Stereoisomers of tetrahydrothiamin pyrophosphate, potent inhibitors of the pyruvate dehydrogenase multienzyme complex from Escherichia coli

Tetrahydrothiamin pyrophosphate, an analogue of thiamin pyrophosphate (TPP) in which the thiazolium ring has been reduced to a thiazolidine ring, was prepared by borohydride reduction of TPP. It consists of four stereoisomers, comprising two diastereomers each of which is a racemic mixture, generated by the introduction of two chiral centers on the thiazolidine ring. The major and minor diastereomers were separated and inferred to be of the cis and trans configurations, respectively, from a study of the nuclear Overhauser effects in the 1H NMR spectrum of the simpler tetrahydrothiamin. Tetrahydro-TPP behaves as a mixture of potent inhibitors of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex from Escherichia coli. The site of binding is probably the TPP-binding site on E1, and the Kd for each of the four stereoisomers was estimated. The cis isomers of tetrahydro-TPP bind more tightly than does TPP, whereas the trans isomers appear to bind with about the same Kd as TPP. Sodium borohydride caused a rapid inhibition of E1 activity in the presence of TPP, believed to be due to reaction of borohydride with enzyme-bound TPP. The experiments are consistent with an enhancement of the reactivity of the thiazole ring of TPP when bound to the catalytic site of E1, which could be due to polarization of the greater than +N=C bond near a hydrophobic or positively charged region of the protein. A spontaneous reactivation occurred after the initial inhibition by borohydride, attributable to a weakly binding inhibitor, not tetrahydro-TPP, being formed at the catalytic site.     

Hydroxyurea retards the progression of G2 cells

Continuous treatment of monolayer cultures of Chinese hamster ovary (CHO) cells with 1.0 mM HU reduced the rate at which G2 cells entered mitosis. Although all cells in G2+M at the start of treatment eventually entered mitosis, early G2 cells, which were exposed to HU the longest, entered mitosis 45 min later than untreated G2 cells. This perturbation of G2 progression should be considered when employing HU as a synchronizing agent in mammalian cell populations.     

The availability of various manganese oxides to plants

Summary A number of oxides of manganese were prepared and their crystalline structures were indentified by X-rays. They were added to four soils on which plants suffered from manganese deficiency. Oxides of the structures manganite, manganous manganite, and pyrolusite cured manganese deficiency of oats and peas. All the successful oxides consisted of very small particles, as shown by electron micrographs.The amount of the oxides dissolved by aqueous quinol during an hour at room temperature was correlated with their ability to supply plants with manganese.     

A comparison of two methods for constructing evolutionary distances from a weighted contribution of transition and transversion differences

Since the initial work of Jukes and Cantor (1969), a number of procedures have been developed to estimate the expected number of nucleotide substitutions corresponding to a given observed level of nucleotide differentiation assuming particular evolutionary models. Unlike the proportion of different sites, the expected number of substitutions that would have occurred grows linearly with time and therefore has had great appeal as an evolutionary distance. Recently, however, a number of authors have tried to develop improved statistical approaches for generating and evaluating evolutionary distances (Schoniger and von Haeseler 1993; Goldstein and Polock 1994; Tajima and Takezaki 1994). These studies clearly show that the estimated number of nucleotide substitutions is generally not the best estimator for use in reconstruction of phylogenetic relationships. The reason for this is that there is often a large error associated with the estimation of this number. Therefore, even though its expectation is correct (i.e., on average the expected number of substitutions is proportional to time- -but see Tajima 1993), it is not expected to be as useful as estimators designed to have a lower variance.      

Prevalence of risk factors for cardiovascular disease in Canadians 55 to 74 years of age: results from the Canadian Heart Health Surveys, 1986-1992

BACKGROUND: By 2016, the proportion of Canadians older than 65 years of age will increase to 16%, and there will be an increase in the absolute number of cases of cardiovascular disease in older Canadians. The Canadian Heart Health Surveys database provides information about this population upon which health policy related to cardiovascular disease can be based. This paper presents for the first time population-based data on the risk factors for cardiovascular disease in older Canadians. METHODS: Canadians from all 10 provinces participated in surveys of cardiovascular risk factors; health insurance registries were used as sampling frames. In each province, probability samples of 2200 adults 18 to 74 years old not living in institutions, on reserves or in military camps were asked to participate in interviews and to undergo testing at clinics for major risk factors for cardiovascular disease. RESULTS: A total of 2739 men (response rate 70%) and 2617 women (response rate 66%) aged 55 to 74 years participated in the survey and also provided follow-up clinical measurements at the clinic. Overall, 52% of participants were hypertensive, 26% had isolated systolic hypertension, and 30% had a total blood cholesterol level of 6.2 mmol/L or greater. Rates of current smoking were lower in women than men (17% v. 22%). Overall, 87% of men and 78% of women who were current smokers smoked at least 10 cigarettes per day. Only slightly more than half of participants exercised at least once a week for at least 15 minutes, and almost half had a body mass index of 27 or greater. In only 4% was no major risk factor for cardiovascular disease detected. INTERPRETATION: Significant numbers of older Canadians have one or more major risk factors for cardiovascular disease. Many of these risk factors are amenable to modification.     

gamma-aminobutyric acid increases the water accessibility of M3 membrane-spanning segment residues in gamma-aminobutyric acid type A receptors

gamma-Aminobutyric acid type A (GABA(A)) receptors are members of the ligand-gated ion channel gene superfamily. Using the substituted cysteine accessibility method, we investigated whether residues in the alpha(1)M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from alpha(1)Ala(291) to alpha(1)Val(307). The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS(-) was assayed electrophysiologically. Cysteines substituted for alpha(1)Ala(291) and alpha(1)Tyr(294) reacted with pCMBS(-) applied both in the presence and in the absence of GABA. Cysteines substituted for alpha(1)Phe(298), alpha(1)Ala(300), alpha(1)Leu(301), and alpha(1)Glu(303) only reacted with pCMBS(-) applied in the presence of GABA. We infer that the pCMBS(-) reactive residues are on the water-accessible surface of the protein and that GABA induces a conformational change that increases the water accessibility of the four M3 residues, possibly by inducing the formation of water-filled crevices that extend into the interior of the protein. Others have shown that mutations of alpha(1)Ala(291), a water-accessible residue, alter volatile anesthetic and ethanol potentiation of GABA-induced currents. Water-filled crevices penetrating into the interior of the membrane-spanning domain may allow anesthetics and alcohol to reach their binding sites and thus may have implications for the mechanisms of action of these agents.     

Thermotolerance and intracellular pH in two Chinese hamster cell lines adapted to growth at low pH

As an in vitro model for the low extracellular pH (pH_e) which has frequently been observed in tumors, cell lines have been grown in a low-pH medium in order to allow cell adaptation to that milieu. Two Chinese hamster cell lines [Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa)] were compared, both of which acquired thermotolerance during 42°C heating in pH_e = 7.3 buffer, but not in pH_e = 6.7 medium unless grown at that pH long enough to become adapted. CHO cells, even when acutely acidified, showed higher intracellular pH (pH_i) values in a suspension assay than OvCa cells, which confirmed the danger of comparing absolute values of pH_i between cell lines. Despite this fundamental difference, relative changes in pH_i were similar in that both lines showed a higher pH_i in adapted than in unadapted cells, over the range of pH_e values tested. The upregulation of pH_i was statistically significant, but the two lines differed in the time frame over which adaptation occurred. OvCa cells acquired an enhanced ability to develop tolerance to 42° heat at pH_e = 6.7 in 4 days, but the CHO cells acquired this ability more progressively, achieving a maximum ability at approximately 100 days. In contrast, both lines were able to upregulate their pH_i within 4 hours of being exposed to pH 6.7 medium. A further indication of different biochemical mechanisms at work was the opposite effects seen on pH_i in the two cell lines upon the removal of extracellular CO₂/HCO⁻₃. The differential between adapted and unadapted OvCa cells was enhanced by removal of bicarbonate, whereas CHO cells seemed less stable and the data with greater scatter failed to show any difference between adapted and unadapted cells. © 1996 Wiley-Liss, Inc.