Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.     

Legionella contamination of dental-unit waters.

Water samples collected from 28 dental facilities in six U.S. states were examined for the presence of Legionella pneumophila and other Legionella spp. by the PCR-gene probe, fluorescent-antibody microscopic, and viable-plate-count detection methods. The PCR and fluorescent-antibody detection methods, which detect both viable and viable nonculturable Legionella spp., gave higher counts and rates of detection than the plate count method. By the PCR-gene probe detection method, Legionella spp. were detected in 68% of the dental-unit water samples and L. pneumophila was detected in 8%. Concentrations of Legionella spp. in dental-unit water reached 1,000 organisms per ml or more in 36% of the samples, and 19% of the samples were in the category of 10,000/ml or above. L. pneumophila, when present in dental-unit water, never reached concentrations of 1,000/ml or more. Microscopic examination with fluorescent-antibody staining indicated that the contamination was in the dental-unit water lines rather than in the handpieces. Legionella spp. were present in 61% of potable water samples collected for comparative analysis from domestic and institutional faucets and drinking fountains; this percentage was not significantly different from the rate of detection of Legionella spp. in dental-unit water. However, in only 4% of the potable water samples did Legionella spp. reach concentrations of 1,000 organisms per ml, and none was in the 10,000 organisms-per-ml category, and so health-threatening levels of Legionella spp. in potable water were significantly lower than in dental-unit water. L. pneumophila was found in 2% of the potable water samples, but only at the lowest detectable level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular evolution of mitochondrial 12S RNA and cytochrome b sequences in the pantherine lineage of Felidae

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among 17 Felidae species, notably 15 in the previously described pantherine lineage. The polymerase chain reaction (PCR) was used to generate sequences of 358 base pairs of the mitochondrial 12S RNA gene and 289 base pairs of the cytochrome b protein coding gene. DNA sequences were compared within and between 17 felid and five nonfelid carnivore species. Evolutionary trees were constructed using phenetic, cladistic, and maximum likelihood algorithms. The combined results suggested several phylogenetic relationships including (1) the recognition of a recently evolved monophyletic genus Panthera consisting of Panthera leo, P. pardus, P. onca, P. uncia, P. tigris, and Neofelis nebulosa; (2) the recent common ancestry of Acinonyx jubatus, the African cheetah, and Puma concolor, the American puma; and (3) two golden cat species, Profelis temmincki and Profelis aurata, are not sister species, and the latter is strongly associated with Caracal caracal. These data add to the growing database of vertebrate mtDNA sequences and, given the relatively recent divergence among the felids represented here (1-10 Myr), allow 12S and cytochrome b sequence evolution to be addressed over a time scale different from those addressed in most work on vertebrate mtDNA.      

Monitoring genetically engineered microorganisms in freshwater microcosms

Summary The effectiveness of gene probe methods for tracking genetically engineered microorganisms (GEMs) in the environment was tested by inoculating nutrient-supplemented freshwater microcosms withAlcaligenes A5 (a naturally occurring 4-chlorobiphenyl degrader) orPseudomonas cepacia AC1100 (a genetically engineered 2, 4, 5 T-degrader) and following the fates of the introduced bacterial populations. Colony hybridization of the viable heterotrophic bacterial populations and dot blot hybridization of DNA recovered from the total microcosm microbial communities showed persistence of bothAlcaligenes A5 andP. cepacia AC1100 in the microcosms in the presence and absence of the xenobiotic substrates that these organisms biodegrade. Although there was a gradual decline in the added populations, both of the bacterial populatins were still detected in the microcosms two months after their introduction into the microcosms. Addition of 2, 4, 5-T enhanced the survival ofP. cepacia AC1100 — and 4-chlorobiphenyl addition resulted in increased levels ofAlcaligenes A5. The results indicate that both organisms may persist for very long periods in freshwater habitats.     

On the active site of elastase: Partial mapping by means of specific peptide substrates

RNase-S peptide as well as some related octa- and hexapeptides were found to be highly, reactive substrates of porcine elastase (e.g. Ala(4)-Lys-Phe: K(m) = 4500 M(-1), k(cat) = 32 sec(-1), C = 1.4 x 10(5) M(-1) sec(-1)). Comparison of the various peptides led to the conclusion that the active site of porcine elastase is composed of 6-7 subsites (c.f. [1]). Preliminary mapping shows that subsites S(2), S'(1) and S'(2) have hydrophobic character. Occupation of subsite S(4) by the substrate is important for efficient hydrolysis. Binding at this subsite was found to be stereospecific.     

Role of increased glomerular filtration rate in atrial natriuretic factor-induced natriuresis in the rat

One of the major renal hemodynamic actions of atrial natriuretic factor (ANF) is to increase glomerular filtration rate (GFR). To assess the role of this effect on ANF-induced natriuresis (UNaV), diuresis (V) and kaliuresis (UKV) we performed late clamp experiments in six rats. After control periods (C), synthetic ANF (auriculin A) was infused i.v. (2 micrograms X min-1/kg body wt) throughout the experiment (150 min). After pre-clamp periods, the perfusion pressure of the left kidney (LK) was reduced to 75-80 mmHg. The right kidney (RK) served as a time control. In LK, before the late clamp, ANF increased (p less than 0.01) GFR from 1.5 +/- 0.1 to 1.8 +/- 0.1 ml/min, V from 17 +/- 5 to 53 +/- 5 microliters/min, and UNaV from 2.1 +/- 0.6 to 10.0 +/- 0.9 microEq/min. Almost identical increases occurred in the RK. The late clamp returned all parameters in LK to C values (p greater than 0.05): GFR to 1.4 +/- 0.1 ml/min, V to 6.3 +/- 1.2 microliter/min, and UNaV to 1.0 +/- 0.3 microEq/min. The late clamp also reversed the ANF-induced increase in UKV. In the RK, GFR (1.8 +/- 0.1 ml/min), V (38 +/- 4 microliter/min) and UNaV (7.8 +/- 0.8 microEq/min) remained elevated (p less than 0.01 vs. C) to the end of the experiment. These data demonstrate that upon return of GFR to control levels, the ANF-induced diuresis, natriuresis and kaliuresis is abolished. The results support our previous view that the increase in GFR together with a decrease in inner-medullary hypertonicity account wholly or in great part for the natriuretic action of ANF.     

Denitrification and Nitrogen Fixation in Alaskan Continental Shelf Sediments

Rates of nitrogen fixation and denitrification were measured in Alaskan continental shelf sediments. In some regions, rates of nitrogen fixation and denitrification appeared to be equal; in other areas, rates were significantly different. Potential rates of denitrification were found to be limited primarily by the available nitrate substrate. Major regional differences in rates of denitrification were not statistically significant, but significant differences were found for nitrogen fixation rates in different regions of the Alaskan continental shelf. Estimated net losses of nitrogen from Bering Sea sediments were calculated as 1.8 × 10¹² g of N/yr. Experimental exposure of continental shelf sediments to petroleum hydrocarbons reduced rates of nitrogen fixation and denitrification in some cases but not others. Long-term exposure was necessary before a reduction in nitrogen fixation rates was observed; unamended rates of denitrification but not potential denitrification rates (NO₃⁻ added) were depressed after exposure to hydrocarbons.     

A possible molecular mechanism for beta-receptor desensitization: experiments and hypotheses

β-Receptor desensitization in intact NRK-S cells and in a crude membrane preparation derived from these cells was found not to involve methylation, cAMP dependent phosphorylation, Ca⁺⁺ dependent phosphorylation or ADP ribosylation, but is absolutely dependent on ATP in cell-free systems. Also, the desensitized state could not be relieved by protein phosphatases or alkaline phosphatases. Depletion of intact cells from ATP by prolonged incubation with 2-deoxy-glucose and NaN₃ did not inhibit the rapid onset of desensitization by incubating the cells with β-agonists. In order to rationalize the two seemingly contradictory findings, namely, the absolute requirement of ATP in the cell-free desensitization system and the inability to reverse the desensitized state by a variety of phosphatases, we propose that the uncoupling reaction responsible involve for establishing the desensitized state does not Our working hypothesis suggests instead, that the uncoupling reaction involves a covalent modification of the receptor protein and is catalyzed by a receptor modulator protein which is under the control of the ATP dependent phosphorylation.     

Effect of inhibition of oxygen free radical on ovulation and progesterone production by the in-vitro perfused rabbit ovary

The potential role of oxygen free radicals in hCG-induced ovulation was investigated using the free radical scavenging enzymes superoxide dismutase (SOD) and/or catalase with the in-vitro perfused rabbit ovary preparation. SOD (25 micrograms/ml) and SOD + catalase (25 micrograms/ml) significantly reduced the % of large follicles that ovulated during perfusion (P less than 0.005). Neither maturity nor degeneration of ovulated ova and follicular oocytes was affected by SOD and/or catalase. Progesterone concentration in the perfusate was significantly increased in the SOD + catalase treatment group (P less than 0.01). These results indicate a significant role for oxygen free radicals in the process of ovulation.     

Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts.

A method was developed for the detection of Giardia cysts by using the polymerase chain reaction (PCR) and the giardin gene as the target. DNA amplification by PCR, using giardin DNA as the target, resulted in detection of both live and dead cysts. When giardin mRNA was used as the target, the ability to amplify cDNA by PCR depended on the mode of killing. Cysts killed by freezing were not detected by PCR when giardin mRNA was the target. Cysts killed by heating or exposure to monochloramine, however, gave positive detection signals for both DNA and giardin mRNA targets. The amount of giardin mRNA and total RNA was significantly increased in live cysts following the induction of excystation. Cysts killed by freezing, heating, or exposure to monochloramine did not show a change in RNA content. The detection of the giardin gene by PCR permits a sensitive and specific diagnosis for Giardia spp. Discrimination between live and dead cysts can be made by measuring the amounts of RNA or PCR-amplified product from the giardin mRNA target before and after the induction of excystation.