Enzymes of glucose and methanol metabolism in the actinomycete Amycolatopsis methanolica.

The actinomycete Amycolatopsis methanolica was found to employ the normal bacterial set of glycolytic and pentose phosphate pathway enzymes, except for the presence of a PPi-dependent phosphofructokinase (PPi-PFK) and a 3-phosphoglycerate mutase that is stimulated by 2,3-bisphosphoglycerate. Screening of a number of actinomycetes revealed PPi-PFK activity only in members of the family Pseudonocardiaceae. The A. methanolica PPi-PFK and 3-phosphoglycerate mutase enzymes were purified to homogeneity. PPi-PFK appeared to be insensitive to the typical effectors of ATP-dependent PFK enzymes. Nevertheless, strong N-terminal amino acid sequence homology was found with ATP-PFK enzymes from other bacteria. The A. methanolica pyruvate kinase was purified over 250-fold and characterized as an allosteric enzyme, sensitive to inhibition by P(i) and ATP but stimulated by AMP. By using mutants, evidence was obtained for the presence of transketolase isoenzymes functioning in the pentose phosphate pathway and ribulose monophosphate cycle during growth on glucose and methanol, respectively.     

Hydrodynamic characteristics and equilibrium rigidity of pullulan molecules

The hydrodynamic characteristics of the polysaccharide pullulan (polymaltotriose) in water have been investigated and its molecular characteristics have been determined. Experimental values varied over the following ranges: velocity sedimentation coefficient (S): 0.9 < S < 11.2, translational diffusion coefficient (10⁷ cm² s⁻¹): 1.1 < D < 14.7 and intrinsic viscosity (cm³ g⁻¹): 6.7 < [η] < 164, which corresponds to a change in molecular weight (× 10³) in the range 3.9 < M_SD < 644. On the basis of analysis of the literature and our experimental data, excluded volume effects have been shown to have a prevailing influence on the chain length of these polysaccharides. The equilibrium rigidity and hydrodynamic chain diameter of pullulan were evaluated on the basis of the theory of hydrodynamic properties of a wormlike necklace, taking into account excluded volume effects. At low M (< 30 × 10³) the translation friction data (in contrast to viscometric data) cannot be described in the framework of the theory of linear molecules.     

Effects of Ca(II) ions on Mn(II) dynamics in chick glia and rat astrocytes: Potential regulation of glutamine synthetase

Previous studies have demonstrated that in glia and astrocytes Mn(II) is distributed with ca. 30–40% in the cytoplasm, 60–70% in mitochondria. Ca(II) ions were observed to alter both the flux rates and distribution of Mn(II) ions in primary cultues of chick glia and rat astrocytes. External (influxing) Ca(II) ions had the greatest effect on Mn(II) uptake and efflux, compared to internal (effluxing) or internal-external equilibrated Ca(II) ions. External (influxing) Ca(II) ions inhibited the net rate and extent of Mn(II) uptake but enhanced Mn(II) efflux from mitochondria. These observations differ from Ca(II)–Mn(II) effects previously reported with brain (neuronal) mitochondria. Overall, increased cytoplasmic Ca(II) acts to block Mn(II) uptake and enhance Mn(II) release by mitochondria, which serve to increase the cytoplasmic concentration of free Mn(II). A hypothesis is presented involving external L-glutamate acting through membrane receptors to mobilize cell Ca(II), which in turn causes mitochondrial Mn(II) to be released. Because the concentration of free cytoplasmic Mn(II) is poised near the K_d for Mn(II) with glutamine synthetase, a slight increase in cytoplasmic Mn(II) will directly enhance the activity of glutamine synthetase, which catalyzes removal of neurotoxic glutamate and ammonia.     

Histoplasma capsulatum peritonitis associated with continuous ambulatory peritoneal dialysis

This paper reports a case of infection byHistoplasma capsulatum apparently restricted to the peritoneum in a woman submitted to continuous ambulatory peritoneal dialysis. Diagnosis was established by culture of dialysis fluid and peritoneal nodule and by histopathologic examination.     

Quantitative estimation of chimerism in mice using microsatellite markers

An embryonic stem cell line was established from SV129 mouse blastocysts and used to generate chimeric mice by injection into OF1 blastocysts; 18 out of the 30 resulting offspring appeared chimeric as judged from their coat color patterns, and 3 of the 13 males proved to be germ-line chimeras as they transmitted the SV129 agouti phenotype to all or part of their offspring. The degree of chimerism of these males was evaluated for different tissues using polymorphic microsatellite markers amplified by the polymerase chain reaction. It was shown that these new markers can be effectively used to quantitatively estimate levels of chimerism. The CKMM (creatine kinase, muscle) microsatellite system was used to distinguish the SV129 from the OF1 genotype. In all performed tests, the correlation between DNA ratio and signal ratio, expressed as a base 10 logarithm, was shown to exceed or equal 0.98 for known DNA ratios (SV129/OF1) ranging from 1/99 to 99/1. Linear calibration methods were used to predict the % SV129 DNA of a test sample based on the obtained signal ratio. The accuracy of the prediction was evaluated by performing repeated measurements. Differences among three repeated estimates ranged from 2 to 17% for a given sample. Microsatellite systems should be very useful to monitor chimerism involving strains that can not be discerned with coat color or biochemical markers. This will be particularly important when ES methodology becomes available in species other than mice. © 1993 Wiley-Liss, Inc.     

Two genes present on a transposon-like structure in Lactococcus lactis are involved in a Clp-family proteolytic activity

The lactose-protease plasmid pUCL22 of Lactococcus lactis subsp. lactis strain CNRZ270 contained two inverted copies of IS 1076 flanking a region of 3.7 kb. This internal region was sequenced and found to contain two large open reading frames, ORF1 and ORFP in opposite orientations. ORF1 consists of 2289 bp; the deduced 763-amino-acid sequence is similar to the ATPases of the ClpA family. It contains two well-conserved consensus ATP-binding sites. It was named ClpL. ORFP consists of 930 bp encoding a protein of 310 amino acids. No similarity with any known protein was found in GenBank data for ORFP. Increased ATP-dependent proteolytic activity was detected in extracts from Escherichia coli cells expressing the clpL and ORFP genes.     

Abortion of infection during the Rhizobium meliloti—alfalfa symbiotic interaction is accompanied by a hypersensitive reaction

Nodulation, the organogenetic process resulting from the symbiotic interaction between Rhizobium and legumes, is under the feedback control of the plant. However, the autoregulatory mechanisms controlling root nodule formation are poorly understood. In this paper it is shown that alfalfa can react to infection by its symbiont Rhizobium meliloti by eliciting a defence mechanism similar to the hypersensitive reaction (HR) observed in incompatible plant-pathogen interactions. After the first nodule primordia have been induced, an increasing proportion of infection threads abort in a single or a few root cortical cells in which both symbionts simultaneously undergo necrosis. Autofluorescent, cytochemical and immunolocalization assays revealed that phenolic compounds and proteins associated with defence mechanisms in plants have accumulated in the necrotic cells. These results lead to the proposition that the elicitation of a HR is part of the mechanism by which the plant controls infection and, therefore, regulates nodulation.