Thermodynamic study of the formation of adenine nucleotide-manganese complexes. I. "pH stat" titration method results.

The thermodynamics associated with the Mn2- -ATP, Mn-ADP- and Mn-AMP complex formation reactions determined from K potentiometric measurements at I = 0.2 are reported for the temperature range 1--45 degrees C. The K values increase with the length of the phosphate chain and with temperature. The limits and the best conditions for use of the "pH stat" titration method are discussed. Comparison with the results obtained by potentiometric and calorimetric methods in the case of Mg-nucleotide complexes is made.     

Quantitation of cellular deoxyribonucleic acid by flow microfluorometry.

This report characterizes for the first time an easy, reproducible means of standardizing the relative fluorescent units normally reported for flow microfluorometry. Absolute values for deoxyribonucleic acid/cell are obtained by using nucleated red blood cells as references. Cell were selected and characterized for the quantitative analysis of deoxyribonucleic acid per cell over a range from 2 pg/cell to 93 pg/cell using literature values for species having nucleated erythrocytes. Fluorescence staining by either acridine-orange (green wavelength) or propidium iodide (red wavelength) gave linear curves over the entire range investigated only when "gain controls" and current are optimized. The range was equivalent to mammalian cell values from 1 N (=3.5 pg deoxyribonucleic acid/cell) to 28 N (=91 pg deoxyribonucleic acid/cell). The standard curves obtained with nonmammalian erythrocytes were compared to mammalian free-cell preparations of bovine thymus and liver cells which fell at 6.8 and 6.9 pg deoxyribonucleic acid/cell, respectively. The routine use of these easily obtainable red blood cells will allow ready comparisons on the basis of absolute values for deoxyribonucleic acid per cell for work between experiments, work between staining procedures and dye types and work between laboratories.     

Variations in pO2 and pH response to hyperthermia: dependence on transplant site and duration of treatment.

It has been clearly established that changes in intratumor pO2 and pH occur following hyperthermia, and it has been hypothesized that these changes may, in some way, be related to the ultimate response (i.e., cure) of the lesion. The purpose of this study was twofold: first, to examine the changes in intratumor pH during the course of a hyperthermia treatment at biologically related end point "doses"; second, to examine the response of pO2 after treatment in a different lesion transplant site. During hyperthermia treatment of the tumor transplanted in the leg, intratumor pH was found to drop from a control value of 6.74 +/- 0.17 to 6.47 +/- 0.13 within 15 min following the start of treatment. The values then remained relatively constant throughout the remainder of the treatment (either 1 or 2 h at 43.5 degrees C). Following the subcurative (10% tumor cures at 30 days; 60 min at 43.5 degrees C) treatment the pH began to rise immediately, while after the higher dose (60% tumor cures at 30 days; 120 min at 43.5 degrees C) a slight rise in pH was followed by a continuous drop in pH for up to 4 h, as we have reported previously. Oxygen response in the two transplant sites (leg and flank) was found to be remarkably different even though the tumor cure rate was identical for a given hyperthermia "dose" in terms of time and temperature. In the leg, only very low levels of oxygen can be measured in the tumor 24 h after treatment with either "dose" studied (all measured pO2 values less than or equal to 5 mm Hg). In the flank, the tumor response is dependent on hyperthermia "dose." Only 28% of measured oxygen values are less than or equal to 5 mm Hg 24 h following a subcurative "dose," while 4 h following the higher "dose" there is a nonsignificant trend toward hypoxia (approximately 65% of values less than or equal to 5 mm Hg) with a subsequent shift toward reoxygenation. These latter observations are contrary to results reported previously and tend to contradict some current theories regarding the physiological mechanisms associated with hyperthermia treatment.     

A calorimetric determination of maintenance energy requirements during the aerobic growth of Klebsiella aerogenes in batch culture

Theoretical values for the maximum yield, heat output, conversion of substrate energy and number of generations possible for carbon-limited growth of K. aerogenes in batch culture are reported. Values for the maintenance energy and maintenance coefficients are reported for growth in glucose- and -methylglucoside - limited media.     

Lipid phase of transverse tubule membranes from skeletal muscle. An electron paramagnetic resonance study.

The lipid phase of transverse tubule membrane was probed with a variety of fatty acid spin labels. The motion of the probe increased as the distance between the spin label and polar head group increased, in agreement with results reported in other membranes. The value of the order parameter at 37 degrees C for a fatty acid spin label containing the label attached to its fifth carbon atom was closer to values reported for bacterial membranes than to the lower values reported for other mammalian membranes. Order parameters for spin labels containing the label nearer to the center of the bilayer were closer to the values reported in other mammalian membranes than to values reported for bacterial membranes. These results indicate that the lipid segments in the vicinity of the polar head group, and less so those near the center of the bilayer, are motionally more restricted in transverse tubules than in other mammalian membranes. In particular, the lipid phase of the transverse tubule membrane is less fluid than that of the sarcoplasmic reticulum membrane. A possible role of the high cholesterol content of transverse tubules in generating the lower fluidity of its lipid phase is discussed.     

Hydrogen ion binding properties of tuna haemoglobins.

Tunas are very active fish with a high aerobic capacity, but they also regularly perform burst swimming with massive production of lactic acid. The present study examines whether H(+) buffering by tuna haemoglobin (Hb) is elevated to cope with metabolic acidoses (by analogy with the high buffer capacity of tuna white musculature) or whether the Hb-H(+) binding properties resemble other teleosts that have low buffer values and high Haldane effects. H(+) titration of oxygenated and deoxygenated composite Hb from yellowfin tuna, skipjack tuna and bigeye tuna in 0.1 M KCl revealed low Hb-specific buffer values in all three tunas. Values at physiological pH were comparable to those reported in less active species such as carp and eel. The fixed acid Haldane effect was large (maximal uptakes of close to 4 mol H(+) per mol Hb tetramer upon deoxygenation). Thus, the Hb-H(+) binding properties of very active tuna species correspond to other teleosts. Low Hb buffer values may be a pre-requisite for the regulation of red blood cell pH via Na(+)/H(+) exchange. Approximately nine "neutral" groups were titratable in tuna Hbs, suggesting that two alpha-amino groups and seven histidine residues are titrated within each tetramer.     

A probable oxocarbonium ion in the reaction mechanism of small intestinal sucrase and isomaltase.

1-5-D-Gluconolactone is a competitive inhibitor of both sucrase and isomaltase. Substitution of the 1H and 2H at C1 of the glucosyl moiety in p-CL-phenyl-alpha-D-glucopyranoside leads to a decrease in kcat of both sucrase and isomaltase, the k1H/k2H ranging between 1.14 and 1.20. Treatment of the association constants and of the kcat values for a number of p-substituted phenyl-alpha-D-glucopyranosides on the basis of the Hammet-Hansch equation has allowed the estimation of the importance of hydrophilicity-hydrophobicity as well as of the magnitude of the p values for both substrate-enzyme interaction and catalysis in both sucrase and isomaltase. The magnitude of the secondary deuterium effect as well as the low values of p in both sucrase and isomlatase are strongly indicative of the rate-limiting step going through the formation of an oxocarbonium ion. In conjunction with other observations reported previously, the data presented here led to the suggestion of the main lines of a reaction mechanism for the two glucosidases: prptonation of the glycosidic oxygen is followed by the liberation of the "aglycone" with formation of an oxocarbonium ion, which is temporarily stabilized by a carboxylate group.     

Solution properties of synthetic polypeptides. 18. Helix-coil transition of poly-N5-(2-hydroxyethyl)L-glutamine

The helix–coil transition of poly-N⁵-(2-hydroxyethyl)L -glutamine (PHEG) in aqueous isopropanol was examined by means of optical rotatory dispersion (ORD) and intrinsic viscosity [η] measurements. The Zimm–Bragg parameters σ and s for the transition were determined from the ORD data as a function of molecular weight. It was found that the transition was characterized by a relatively low cooperativity; the values of \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt \sigma $\end{document} were in the range from 0.039 to 0.066, depending on the solvent composition. These σ values are much larger than those reported for other polypeptide–solvent systems. The transition enthalpy was negative and its magnitude varied with the solvent composition, with a maximum of 620 cal/mol at 40 wt% isopropanol. The curve of [η] versus helical content for a high-molecular-weight sample exhibited a very broad minimum, and this behavior was attributed to the low cooperativity of the transition.     

Experimental limitations in microdosimetry measurements using the variance technique

Summary Microdosimetry measurements give information about the energy imparted to a volume of given size. With the most frequent method only data for object sizes above 0.2 µm are obtainable. Although restricted to the dose mean values, the variance technique can, however, be applied to object sizes an order of magnitude less. In this report some limiting parameters for this technique are discussed and results down to about 20 nm in a⁶⁰co-ray beam are reported. The resulting data exceed earlier reported values at object sizes below 0.1 µm, but are in good agreement for larger object sizes.     

A Low Repeat Length in Oligodendrocyte Chromatin

The behavior of oligodendrocyte chromatin after micrococcal nuclease digestion of nuclei was assayed in brains of rats of four different ages. During oligodendrocyte differentiation, a decreasing sensitivity of the chromatin to enzymatic attack was observed. On the other hand, the nucleosomal repeat length showed a slight tendency to increase during development. It is worth noting that even the highest values reported here for "oligodendrocyte" chromatin repeat lengths are significantly lower than 200 base pairs, the value previously reported by others for "non-astrocytic glia."     

Predictability of heterozygosity scores and polymorphism information content values for rat genetic markers

Construction of a genetic linkage map of the laboratory rat, Rattus norvegicus, establishes the rat as a genetic model. Allele sizes were reported for 432 simple sequence length polymorphisms (SSLPs) genotyped in 12 different substrains belonging to nine different inbred strains of rats. However, these nine strains represent only a fraction of the more than 140 inbred strains available. If allele sizes are not known, alternative indices of markers' polymorphism content can be used, such as heterozygosity (H) and polymorphism information content (PIC). Here, we have determined heterozygosity scores and PIC values for all markers of the rat genetic linkage map, and we evaluate the predictability of the heterozygosity and the PIC values. Correlation analysis between the nine inbred strains reported for the rat map and ten test strains yielded r=0.42 and r=0.44 for heterozygosity and PIC values, respectively. While the correlation of the indices between the two groups of animals is low, these indices do provide a means of predicting whether a genetic marker will be informative in strains where allele sizes are not known.     

Bacillus thiaminolyticus sp. nov., nom. rev

The name "Bacillus thiaminolyticus" Kuno 1951 was not included on the Approved Lists of Bacterial Names and has lost standing in bacteriological nomenclature. The genetic homogeneity of "Bacillus thiaminolyticus" was assessed by determining guanine-plus-cytosine contents by the buoyant density method and by measuring DNA relatedness by using spectrophotometric reassociation procedures. Of the 26 strains which I studied, 24 had guanine-plus-cytosine contents in the range from 52 to 54 mol%. The consistently high DNA relatedness values of 60 to 100% of these 24 strains to the type strain indicated that the "B. thiaminolyticus" group is genetically homogeneous. Low DNA relatedness values of 20 to 31% showed that "B. thiaminolyticus" is genetically unrelated to Bacillus alvei, "Bacillus aneurinolyticus," "Bacillus apiarius," Bacillus larvae, Bacillus laterosporus, Bacillus macerans, and Bacillus stearothermophilus. In general, the "B. thiaminolyticus" group was highly homogeneous for 49 phenotypic characteristics and clearly distinguishable from B. alvei, with which it was allegedly synonymous. On the basis of these findings, revival of the name Bacillus thiaminolyticus is proposed.     

Effects of pH and thiols on the kinetics of yeast glyoxalase I. An evaluation of the random pathway mechanism.

The disproportionation of alpha-ketoaldehydes, catalyzed by yeast glyoxalase I, has been reported to involve a random pathway mechanism where one branch utilizes the hemimercaptal of glutathione and the alpha-ketoaldehyde in a one-substrate pathway, and the other branch utilizes first glutathione and then the alpha-ketoaldehyde in an ordered two-substrate pathway. The relative importance of the two pathways has been evaluated at 5 degrees in the pH range 3-7, using methylglyoxal and phenylglyoxal as representative aliphatic and aromatic alpha-ketoaldehydes, by comparing initial rates of hemimercaptal formation in the absence of enzyme with initial rates of product formation in the presence of high enzyme concentrations. If the enzyme is not added last, the initial rates of product formation are the same as the initial rates of adduct formation even under conditions where it could be shown that dehydration of the hydrated alpha-ketoaldehyde is not entirely rate determining. If the enzyme is added after hemimercaptal formation, there is a "burst" of product formation equivalent to the amount of hemimercaptal, followed by a slower reaction, consistent with the one-substrate pathway. Additional support for this pathway was obtained from a study of the effects of added thiol reagents on the "burst" kinetics. The broad specificity of yeast glyoxalase I for both aliphatic and aromatic alpha-ketoaldehydes, reflected in Vmax values which are insensitive to the nature of the alpha-ketoaldehyde drops abruptly if the side chain of the alpha-ketoaldehyde is sterically crowded. The hemimercaptal of tert-butylglyoxal has a Vmax 300-fold smaller than Vmax for methylglyoxal; 2,4,6-trimethylphenylglyoxal is essentially inactive as a substrate even though the closely related compound 2,4-dimethylphenylglyoxal is a normal substrate. Analysis of the Vmax and Km (or Ki) values of these alpha-ketoaldehydes suggests that sterically crowded side chains affect both enzyme-substrate formation and the catalytic reaction.     

Reflection coefficients of permeant molecules in human red cell suspensions

The Staverman reflection coefficient, sigma for several permeant molecules was determined in human red cell suspensions with a Durrum stopped-flow spectrophotometer. This procedure was first used with dog, cat, and beef red cells and with human red cells. The stopped-flow technique used was similar to the rapid-flow method used by those who originally reported sigma measurements in human red cells for molecules which rapidly penetrate the red cell membrane. The sigma values we obtained agreed with those previously reported for most of the slow penetrants, except malonamide, but disagreed with all the sigma values previously reported for the rapid penetrants. We were unable to calculate an "equivalent pore radius" with our sigma data. The advantages of our equipment and our experimental procedure are discussed. Our sigma data suggest that sigma is indirectly proportional to the log of the nonelectrolyte permeability coefficient, omega. Since a similar trend has been previously shown for log omega and molar volume of the permeant molecules, a correlatioo was shown between sigma and molar volume suggesting the membrane acts as a sieve.     

Shaking stack model of ion conduction through the Ca(2+)-activated K+ channel.

Motivated by the results of Neyton and Miller (1988. J. Gen. Physiol. 92:549-586), suggesting that the Ca(2+)-activated K+ channel has four high affinity ion binding sites, we propose a physically attractive variant of the single-vacancy conduction mechanism for this channel. Simple analytical expressions for conductance, current, flux ratio exponent, and reversal potential under bi-ionic conditions are found. A set of conductance data are analyzed to determine a realistic range of parameter values. Using these, we find qualitative agreement with a variety of experimental results previously reported in the literature. The exquisite selectivity of the Ca(2+)-activated K+ channel may be explained as a consequence of the concerted motion of the "stack" in the proposed mechanism.     

Phase-locked responses in the Limulus lateral eye. Theoretical and experimental investigation.

The 1:1 phase locking of the neural discharge to sinusoidally modulated stimuli was investigated both theoretically and experimentally. On the theoretical side, a neural encoder model, the self-inhibited leaky integrator, was considered, and the phase of the locked impulse was computed for each frequency in the locking range by imposing the condition that the "leaky integral" u(t) of the driving signal should reach the threshold for the first time one stimulus period after the preceding impulse. As u(t) can be a nonmonotonic function, this approach leads to results that sometimes differ from those reported in the literature. It turns out that the phase excursion is often much smaller than the values of about 180 degrees predicted from previous analysis. Moreover, our analysis shows a peculiar effect; the phase locking frequency range narrows when the input modulation depth increases. The theoretical predictions are then compared with phase-locked discharge patterns recorded from visual cells of the Limulus lateral eye, stimulated by sinusoidally modulated light or depolarizing current. The phases of the locked spikes at each of a number of modulation frequencies have been measured. The predictions offered by the model fit the experimental data, although there are some difficulties in determining the effective driving signal.     

Calculation of static hyperpolarisabilities of donor-acceptor systems using a desktop molecular modelling package

A method for calculating the first static hyperpolarisability (₀) values of donor-acceptor molecules using a desktop molecular modelling package is presented. The ₀ values are deduced from a two-state model using geometries from AM1 and dipole moments from ZINDO/s calculations. The estimated ₀ values are in reasonable agreement with reported EFISH values. This method involves the use of a conventional PC and a molecular modelling package and does not require powerful workstations or long computational times.     

Limnological investigations on the Rosetta branch of the Nile. I. Environmental conditions

SUMMARY.

• 1 Seasonal and local variations of some environmental conditions in the Rosetta branch of the River Nile are reported. The lowest air and water temperatures were recorded in January (winter) and the highest in July (summer). Slight decrease in water temperatures with depth is related to solar heating and mixing processes. The pH values, averaging between pH 7.5 and 8.2, showed seasonal changes which, in the main, may be related to photosynthesis.
• 2 The seasonal average chlorosity values showed very wide variation, due to fluctuations in the discharge rate of the River Nile and its effect on the estuarine sea water. Sharp increase in chlorosity values with depth at the estuarine stations was produced by the less dense river water, flowing over denser tongues of sea water. In January, however, the maximum discharge flushed out the sea water.
• 3 Minimum oxygen saturation values occurred in January and the highest values of dissolved organic matter were obtained in July. Both of these will be shown in a later publication to be related to phytoplankton abundance. Increase in dissolved organic matter near the bottom was common and is interpreted as due to the more complete decomposition of descending dead organisms and the release of organic matter from the bottom deposits.

     

Evaluation of dopaminergic tone in postmenopausal women: effects of piribedil on anterior pituitary hormones.

In order to evaluate the relationships between gonadal steroid hormones and central dopaminergic (DA) tone, we have administered a "weak" dopamine agonist drug (piribedil) in 12 normal women, who were postmenopausal for at least 5 yrs, and we have studied the effects on anterior pituitary hormone release. We observed a decrease of plasma PRL levels and an increase of plasma GH values with all doses (40, 60, 100 mg p.o.) of the drug employed. No consistent changes in plasma FSH, LH, ACTH and TSH were observed and no side effects were reported. These results were greatly different from those previously described in premenopausal women in whom dose-related effects were observed and were similar to those observed in normal male subjects. The differences in the response to piribedil observed in women before and after the menopause could be due to a different sexual steroid environment.     

Thermodynamic study of the formation of adenine nucleotide-manganese complexes. II. Calorimetric results.

Enthalpic variations in the formation of adenine nucleotide-manganese complexes, as measured by microcalorimetry, are reported. All the results are obtained in the temperature range 6--30 degrees C at I =0.2 and pH values 7.00 or 7.50. All the reactions are endothermic and the deltaH values increase with the length of the phosphate chain and with temperature. The deltaH values are compared with those previously obtained for adenine nucleotide-manganesium complexes. The comparison between calorimetric and potentiometric deltaH values is made. The divergence observed between these results at low temperature leads us to assume the formation of nucleotide aggregates induced by the presence of manganese ions. This hypothesis is confirmed by differential ultraviolet spectra.