The monomeric glutamyl-tRNA synthetase from Bacillus subtilis 168 and its regulatory factor. Their purification, characterization, and the study of their interaction

The glutamyl-tRNA synthetase from Bacillus subtilis has been purified to homogeneity. It is a monomer of Mr = 65,500 whose NH2-terminal sequence is Met-Asn-Glu-Val-Arg-Val-Arg-Tyr-Ser-Pro-Ser-Pro-Thr-Gly-His-Leu. The number of tryptic peptides indicates the absence of a significant amount of sequence duplication. Under certain conditions, this monomeric enzyme is co-purified with a polypeptide beta of Mr = 46,000, which increases the affinity of the enzyme about 10-fold for glutamate and for ATP, and stabilizes it against heat inactivation. gamma-Globulins prepared against the monomeric enzyme can inhibit completely the glutamyl-tRNA synthetase activity of a B. subtilis extract and precipitate from this extract both the monomeric enzyme and the regulatory factor beta. These anti-alpha immunoglobulins do nt precipitate pure beta. These results show that the glutamyl-tRNA synthetase of B. subtilis has a structure similar to that of the Escherichia coli enzyme (Lapointe, J., and S?ll, D. (1972) J. Biol. Chem. 247, 4966-4974) and indicate that the beta factor has a function in the regulation of glutamyl-tRNA biosynthesis in vivo.     

Rhythmic melatonin secretion in different teleost species: an in vitro study

The rhythmic production of melatonin is governed by intrapineal oscillators in all fish species so far investigated except the rainbow trout. To determine whether the latter represents an exception among fish, we measured in vitro melatonin secretion in pineal organs of nine wild freshwater and six marine teleost species cultured at constant temperature and under different photic conditions. The results demonstrate that pineal organs of all species maintain a rhythmic secretion of melatonin under light:dark cycles and complete darkness, and strongly suggest that most fish possess endogenous intrapineal oscillators driving the rhythm of melatonin production, with the exception of the rainbow trout.Abbreviations LD light:dark - DD dark:dark - NAT N-acetyltransferase - RIA radioimmunoassay     

Ecological optima and tolerances of Thelypteris limbosperma,Athyrium distentifolium,and Matteuccia struthiopteris along environmental gradients in Western Norway

The distribution and abundance of Thelypteris limbosperma, Athyrium distentifolium, and Matteuccia struthiopteris are modelled statistically in relation to 14 environmental variables along the major climatic, topographic, and edaphic gradients in western Norway. The data are from 624 stands from which measurements or estimates of mean January and mean July temperatures, humidity, altitude, aspect, and slope are available. From 182 of these stands eight soil variables have also been measured. The species responses are quantified by two numerical methods: Gaussian logit regression and weighted averaging (WA) regression. The estimated WA optima suggest that A. distentifolium has an ecological preference for low July and January temperatures, high altitudes, and soils of low-medium pH and base content. The species shows statistically significant Gaussian responses with summer temperature, humidity (= Martonnes humidity index), altitude, slope, aspect, pH, cation exchange capacity, and base saturation with optima of 8.7 °C, 188.9, 1220 m, 28°, 29°, 4.8, 13.77 mEq 100 g dry soil^-1, and 13.4%, respectively. These suggest that the occurrence and relative abundance of A. distentifolium are well predicted by summer temperature, topography, and soil pH and base status. T. limbosperma has WA optima that suggest that it favours moderately high winter and summer temperatures, high humidity, medium altitude, and soils of low pH and base content. It has significant Gaussian responses to summer temperature (optimum =12.6 °C), winter temperature (-1.8 °C), humidity (179.2), altitude (459.5 m), slope (22.5°), and Na (0.7 mg 100 g dry soil^-1). These suggest that climatic factors, altitude, and slope are significant predictors for its occurrence and abundance. M. struthiopteris has high WA optima for summer temperature, pH, Ca, Mg, K, Na, cation exchange capacity (CEC), and base saturation, and a low optima for humidity and winter temperature. Of these, summer temperature (16.0 °C), Ca (63.1 mg 100 g dry soil^-1), Mg (41.0 mg 100 g dry soil^-1), K (23.6 mg 100 g dry soil^-1), Na (5.0 mg 100 g dry soil^-1), CEC (60.7 mEq 100 g dry soil^-1), and base saturation (56.3%) have significant Gaussian logit responses, as do aspect (150.2°) and loss-on-ignition (9.4%). These results suggest that the occurrence and relative abundance of M. struthiopteris are well predicted by high soil base cations, a generally southern aspect, low organic content in the soil, and high July temperatures.     

Quantitative vegetation-environment relationships in west norwegian tall-fern vegetation

The aims of this paper are to detect floristic variation within different types of tall-fern dominated vegetation and to interpret these patterns in terms of environmental variables. Numerical approaches have been applied to a large and varied vegetational data-set with associated environmental data from stands dominated by Athyrium distentifolium, Thelypteris limbosperma , and Matteuccia struthiopteris in different parts of western Norway. The numerical procedures of two-way indicator species analysis, simple discriminant functions, and canonical correspondence analysis have been used, and the strengths and weaknesses of these as tools in discerning vegetational-environmental relationships are discussed. For each of the 96 quadrats investigated, 17 environmental variables were measured. The investigation shows that some of the observed differences in vegetational composition can be explained in terms of relatively simple soil and climatic variables measured for each quadrat. The ferns appear to be ecologically well separated. T. limbosperma-dominaled stands are mainly characterised by low soil fertility, high January temperature, and high humidity. A. distentifolium-dominated stands are associated with low winter temperatures, and M. struthiopteris-dominated stands have high soil fertility and high summer temperatures.     

Thermodynamic determination of the Na+: glucose coupling ratio for the human SGLT1 cotransporter.

Phlorizin-sensitive currents mediated by a Na-glucose cotransporter were measured using intact or internally perfused Xenopus laevis oocytes expressing human SGLT1 cDNA. Using a two-microelectrode voltage clamp technique, measured reversal potentials (Vr) at high external alpha-methylglucose (alpha MG) concentrations were linearly related to In[alpha MG]o, and the observed slope of 26.1 +/- 0.8 mV/decade indicated a coupling ratio of 2.25 +/- 0.07 Na ions per alpha MG molecule. As [alpha MG]o decreased below 0.1 mM, Vr was no longer a linear function of In[alpha MG]o, in accordance with the suggested capacity of SGLT1 to carry Na in the absence of sugar (the "Na leak"). A generalized kinetic model for SGLT1 transport introduces a new parameter, Kc, which corresponds to the [alpha MG]o at which the Na leak is equal in magnitude to the coupled Na-alpha MG flux. Using this kinetic model, the curve of Vr as a function of In[alpha MG]o could be fitted over the entire range of [alpha MG]o if Kc is adjusted to 40 +/- 12 microM. Experiments using internally perfused oocytes revealed a number of previously unknown facets of SGLT1 transport. In the bilateral absence of alpha MG, the phlorizin-sensitive Na leak demonstrated a strong inward rectification. The affinity of alpha MG for its internal site was low; the Km was estimated to be between 25 and 50 mM, an order of magnitude higher than that found for the extracellular site. Furthermore, Vr determinations at varying alpha MG concentrations indicate a transport stoichiometry of 2 Na ions per alpha MG molecule: the slope of Vr versus In[alpha MG]o averaged 30.0 +/- 0.7 mV/decade (corresponding to a stoichiometry of 1.96 +/- 0.04 Na ions per alpha MG molecule) whenever [alpha MG]o was higher than 0.1 mM. These direct observations firmly establish that Na ions can utilize the SGLT1 protein to cross the membrane either alone or in a coupled manner with a stoichiometry of 2 Na ions per sugar, molecule.     

Increasing the effectiveness of clinical supervision.

Effective use of preceptors in the clinical training and supervision of residents involves four essential steps: careful screening of the preceptor''s practice to ensure it reflects the goals of the residency program and teaching the preceptor about the goals; setting a realistic contract for learning between resident and preceptor; teaching the preceptor to use constructive feedback techniques in the day-to-day supervision of residents; and developing the preceptor''s skills in the reliable and valid evaluation of the resident''s performance. Clinical preceptors must be trained to become effective teachers and evaluators in residency programs.     

Kinetics of carrier-mediated ion transport in two new types of solvent-free lipid bilayers.

In contrast with the usual glyceryl-monooleate/decane (GMO-D) bilayer lipid membranes, new membranes, formed from a mixture of GMO in squalene (GMO-S) or from a mixture of GMO in triolein (GMO-T), seem to be almost solvent free. Our results from voltage-jump relaxation studies, using these "solvent-free" membranes with the homologue carriers, nonactin, monactin, dinactin, trinactin, and tetranactin, are compared with the corresponding ones for GMO-D membranes. With all homologues, solvent-free membranes show an increase of the free carrier translocation rate, ks, by a factor of 2.5, a decrease in the dissociation rate constant of the complex, kDi, by a factor of 1.5 and no significant change in its formation rate constant, kRi. However, the principal effect of the absence of solvent in these membranes is an increase by a factor of approximately 10 of the translocation rate constant for moving the complex across the membrane, kis. This increase varies regularly from a factor of 7-15 with decreasing carrier size, and is always larger for GMO-T than for GMO-S membranes. These solvent-free effects are interpreted in terms of modifications of electrostatic and hydrophobic energy profiles in the membrane.     

Regulation of basolateral membrane potential after stimulation of Na+ transport in proximal tubules

Summary We have previously shown that stimulation of apical Na-coupled glucose and alanine transport produces a transient depolarization of basolateral membrane potential (V _bl) in rabbit proximal convoluted tubule (PCT. Sl segment). The present study is aimed at understanding the origin of the membrane repolarization following the intial effect of addition of luminal cotransported solutes. Luminal addition of 10–15mMl-alanine produced a rapid and highly significant depolarization ofV _bl (20.3±1.1 mV,n=15) which was transient and associated with an increase in the fractional K⁺ conductance of the basolateral membrane (t _K) from 8 to 29% (P<0.01,n=6). Despite the significant increase int _K, the repolarization was only slightly reduced by the presence of basolateral Ba²⁺ (2mM,n=6) or quinine (0.5 mM,n=5). The repolarization was greatly reduced in the presence of 0.1 mM 4-acetamino-4isothiocyamostilbene-2,2-disulfonic acid (SITS) and blunted by bicarbonate-free solutions. Intracellular pH (pH _i ) determined with the fluorescent dye 2, 7-bis-2-carboxyethyl-5(and-6)-carboxyfluorescein (BCECF), averaged 7.39±0.02 in control solution (n=9) and increased to 7.50±0.03 in the first 15 sec after the luminal application of alanine. This was followed by a significant acidification averaging 0.16±0.01 pH unit in the next 3 min. In conclusion, we believe that, contrary to other leaky epithelia, rabbit PCT can regulate its basolateral membrane potential not only through an increase in K⁺ conductance but also through a cellular acidification reducing the basolateral HCO ₃ ^– exit through the electrogenic Na-3(HCO₃) cotransport mechanism.     

Elucidation of the complete Azorhizobium nicotinate catabolism pathway.

A complete pathway for Azorhizobium caulinodans nicotinate catabolism has been determined from mutant phenotype analyses, isolation of metabolic intermediates, and structural studies. Nicotinate serves as a respiratory electron donor to O2 via a membrane-bound hydroxylase and a specific c-type cytochrome oxidase. The resulting oxidized product, 6-hydroxynicotinate, is next reduced to 1,4,5,6-tetrahydro-6-oxonicotinate. Hydrolytic ring breakage follows, with release of pyridine N as ammonium. Decarboxylation then releases the nicotinate C-7 carboxyl group as CO2, and the remaining C skeleton is then oxidized to yield glutarate. Transthioesterification with succinyl coenzyme A (succinyl-CoA) yields glutaryl-CoA, which is then oxidatively decarboxylated to yield crotonyl-CoA. As with general acyl beta oxidation, L-beta-hydroxybutyryl-CoA, acetoacetyl-CoA, and finally two molecules of acetyl-CoA are produced. In sum, nicotinate is catabolized to yield two CO2 molecules, two acetyl-CoA molecules, and ammonium. Nicotinate catabolism stimulates Azorhizobium N2 fixation rates in culture. Nicotinate catabolism mutants still able to liberate pyridine N as ammonium retain this capability, whereas mutants so blocked do not. From, mutant analyses and additional physiological tests, N2 fixation stimulation is indirect. In N-limited culture, nicotinate catabolism augments anabolic N pools and, as a consequence, yields N2-fixing cells with higher dinitrogenase content.