoma viral (v-kit) oncogene homolog (KIT), albumin (ALB), phosphodiesterase cyclic GMP (rod receptor) beta polypeptide (PDEB), and complement component 1 (IF), were assigned to sheep Chromosome (Chr) 6 by Southern hybridization to a panel of chromosomally characterized sheep x hamster cell hybrids. By isotopic in situ hybridization, CSN2 was regionally localized to sheep Chr (OOV) 6q22–q31, anchoring this syntenic group of markers on to OOV6 and confirming its homology at a molecular and cytological level with cattle Chr 6. The assignment of these loci, from PDEB (located on human Chr 4p16.3) to IF (on HSA4q24–q25), and the observation that interleukin 2 (IL2, on HSA 4q26–q27) and tryptophan 2,3-dioxygenase (TDO2, on HSA4q31) are not located on OOV6, is further evidence of the close evolutionary relationship of sheep and cattle and the conserved synteny in these species of this extensive region of human Chr 4. On the basis of this conserved synteny, and the similar G- and Q-banding patterns of this chromosome in cattle and sheep, we propose that this sheep chromosome be numbered as 6, not 4 as recommended by ISCNDA (1990).     

The Chlamydia trachomatis Mip-like protein is a lipoprotein.

The Mip-like protein of Chlamydia trachomatis is similar to the Mip protein of Legionella pneumophila and may be equally important for the initiation of intracellular infection. This article presents data which identify the chlamydial Mip-like protein as a lipoprotein. The amino acid sequence of the Mip-like protein contains a signal peptidase II recognition sequence, as is seen in procaryotic lipoproteins. Palmitic acid was incorporated into the recombinant chlamydial Mip-like protein. Globomycin, known to inhibit signal peptidase II, inhibited processing of the recombinant Mip-like protein. Labelling of chlamydial organisms with palmitic acid revealed incorporation into the native Mip-like protein.