Enantiopurity and absolute configuration determination of arene cis‐dihydrodiol metabolites and derivatives using chiral boronic acids

The relative merits of the methods employed to determine enantiomeric excess (ee) values and absolute configurations of chiral arene and alkene cis‐1,2‐diol metabolites, including boronate formation, using racemic or enantiopure (+) and (?)‐2‐(1‐methoxyethyl)phenylboronic acid (MEPBA), are discussed. Further applications of: 1) MEPBA derived boronates of chiral mono‐ and poly‐cyclic arene cis‐dihydrodiol, cyclohex‐2‐en‐1‐one cis‐diol, heteroarene cis/trans‐2,3‐diol, and catechol metabolites in estimating their ee values, and 2) new chiral phenylboronic acids, 2‐[1‐methoxy‐2,2‐dimethylpropyl]phenyl boronic acid (MDPBA) and 2‐[1‐methoxy‐1‐phenylmethyl]phenyl boronic acid (MPPBA) and their advantages over MEPBA, as reagents for stereochemical analysis of arene and alkene cis‐diol metabolites, are presented.     

The proteins of polytene chromosomes of Drosophila hydei

It is reported that chromatin can be prepared from highly purified polytene nuclei from the salivary glands of third instar larvae of Drosophila hydei; such chromatin differs from that of diploid nuclei mainly by deficiencies in certain nonhistone chromosomal proteins. It is suggested that these proteins are important components of constitutive heterochromatin, which is severely underrepresented in polytene chromosomes. Chromosome morphology, including the pattern of induced puffs, is maintained throughout the mass isolation of glands and sucrose gradient purification of nuclei, as indicated by studies on temperature-shocked and control larvae. No significant alteration in the chromosomal proteins following puff induction by heat shock could be detected on analysis of the isolated protein fractions by disc gel electrophoresis. More sensitive techniques must be developed to study the apparent rearrangement or accumulation of protein at puff sites, and to elucidate the role of this protein in gene activation.     

Host genes affect intestinal colonisation of newly hatched chickens by Campylobacter jejuni

Campylobacter jejuni is the leading cause of food-borne gastro-enteritis and infection can be followed by severe clinical complications, such as the autoimmune neuropathy Guillain–Barré syndrome. Poultry meat is considered to be a common source of infection, with most flocks infected from 2 to 3 weeks of age. We have examined the effect of host genetics on the colonisation levels of C. jejuni in chickens. Chicks from different inbred lines were challenged with 10⁷ to 10⁸ cfu of C. jejuni 14N or C. jejuni 81–176 on the day of hatch and levels of bacterial colonisation measured over a period of 2–3 weeks. We consistently observed a 10- to 100-fold difference between four inbred lines in the number of C. jejuni organisms present in the cloaca or in the caeca, with the greatest differences detected between line N, which carried relatively high bacterial levels, and line 6₁, which carried relatively low numbers of bacteria. Amongst the four lines studied, major histocompatibility complex did not appear to be a major factor in determining the resistance. The difference in numbers of cloacal bacteria was observed as soon as 24 h after challenge and was still present at the end of the experiment. Lines N and 6₁ were chosen to analyse the mode of inheritance of the genetic differences in response to this infection. Challenge of progeny from reciprocal (N×6₁) and (6₁×N) F1 crosses and from (N×6₁) F1×N backcrosses with C. jejuni 14N revealed that the difference in bacterial numbers was inherited in a manner consistent with the resistance (low bacterial numbers) controlled by a single autosomal dominant locus. These data suggest that it might be possible to identify the genes responsible by genetic mapping and candidate gene analysis.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Optimizing healthy gestational weight gain in women at high risk of gestational diabetes: A randomized controlled trial

Objective:

Optimizing gestational weight gain (GWG) in early pregnancy is of clinical and public health importance, especially in higher risk pregnancies.

Design and Methods:

In a robustly designed, randomized controlled trial, 228 pregnant women at risk of developing gestational diabetes mellitus (GDM) were allocated to either control (written health information only) or intervention (four‐session lifestyle program). All women received standard maternal care. Measures were completed at 12‐15 and 26‐28 weeks gestation. Measures included anthropometrics (weight and height), physical activity (pedometer and International Physical Activity Questionnaire), questionnaires (risk perception), and GDM screening.

Results:

The mean (SD) age [31.7 (4.5) and 32.4 (4.7) years] and body mass index [BMI; 30.3 (5.9) and 30.4 (5.6) kg/m²] were similar between control and intervention groups, respectively. By 28 weeks, GWG was significantly different between control and intervention groups [6.9 (3.3) vs. 6.0 (2.8) kg, P < 0.05]. When stratified according to baseline BMI, overweight women in the control group gained significantly more weight compared to overweight women in the intervention group [7.8 (3.4) vs. 6.0 (2.2) kg, P < 0.05], yet in obese women, GWG was similar in both groups. Physical activity levels declined by 28 weeks gestation overall (P < 0.01); however, the intervention group retained a 20% higher step count compared to controls [5,203 (3,368) vs. 4,140 (2,420) steps/day, P < 0.05]. Overall, GDM prevalence was 22%, with a trend toward less cases in the intervention group (P = 0.1).

Conclusions:

Results indicate that a low‐intensity lifestyle intervention, integrated with antenatal care, optimizes healthy GWG and attenuates physical activity decline in early pregnancy. Efficacy in limiting weight gain was greatest in overweight women and in high‐risk ethnically diverse women.     

Mus308 Mutants of Drosophila Exhibit Hypersensitivity to DNA Cross-Linking Agents and Are Defective in a Deoxyribonuclease

Mutagen-sensitive strains that identify 16 different Drosophila genes have been screened for alterations in DNA metabolic enzymes. A characteristic defect in an acid-active deoxyribonuclease was observed in strains carrying the six available mutant alleles of the mus308 gene. Since that enzyme is detected at normal levels in a mutant strain that is deficient in the previously identified enzymes DNase 1 and DNase 2, it represents a new Drosophila nuclease that is designated Nuclease 3. The mus308 mutants were originally distinguished from all other mutagen-sensitive mutants of Drosophila because they exhibit hypersensitivity to the DNA cross-linking agent nitrogen mustard without expressing a concurrent sensitivity to the monofunctional agent methyl methanesulfonate. Further observations of hypersensitivity to the mutagens trimethylpsoralen, diepoxybutane and cis-platinum now establish a more general sensitivity of these mutants to agents capable of generating DNA cross-links. In spite of the hypersensitivity of the mus308 mutants to DNA cross-linking agents, the initial incision step of DNA cross-link repair is normal in mus308 cells as assayed by the alkaline elution procedure. The Drosophila mus308 mutants show promise of providing a useful model for analogous defects in other organisms including man.     

Excision dynamics of <Emphasis Type="Italic">Vibrio</Emphasis> pathogenicity island-2 from <Emphasis Type="Italic">Vibrio cholerae:</Emphasis> role of a recombination directionality factor VefA

Background  

Vibrio Pathogenicity Island-2 (VPI-2) is a 57 kb region present in choleragenic V. cholerae isolates that is required for growth on sialic acid as a sole carbon source. V. cholerae non-O1/O139 pathogenic strains also contain VPI-2, which in addition to sialic acid catabolism genes also encodes a type 3 secretion system in these strains. VPI-2 integrates into chromosome 1 at a tRNA-serine site and encodes an integrase intV2 (VC1758) that belongs to the tyrosine recombinase family. IntV2 is required for VPI-2 excision from chromosome 1, which occurs at very low levels, and formation of a non-replicative circular intermediate.     

l-Arginine Effects on Na+ Transport in M-1 Mouse Cortical Collecting Duct Cells—A Cationic Amino Acid Absorbing Epithelium

The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using continuous short circuit current (I _SC ) measurements in HCO₃ ⁻/CO₂ buffered solution. Steady state I _SC averaged 73.8 ± 3.2 μA/cm² (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na⁺ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y⁺ and a basal amino acid transport system y⁺L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I _SC averaging 36.6 ± 5.4 μA/cm² (n= 19) and 32.0 ± 7.2 μA/cm² (n= 8), respectively. The response was preserved in the absence of bath Cl⁻ (n= 4), but was abolished either in the absence of apical Na⁺ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I _SC response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na⁺ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract passive cationic amino acid leak into the CCD in the presence of electrogenic Na⁺ transport, consistent with reports of stimulated expression of Na⁺ and cationic amino acid transporters by aldosterone. Received: 11 September 2000/Revised: 6 December 2000     

The neurotrophin receptors,trkB and p75, differentially regulate motor axonal regeneration

Neurotrophic factors that support neuronal survival are implicated in axonal regeneration after injury. Specifically, a strong role for BDNF in motor axonal regeneration has been suggested based on its pattern of expression after injury, as well as the expression of its receptors, trkB and p75. Despite considerable in vitro evidence, which demonstrate specific and distinct physiological responses elicited following trkB and p75 activation, relatively little is known about the function of these receptors in vivo. To investigate the roles of the trkB and p75 receptors in motor axonal regeneration, we have used a tibial (TIB)‐ common peroneal (CP) cross suture paradigm in p75 homozygous (?/?) knockout mice, trkB heterozygous (+/?) knockout mice, as well as in their wild‐type controls. Contralateral intact TIB motoneurons, and axotomized TIB motoneurons that regenerated their axons 10 mm into the CP distal nerve stump were identified by fluorescent retrograde tracers and counted in the T11‐L1 spinal segments. Regeneration was evaluated 2, 3, 4, 6, and 8 weeks after nerve repair. Compared to wild‐type animals, there are significantly fewer intact TIB motoneurons in p75 (?/?), but not trkB (+/?) mice. The number of motoneurons that regenerated their axons was significantly increased in the p75 (?/?) knockout mice, but significantly attenuated in the trkB (+/?) mice compared to wild‐type controls. These results suggest that p75 is important for motoneuronal survival during development, but p75 expression after injury serves to inhibit motor axonal regeneration. In addition, full expression of trkB is critical for complete axonal regeneration to proceed. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 314–325, 2001     

Effects of temperature and biostimulation on oil-degrading microbial communities in temperate estuarine waters

Improved strategies for oil-spill remediation will follow a better understanding of the nature, activities and regulating parameters of petroleum hydrocarbon-degrading microbial communities in temperate marine environments. The addition of crude oil to estuarine water resulted in an immediate change in bacterial community structure, increased abundance of hydrocarbon-degrading microorganisms and a rapid rate of oil degradation, suggesting the presence of a pre-adapted oil-degrading microbial community and sufficient supply of nutrients. Relatively rapid degradation was found at 4°C, the lowest temperature tested; and it was temperature rather than nutrient addition that most influenced the community structure. A detailed phylogenetic analysis of oil-degrading microcosms showed that known hydrocarbonoclastic organisms like Thalassolituus and Cycloclasticus , as well as proposed oil degraders like Roseobacter , were present at both 4°C and 20°C, demonstrating the thermo-versatility of such organisms. Clones related to Oleispira antarctica (98% 16S rRNA similarity), a psychrophilic alkane degrader, were dominant in the 4°C oil-degrading community, whereas other clones constituting a different clade and showing 94% similarity 16S rRNA with O. antarctica were found in situ. These findings demonstrate the potential for intrinsic bioremediation throughout the course of the year in temperate estuarine waters, and highlight the importance of both versatile psychrotolerant and specialized psychrophilic hydrocarbon-degrading microbes in effecting this process at low temperatures.