Dependence of cytoplasmic calcium transients on the membrane potential in isolated nerve endings of the guinea pig

The relation of changes in internal, free Ca2+, measured with arsenazo III, to the membrane potential, measured with the cyanine dye di-S-C2(5) or 86Rb+ distribution ratio, was studied in isolated guinea pig cortical nerve endings. Depolarization of the plasma membrane with veratridine or gramicidin as well as addition of ionophore A23187 led to an increase in cytosolic Ca2+. Only the response to veratridine was inhibited by tetrodotoxin. The dependence of the depolarization-induced increase in intraterminal, free Ca2+ on the membrane potential between about -50 to 0 mV was sigmoidal. A maximal increase in cytosolic Ca2+ was reached when the membrane potential was depolarized from the resting level, about -64 mV, to about -40 mV. These results show that in isolated nerve endings the activation of voltage-sensitive Ca2+ channels concomitantly leads to an increase in cytosolic, free Ca2+. Comparison of the results of the present study with the previous electrophysiological observations indicate that Ca2+ channels in synaptosomes, presynaptic nerve terminals of the squid giant synapse and cardiac cells have essentially similar voltage dependency.     

The Unique Chemistry of Eastern Mediterranean Water Masses Selects for Distinct Microbial Communities by Depth

The waters of the Eastern Mediterranean are characterized by unique physical and chemical properties within separate water masses occupying different depths. Distinct water masses are present throughout the oceans, which drive thermohaline circulation. These water masses may contain specific microbial assemblages. The goal of this study was to examine the effect of physical and geological phenomena on the microbial community of the Eastern Mediterranean water column. Chemical measurements were combined with phospholipid fatty acid (PLFA) analysis and high-throughput 16S rRNA sequencing to characterize the microbial community in the water column at five sites. We demonstrate that the chemistry and microbial community of the water column were stratified into three distinct water masses. The salinity and nutrient concentrations vary between these water masses. Nutrient concentrations increased with depth, and salinity was highest in the intermediate water mass. Our PLFA analysis indicated different lipid classes were abundant in each water mass, suggesting that distinct groups of microbes inhabit these water masses. 16S rRNA gene sequencing confirmed the presence of distinct microbial communities in each water mass. Taxa involved in autotrophic nitrogen cycling were enriched in the intermediate water mass suggesting that microbes in this water mass may be important to the nitrogen cycle of the Eastern Mediterranean. The Eastern Mediterranean also contains numerous active hydrocarbon seeps. We sampled above the North Alex Mud Volcano, in order to test the effect of these geological features on the microbial community in the adjacent water column. The community in the waters overlaying the mud volcano was distinct from other communities collected at similar depths and was enriched in known hydrocarbon degrading taxa. Our results demonstrate that physical phenomena such stratification as well as geological phenomena such as mud volcanoes strongly affect microbial community structure in the Eastern Mediterranean water column.     

Comparative assessment of inorganic pyrophosphate and pyrophosphatase levels of Escherichia coli, Clostridium pasteurianum, and Clostridium thermoaceticum

Abstract Pyrophosphatase (PP_iase) specific activities were much higher in anaerobic cultures of Escherichia coli (0.54 units) than in Clostridium pasteurianum (0.067 units) and Clostridium thermoaceticum (0.017 units) (1 unit = 1 μ mole PP_i hydrolyzed/min per mg cell dry wt.), and were fairly constant throughout the growth of all three organisms. Conversely, intracellular levels of pyrophosphate (PP_i) were very low and constant in E. coli throughout growth (0.3 mM), while those of C. pasteurianum and C. thermoaceticum were higher (1.44 and 0.8 mM, respectively) and peaked sharply during mid log-phase of growth. PP_iase and intracellular PP_i remained relatively constant in E. coli when grown aerobically or anaerobically, and when growth was in medium containing PP_i as the sole source of supplemental phosphorus.     

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach     

Occurrence of esterified triterpenoid alcohols in the leaves of Pilosella officinarum

The mature winter leaves of Pilosella officinarum coll. Schultz and Schultz contained 29 mg esterified triterpenoid alcohols (g dry weight)⁻¹, of which over 80% was esterified with long-chain (C16-C18) fatty acids. The major fatty acids were 16:0, 18:2ω6 and 18:3ω3 and their amounts varied according to the season and stage of leaf development. In mature leaves the amount of triterpenoid alcohols esterified with long-chain fatty acids varied only slightly with the season. The changes in the fatty acid proportions in late winter-early spring, however, indicated a turnover in the esterified triterpenoid alcohol pool. In late winter after the snow had melted a 50% decrease occurred in the amount of triterpenoid alcohols esterified with short-chain fatty acids. The amount of esterified triterpenoid alcohols in immature leaves was relatively low [4 mg (g dry weight)⁻¹].
In the mature leaves esterified triterpenoid alcohols were found in globules whose appearance varied greatly according to the season. The globules were partly dissolved in late winter, and this together with the activation of respiration, suggests that the globules function as short-term energy reservoirs. Relationships between the appearance of lipid globules and the amount of esterified triterpenoid alcohols are discussed.     

A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase.

Nebularine undergoes hydration at the active site of adenosine deaminase, in a reaction analogous to a partial reaction in the displacement of ammonia from adenosine by water, to generate an inhibitory complex that captures much of the binding affinity expected of an ideal transition-state analogue. Enzyme affinities of several compounds related to nebularine 1,6-hydrate, and to its stable analog 2'-deoxycoformycin, were compared in an effort to identify the structural origins of strong binding. Binding of the stable transition-state analog inhibitor 2'-deoxycoformycin was rendered 9.8 kcal/mol less favorable by removal of substituent ribose, 9.7 kcal/mol less favorable by inversion of the 8-hydroxyl substituent of the diazepine ring, and 10.0 kcal/mol less favorable by removal of atoms 4-6 of the diazepine ring. Binding of the unstable transition-state analog nebularine hydrate was rendered at least 9.9 kcal/mol less favorable by removal of the 6-hydroxyl group and 10.2 kcal/mol less favorable by removal of atoms 1-3 of the pyrimidine ring. In each case, the enzyme exhibited only modest affinity (Kd greater than or equal to 10(-2) M) for the "missing piece", indicating that incorporation of 2 binding determinants within a single molecule permits an additional 7-12 kcal/mol of intrinsic binding energy to be manifested as observed binding energy. These results are consistent with earlier indications that adenosine deaminase may use 10.5 kcal/mol of the intrinsic free energy of binding of the two substrates to place them in positions appropriate for reaction at the active site, overcoming the unfavorable entropy change of -35 eu for the equilibrium of 1,6-hydration of purine ribonucleoside and reducing the equilibrium constant for attainment of the transition state in deamination of adenosine. Thus, adenosine deaminase may achieve up to 8 orders of magnitude of its catalytic power by converting the nonenzymatic, bimolecular, hydration reaction to a monomolecular reaction at its active site. Several new 6-substituted 1,6-dihydropurine ribonucleosides, prepared by photoaddition of formate and by low-temperature addition of organolithium reagents to a derivative of purine ribonucleoside, exhibited Ki values of 9-1400 microM against adenosine deaminase, in accord with the active site's considerable tolerance of bulky leaving groups in substrates. Inhibition by one diastereomer of 6-carboxy-1,6-dihydropurine ribonucleoside was found to be time-dependent, progressing from a weakly bound to a more strongly bound complex.     

Serum high density lipoprotein cholesterol,alcohol, and coronary mortality in male smokers.

OBJECTIVE--To determine whether the increase in mortality from coronary heart disease with high concentration (> 1.75 mmol/l) of high density lipoprotein cholesterol could be due to alcohol intake. DESIGN--Cohort study. SETTING--Placebo group of the alpha tocopherol, beta carotene cancer prevention (ATBC) study of south western population in Finland. PARTICIPANTS--7052 male smokers aged 50-69 years enrolled to the ATBC study in the 1980s. MAIN OUTCOME MEASURES--The relative and absolute rates adjusted for risk factors for clinically or pathologically verified deaths from coronary heart disease for different concentrations of high density lipoprotein cholesterol with and without stratification for alcohol intake. Similar rates were also calculated for different alcohol consumption groups. RESULTS--During the average follow up period of 6.7 years 258 men died from verified coronary heart disease. Coronary death rate steadily decreased with increasing concentration of high density lipoprotein cholesterol until a high concentration. An increase in the rate was observed above 1.75 mmol/l. This increase occurred among those who reported alcohol intake. Mortality was associated with alcohol intake in a J shaped dose response, and those who reported consuming more than five drinks a day (heavy drinkers) had the highest death rate. Mortality was higher in heavy drinkers than in non-drinkers or light or moderate drinkers in all high density lipoprotein categories from 0.91 mmol/l upward. CONCLUSIONS--Mortality from coronary heart disease increases at concentrations of high density lipoprotein cholesterol over 1.75 mmol/l. The mortality was highest among heavy drinkers, but an increase was found among light drinkers also.     

A site-directed mutagenesis study on Escherichia coli inorganic pyrophosphatase. Glutamic acid-98 and lysine-104 are important for structural integrity, whereas aspartic acids-97 and -102 are essential for catalytic activity

Analysis of the conservation of functional residues between yeast and Escherichia coli inorganic pyrophosphatases (PPases) suggested that Asp-97, Glu-98, Asp-102, and Lys-104 are important for the action of E. coli PPase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K., & Cooperman, B. S. (1990) Biochim. Biophys. Acta 1038, 338-345]. We replaced these four residues by oligonucleotide-directed mutagenesis, giving variant PPases DV97, DE97, EV98, DV102, DE102, KI104, and KR104. PPase variants DV97, DV102, and KI104 had no enzyme activity, whereas PPase variants DE97, EV98, DE102, and KR104 had 22%, 33%, 3%, and 3% of the wild-type PPase activity, respectively. This suggests that Asp-97, Asp-102, and Lys-104 are essential for the catalytic activity of E. coli PPase. PPase variants DV98 and KR104 also had an increased sensitivity to heat denaturation; incubation of these mutant PPases at 75 degrees C for 15 min in the presence of 5 mM magnesium ion decreased the activity to 20% and 1%, respectively, of the initial value while 74% of the activity was observed with wild-type PPase. Furthermore, these thermolabile mutant PPases displayed the most profound conformational changes of the PPase variants examined, as demonstrated by the binding of the fluorescent dye Nile red that monitors the hydrophobicity of protein surfaces. Accordingly, Glu-98 and Lys-104 seem to be important for the structural integrity of E. coli PPase.     

Activity changes of inorganic pyrophosphatase of Streptococcus faecalis during batch culture

The state of inorganic pyrophosphatase (EC 3.6.1.1) from Streptococcus faecalis ATCC 8043 was studied in different phases of batch culture. The degree of inactivation (i.e. the ratio of activities observed before and after incubation at 37 degrees C without cysteine) was highest, and the degree of activation (i.e. the ratio of activities after and before incubation in the presence of cysteine) was lowest, in samples taken during the early-exponential growth phase. During the various phases of batch culture, the specific activity before incubation and the degree of inactivation changed in parallel, whereas the specific activity observed after incubation remained nearly constant. During the early-exponential phase of growth almost all the enzyme was in the high-activity form, whereas during the stationary phase the highly active and the less active forms existed in equal amounts. These findings suggest that inorganic pyrophosphatase in S. faecalis is synthesized constitutively and is primarily regulated at the level of activity.