Pyruvate carboxylase from Aspergillus nidulans. Effects of regulatory modifiers on the structure of the enzyme

Pigment-binding protein of the facultatively phototrophic bacterium Rhodospeudomonas capsulata could be selectively synthesized in toluene-treated cells as well as in homologous and heterologous cell-free translation systems by isolated polysomes. It is shown that the pigment-binding polypeptides of the light-harvesting complexes are encoded by messenger RNA of extreme longevity. The dependence of their synthesis on the concomitant synthesis of tetrapyrroles was demonstrated in the toluene-treated cells. The large Mr-28 000 polypeptide of the reaction center and the Mr-10 000 pigment-binding polypeptide of the light-harvesting complex II were found to be synthesized by free (water-soluble) polysomes without a cleavable 'leader' or 'signal' peptide [reviewed by W. Wickner (1979) Annu. Rev. Biochem. 48, 23-45]. The Mr-10 000 polypeptide, as synthesized in vitro, was studied in more detail. Unlike the membrane-assembled polypeptide in vivo it was insoluble in an organic solvent mixture (chloroform/methanol 1:1, v/v). After detergent denaturation in the presence of membrane isolated from the organism it became organic-solvent-soluble. Obviously the polypeptide could be induced to assume alternative conformations in which its apolar residues were either exposed to the solvent or buried within. These findings, in agreement with Wickner's hypothesis, indicate that the Mr-10 000 polypeptide may enter the lipid bilayer by a 'membrane-triggered' conformational change.     

Review article number 6 : Plant molluscicides

A review on the application of plant molluscicides in the control of schistosomiasis is presented. Laboratory bioassays are discussed, together with criteria for activity. An attempt has been made to provide a comprehensive list of known molluscicidal natural products.     

Gene flow and range expansion in a mountain‐dwelling passerine with a fragmented distribution

We studied gene flow and bottleneck events in the population history of locally isolated citril finches endemic to European mountains. For the present study, we used two genetic markers with different rates of evolution: a fast evolving mitochondrial marker (ATPase6/8) and a more slowly evolving nuclear marker (02401). Populations north of the Pyrenees showed in general fewer haplotypes and a considerable lower nucleotide and gene diversity than the Iberian populations. Unexpectedly, we found very little genetic variability in the fast evolving mitochondrial marker, arguing for a strong and relatively recent bottleneck event in the species population history. This pattern potentially reflects a sudden decrease of crucial resources during Mid‐Holocene (mountain pine, Scots pine, and black pine) and a subsequent breakdown of the population. The bottleneck could also have been caused or coincide with a selective sweep in the mitochondrion. By contrast, the slowly evolving nuclear marker showed a much higher variability. This marker probably reflects major gene flow along a potential expansion pathway from the Eastern Pyrenees, northwards to the populations of Central Europe, and southwards to the more fragmented populations of central and southern Spain. The population of the Western Pyrenees (Navarra) appears to be cut‐off from this major gene flow and our data indicate a certain degree of partial isolation, probably reflecting more ancient events (e.g. the separation in distinct refuge sites during the last glacial maximum). © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 707–721.     

The Dilated Cardiomyopathy-Causing Mutation ACTC E361G in Cardiac Muscle Myofibrils Specifically Abolishes Modulation of Ca2+ Regulation by Phosphorylation of Troponin I

Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca²⁺ sensitivity and increases the rate of Ca²⁺ release from troponin C and the rate of relaxation in cardiac muscle. In vitro experiments indicate that mutations that cause dilated cardiomyopathy (DCM) uncouple this modulation, but this has not been demonstrated in an intact contractile system. Using a Ca²⁺-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic parameters of Ca²⁺ regulation of contractility in single transgenic mouse heart myofibrils. We used propranolol treatment of mice to reduce the level of troponin I and myosin binding protein C (MyBP-C) phosphorylation in their hearts before isolating the myofibrils. In nontransgenic mouse myofibrils, the Ca²⁺ sensitivity of force was increased, the fast relaxation phase rate constant, k_REL, was reduced, and the length of the slow linear phase, t_LIN, was increased when the troponin I phosphorylation level was reduced from 1.02 to 0.3 molPi/TnI (EC₅₀ P/unP = 1.8 ± 0.2, p < 0.001). Native myofibrils from ACTC E361G transgenic mice had a 2.4-fold higher Ca²⁺ sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca²⁺ sensitivity and relaxation parameters of ACTC E361G myofibrils did not depend on the troponin I phosphorylation level (EC₅₀ P/unP = 0.88 ± 0.17, p = 0.39). Nevertheless, modulation of the Ca²⁺ sensitivity of ACTC E361G myofibrils by sarcomere length or {"type":"entrez-protein","attrs":{"text":"EMD57033","term_id":"451702631","term_text":"EMD57033"}}EMD57033 was indistinguishable from that of nontransgenic myofibrils. Overall, EC₅₀ measured in different conditions varied over a 7-fold range. The time course of relaxation, as defined by t_LIN and k_REL, was correlated with EC₅₀ but varied by just 2.7- and 3.3-fold, respectively. Our results confirm that troponin I phosphorylation specifically alters the Ca²⁺ sensitivity of isometric tension and the time course of relaxation in cardiac muscle myofibrils. Moreover, the DCM-causing mutation ACTC E361G blunts this phosphorylation-dependent response without affecting other parameters of contraction, including length-dependent activation and the response to {"type":"entrez-protein","attrs":{"text":"EMD57033","term_id":"451702631","term_text":"EMD57033"}}EMD57033.     

Genetic Diversity and Temporal Variation in the Cyanophage Community Infecting Marine Synechococcus Species in Rhode Island's Coastal Waters

The cyanophage community in Rhode Island's coastal waters is genetically diverse and dynamic. Cyanophage abundance ranged from over 10⁴ phage ml⁻¹ in the summer months to less then 10² phage ml⁻¹ during the winter months. Thirty-six distinct cyanomyovirus g20 genotypes were identified over a 3-year sampling period; however, only one to nine g20 genotypes were detected at any one sampling date. Phylogenetic analyses of g20 sequences revealed that the Rhode Island cyanomyoviral isolates fall into three main clades and are closely related to other known viral isolates of Synechococcus spp. Extinction dilution enrichment followed by host range tests and PCR restriction fragment length polymorphism analysis was used to detect changes in the relative abundance of cyanophage types in June, July, and August 2002. Temporal changes in both the overall composition of the cyanophage community and the relative abundance of specific cyanophage g20 genotypes were observed. In some seawater samples, the g20 gene from over 50% of isolated cyanophages could not be amplified by using the PCR primer pairs specific for cyanomyoviruses, which suggested that cyanophages in other viral families (e.g., Podoviridae or Siphoviridae) may be important components of the Rhode Island cyanophage community.     

3 Beta-hydroxysteroid dehydrogenase-isomerase activity in bovine adrenocortical cells in culture: lack of response to ACTH treatment

Primary cultures of bovine adrenocortical cells (BAC) were used to determine whether the adrenal microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase complex (3 beta-HSD), like the 17 alpha-hydroxylase (17-OHase), responded to ACTH treatment with an increase in activity. Both enzymes influence the steroidogenic path leading to 17 alpha-hydroxyprogesterone formation and thus could affect adrenal androgen biosynthesis. 3 beta-HSD Activity in postmitochondrial supernatant fluid, homogenates or cell monolayers remained unchanged after cells had been maintained in 1 microM ACTH up to 48 h. Since ACTH exposure led to a marked increase in 17-OHase activity over the same time period, it is concluded that, under the conditions used, the 3 beta-HSD-isomerase complex in BAC is nonresponsive to tropic hormone treatment.     

Some properties of human small heat shock protein Hsp20 (HspB6).

Human heat shock protein of apparent molecular mass 20 kDa (Hsp20) and its mutant, S16D, mimicking phosphorylation by cyclic nucleotide-dependent protein kinases, were cloned and expressed in Escherichia coli. The proteins were obtained in a homogeneous state without utilization of urea or detergents. On size exclusion chromatography at neutral pH, Hsp20 and its S16D mutant were eluted as symmetrical peaks with an apparent molecular mass of 55-60 kDa. Chemical crosslinking resulted in the formation of dimers with an apparent molecular mass of 42 kDa. At pH 6.0, Hsp20 and its S16D mutant dissociated, and were eluted in the form of two peaks with apparent molecular mass values of 45-50 and 28-30 kDa. At pH 7.0-7.5, the chaperone activity of Hsp20 (measured by its ability to prevent the reduction-induced aggregation of insulin or heat-induced aggregation of yeast alcohol dehydrogenase) was similar to or higher than that of commercial alpha-crystallin. Under these conditions, the S16D mutant of Hsp20 possessed lower chaperone activity than the wild-type protein. At pH 6.0, both alpha-crystallin and Hsp20 interacted with denatured alcohol dehydrogenase; however, alpha-crystallin prevented, whereas Hsp20 either did not affect or promoted, the heat-induced aggregation of alcohol dehydrogenase. The mixing of wild-type human Hsp27 and Hsp20 resulted in a slow, temperature-dependent formation of hetero-oligomeric complexes, with apparent molecular mass values of 100 and 300 kDa, which contained approximately equal amounts of Hsp27 and Hsp20 subunits. Phosphorylation of Hsp27 by mitogen activated protein kinase-activated protein kinase 2 was mimicked by replacing Ser15, 78 and 82 with Asp. A 3D mutant of Hsp27 mixed with Hsp20 rapidly formed a hetero-oligomeric complex with an apparent molecular mass of 100 kDa, containing approximately equal quantities of two small heat shock proteins.     

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.     

Measuring the Impact of Antiretroviral Therapy Roll-Out on Population Level Fertility in Three African Countries

Background

UNAIDS official estimates of national HIV prevalence are based on trends observed in antenatal clinic surveillance, after adjustment for the reduced fertility of HIV positive women. Uptake of ART may impact on the fertility of HIV positive women, implying a need to re-estimate the adjustment factors used in these calculations. We analyse the effect of antiretroviral therapy (ART) provision on population-level fertility in Southern and East Africa, comparing trends in HIV infected women against the secular trends observed in uninfected women.

Methods

We used fertility data from four community-based demographic and HIV surveillance sites: Kisesa (Tanzania), Masaka and Rakai (Uganda) and uMkhanyakude (South Africa). All births to women aged 15–44 years old were included in the analysis, classified by mother’s age and HIV status at time of birth, and ART availability in the community. Calendar time period of data availability relative to ART Introduction varied across the sites, from 5 years prior to ART roll-out, to 9 years after. Calendar time was classified according to ART availability, grouped into pre ART, ART introduction (available in at least one health facility serving study site) and ART available (available in all designated health facilities serving study site). We used Poisson regression to calculate age adjusted fertility rate ratios over time by HIV status, and investigated the interaction between ART period and HIV status to ascertain whether trends over time were different for HIV positive and negative women.

Results

Age-adjusted fertility rates declined significantly over time for HIV negative women in all four studies. However HIV positives either had no change in fertility (Masaka, Rakai) or experienced a significant increase over the same period (Kisesa, uMkhanyakude). HIV positive fertility was significantly lower than negative in both the pre ART period (age adjusted fertility rate ratio (FRR) range 0.51 95%CI 0.42–0.61 to 0.73 95%CI 0.64–0.83) and when ART was widely available (FRR range 0.57 95%CI 0.52–0.62 to 0.83 95%CI 0.78–0.87), but the difference has narrowed. The interaction terms describing the difference in trends between HIV positives and negatives are generally significant.

Conclusions

Differences in fertility between HIV positive and HIV negative women are narrowing over time as ART becomes more widely available in these communities. Routine adjustment of ANC data for estimating national HIV prevalence will need to allow for the impact of treatment.     

In vitro activity of 15-azasterol (A25822B) against chalkbrood pathogen Ascosphaera apis in the honey bee

The antifungal agent 15-azasterol A25822B was examined for effects on the growth and development of Ascosphaera apis. The minimum inhibition concentration (MIC) of azasterol against A. apis was 1 m. Growth and development of A. apis was completely controlled at this concentration. At a concentration of 0.01 m growth of A. apis was retarded and although sporocysts were formed developing spores were not be able to reach maturation. A major effect of azasterol at this low concentration was the accumulation of lipid in the hyphae, sporocysts and immature spores. In addition it caused a conformational change in mitochondria and damage to the spore membrane structure. On the basis of these results, further investigations of azasterol for the treatment of chalkbrood disease in the honey bee are warranted.Work was performed during sabbatical leave at the University of California, Davis.