Significance of low-temperature growth associated with the fecal coliform response, indole production, and pectin liquefaction in Klebsiella

In the genus Klebsiella, the growth respnse in nutient broth at 10 degrees C correlates inversely with the operational definition of a fecal coliform and not merely with the ability to grow at 44.5 degrees C. Of the fecal coliform-positive Klebsiella, 97% did not grow at 10 degrees C after 72 h of incubation. Conversely, 97% of the fecal coliform-negative isolates grew at 10 degrees C. The amount of growth at 10 degrees C varied among the fecal coliform-negative isolates and was found to correlate with indole production and pectin liquefaction. Low-temperature growth associated with specific biochemical tests can be used to differentiate several groups in the genus Klebsiella. Three main groups were discerned. Group I consists of indole-negative, pectin-nonliquefying, fecal coliform-positive isolates that do not grow at 10 degrees C. Group II isolates are differentiated from group I by a fecal-coliform-negative response and growth at 10 degrees C. Group III are indole-positive, pectin-liquefying, fecal coliform-negative isolates that grow at 10 degrees C. In our culture collection, isolates of group I are most frequently of human/animal clinical origins, whereas isolates of groups II and III are predominantly derived from the environment.     

Calibration of a flow cytometric assay of glucose-6-phosphate dehydrogenase activity.

The reduction of tetrazolium salts to colored formazans is a reaction which has been exploited both in histo- and cytochemistry. Tetrazolium salts forming fluorescent formazans prove suitable for measuring defined cellular dehydrogenase activities in automated processes. This study considers an important aspect of formazan measurement in flow cytometry, namely, calibration. Calibration is performed by correlating the number (and fluorescence intensity) of formazan-bearing cells measured by flow cytometry with simultaneously performed biochemical analyses of the same material. The method is demonstrated by an example of glucose-6-phosphate dehydrogenase. Using the data of a typical experiment, the enzyme activity is expressed in femtomol of hydrogen transferred per cell during incubation time. Furthermore, through spatially resolved double excitation of formazan and nuclear DAPI fluorescence, an independent analysis of cell cycle and cellular enzymatic activity is established.     

Fetal dexamethasone exposure impairs cellular development in neonatal rat heart and kidney: effects on DNA and protein in whole tissues

Fetal glucocorticoid exposure causes postnatal growth retardation. To examine the mechanisms underlying effects on specific organ systems, we administered 0.2 or 0.8 mg/kg of dexamethasone to pregnant rats on gestational days 17, 18, and 19 and assessed three biochemical markers of cell development in heart and kidney of the offspring: DNA content per organ as an index of total cell numbers, DNA per g tissue as an index of cell packing density, and protein/DNA ratio as an index of relative cell size. In both tissues, DNA content became markedly subnormal during the first postnatal week, the ontogenetic period of rapid cell division. Partial recovery occurred by the end of the first postnatal month. In the heart, cell packing density was subnormal initially and the cells were significantly enlarged. In contrast, packing density was slightly elevated in the kidney; protein/DNA was increased by the low dose of dexamethasone, but markedly decreased by the high dose. These results suggest that tissue growth impairment caused by prenatal dexamethasone treatment reflects primary deficits in cell proliferation that extend to a variety of different cell types; however, consequent effects on cell packing density and cell size are dose-specific, possibly reflecting actions of glucocorticoids selective for certain cell types or phases of cell development.     

Ca2+ transport activities of inside-out vesicles prepared from density-separated erythrocytes from rat and human

The plasma membrane Ca²⁺ ATPase in erythrocytes is vital for the maintenance of intracellular Ca²⁺ levels. Since the cytoplasmic Ca²⁺ concentration is elevated in older erythrocytes, the properties of the Ca²⁺ transport ATPase were examined during cell aging using inside-out vesicles (IOVs) prepared from density-separated, young (less dense, Ey) and old (more dense, Eo) rat and human erythrocytes. The transport of Ca²⁺ and the coupled hydrolysis of ATP were measured using radiolabeled substrates. The calmodulin-independent Ca²⁺ transport activity (Ey, 38.8 vs. Eo, 23.3 nmols/min/mg IOV protein) and the Ca²⁺ dependent ATP phosphohydrolase activity (Ey, 53.5 vs. Eo, 48.8 nmols/min/mg protein) were greater in IOVs prepared from younger (less dense) rat erythrocytes. The calmodulin-independent Ca²⁺ transport activity in IOVs from human erythrocytes was 12.9 nmols/min/mg IOV protein for Ey and 10.7 nmols/min/mg IOV protein for Eo. Inside-out vesicles from older (more dense) cells exhibited a lower pumping efficiency as determined by the calculated stoichiometry, molecule of Ca²⁺ transported per molecule of ATP hydrolyzed (rat: Ey, 0.74 vs. Eo, 0.49; human: Ey, 1.22 vs. Eo, 0.77). The enzymatic activity of rat and human Ey IOVs was labile. The Ca²⁺ transport activity in Ey but not Eo IOVs rapidly declined during cold storage (4°C). The decrease in Ca²⁺ transport activity during aging may accentuate the age-related decline in several erythrocytic properties.Abbreviations IOV Inside-Out Vesicles - Ey Erythrocytes enriched with young (less dense) cells - Eo Erythrocytes enriched with old (more dense) cells - ACEase Acetylcholinesterase     

Predictive model of conjugative plasmid transfer in the rhizosphere and phyllosphere.

A computer simulation model was used to predict the dynamics of survival and conjugation of Pseudomonas cepacia (carrying the transmissible recombinant plasmid R388:Tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. Plasmid transfer rates were derived for a mass action model, and donor and recipient survival were modeled as exponential growth and decay processes or both. Rate parameters were derived from laboratory studies in which donor and recipient strains were incubated in test tubes with a peat-vermiculite solution or on excised radish or bean leaves in petri dishes. The model predicted donor, recipient, and transconjugant populations in hourly time steps. It was tested in a microcosm planted with radish seeds and inoculated with donor and recipient strains and on leaf surfaces of radish and bean plants also growing in microcosms. Bacteria were periodically enumerated on selective media over 7 to 14 days. When donor and recipient populations were 10(6) to 10(8) CFU/g (wet weight) of plant or soil, transconjugant populations of about 10(1) to 10(4) were observed after 1 day. An initial rapid increase and a subsequent decline in numbers of transconjugants in the rhizosphere and on leaf surfaces were correctly predicted.     

Broth medium for enrichment of Vibrio fluvialis from the environment.

A medium was designed for the enrichment and enumeration of Vibrio fluvialis from environmental samples. The medium contains 1% peptone plus 4% sodium chloride and 5 micrograms of novobiocin per ml, pH 8.5. This V. fluvialis enrichment medium (FEM) was tested, in comparison with alkaline peptone (AP), in field samplings. A total of 177 samples (estuarine waters and sediment, sewage, and crabs) collected over a 14-month period were examined with FEM and with AP broth. Results showed that FEM was more effective than AP in detecting V. fluvialis, particularly from water and sewage samples with low salinities (less than 6%). The best recovery of V. fluvialis occurred when both enrichment media were used simultaneously.     

Evaluation of a method to measure conjugal transfer of recombinant DNA in soil slurries

Release of recombinant microbes into the environment necessitates an evaluation of their ability to transfer genetic material. The present report evaluates a method to detect conjugal DNA plasmid transfer in soil slurries under various environmental conditions. DonorPseudomonas cepacia containing pR388::Tn1721 andP. cepacia recipient cultures were coincubated in soil slurries containing autoclaved or natural soil and treated with one or more of 14 experimental conditions. Conjugal mating frequency (transconjugants per initial donor) ranged from 4.8×10^–1 to 1.9×10^–7. Highest numbers of transconjugants, 1.5×10⁷ colony forming units/ml soil slurry, were observed following incubation at 35°C with an enriched nutrient supplement added to the soil. Low numbers of transconjugants, 10³ colony forming units/ml soil slurry, were observed when mating pairs were subjected to low nutrient or pH stress even though initial donor and recipient populations were maintained at high levels. This test system provides a simple way to estimate effects of changing environmental factors on plasmid transfer rates and on the survival of recombinant microorganisms. By use of soil collected from sites proposed to receive genetically engineered microorganisms, preliminary risk assessments can be obtained regarding the potential for gene transfer and microorganism survival with this soil slurry test system.     

Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a beta-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of beta-adrenergic receptors: in fact, [125I]pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.     

On the role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II

The role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II has been investigated by means of site-directed mutagenesis and cross-linking. Replacement of Asp-9 resulted in a dramatic increase in proteolytic sensitivity leading to the degradation of the protein forming a 31 kDa fragment with an undefined N-terminus. This fragment was unable to restore oxygen evolution. However, the variants of the 33 kDa protein which remained intact could reconstitute oxygen evolution as effectively as the wild-type protein. Cross-linking experiments with a water-soluble carbodiimide revealed that mutagenesis of residue D9 led to the disruption of an intramolecular salt bridge. Therefore we suggest that the N-terminus of the 33 kDa protein is necessary for maintaining the binding ability of the protein to Photosystem II but might not be involved in binding itself.     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.