Isolation and characterization of several unique lipids from Azotobacter vinelandii cysts.

Unique cyclic compounds were found in the lipid fraction of Azotobacter vinelandii cysts. In addition to two major molecular species which had already been identified, 5-n-alkylresorcinol and its galactoside derivative, five other molecular species (two alkyl side chain homologs of each) were isolated, and their structures were established by infrared, ultraviolet, nuclear magnetic resonance, and mass spectroscopy. These 10 compounds were 6-n-heneicosylresorcylic acid methyl ester and 6-n-tricosylresorcylic acid methyl ester, 5-n-(2-hydroxy)heneicosylresorcinol and 5-n-(2-hydroxy-tricosylresorcinol, 5-n-heneicosyl-4-acetylresorcinol and 5-n-tricosyl-4-acetylresorcinol, 6-n-heneicosyl-4-hydroxypyran-2-one and 6-n-tricosyl-4-hydroxypyran-2-one, and 6-(2-oxotricosyl)-4-hydroxy-pyran-2-one and 6-(2-oxopentacosyl)-4-hydroxypyran-2-one.     

Female Choice, Female Reluctance to Mate and Sexual Selection on Body Size in the Dung Fly Sepsis cynipsea

We investigated the mechanisms of sexual selection in the common dung fly Sepsis cynipsea and how these affect selection on body size at the population level. Because of the presumed costs associated with mating, we predicted that there would be a decrease in the general reluctance of females to mate with any particular male at higher male densities at the mating site, a fresh cow pat, resulting in indirect female choice and a decrease in the strength of sexual selection. In contrast, classical direct female choice and male‐male competition should result in increased selection intensities because more opportunities for choice and competition exist at higher densities. Female reluctance to mate and female assessment of males are expressed in prominent female behaviour to repel mates in several insect species, including S. cynipsea. Laboratory pair‐wise choice experiments showed that large males were more likely to obtain copulations, which also ensued more promptly, suggesting female assessment of male quality (direct female choice). There was a basic influence of male activity but little further effect of male scramble competition on the outcome of mating. Another laboratory experiment showed a decrease in female shaking duration per male, associated with an asymptote in the shaking duration per female, as male density and harassment increased, but did not show the increase in mating frequency predicted by the female reluctance hypothesis. A study estimating sexual selection differentials in the field showed that directional selection for larger males was present overall and was negatively related to seasonally mediated variation in male density. Our study suggests that direct female choice in combination with indirect female choice (due to an interaction of female reluctance to mate and male persistence) is most consistent with the behavioural and selection patterns observed in S. cynipsea, but male effects cannot be definitively excluded.     

Methoxylated fatty acids reported in Rhizobium isolates arise from chemical alterations of common fatty acids upon acid-catalyzed transesterification procedures.

We obtained from a phospholipid extract of wild-type Rhizobium leguminosarum bv. trifolii ANU843 methoxylated fatty acids that had been previously reported as constitutive unusual Rhizobium fatty acids. The use of deuterated reagents and subsequent gas-liquid chromatography-mass spectrometry analyses showed that these methoxylated fatty acid derivatives are the products of chemical alterations of common cyclopropane-containing and unsaturated fatty acids occurring during various acid-catalyzed transesterification treatments aimed at producing the methyl ester derivatives. Similar results were obtained from a phospholipid extract of Escherichia coli K-12. In contrast, these chemical alterations were not induced by an alkaline methanolysis method of transesterification. If an acidic treatment is needed to release the fatty acids from the source molecule, the finding of unusual methoxylated fatty acids should be carefully confirmed with deuterated reagents.     

Kinetics of folding of Escherichia coli OmpA from narrow to large pore conformation in a planar bilayer

The outer membrane protein of Escherichia coli, OmpA, is currently alleged to adopt two native conformations: a major two-domain conformer in which 171 N-terminal residues form a narrow eight beta-barrel pore and 154 C-terminal residues are in the periplasm and a minor one-domain conformer in which all 325 residues create a large pore. However, recent studies in planar bilayers indicate the conformation of OmpA is temperature-sensitive and that increasing temperature converts narrow pores to large pores. Here we examine the reversibility and kinetics of this transition for single OmpA molecules in planar bilayers of diphytanoylphosphatidylcholine (DPhPC). We find that the transition is irreversible. When temperatures are decreased, large pores close down, and when temperatures are stabilized they reopen in the large pore conformation, with gradually increasing open time. Large pores are converted to narrow pores only by denaturing agents. The transition from narrow to large pores requires temperatures >or= 26 degrees C and is a biphasic process with rates that rise steeply with temperature. The first phase, a flickering stepwise transition from a low-conductance to a high-conductance state requires approximately 7 h at 26 degrees C but only approximately 13 min at 42 degrees C, signifying an activation energy of 139 +/- 12 kJ/mol. This is followed by a gradual increase in conductance and open probability, interpreted as optimization of the large pore structure. The results indicate that the two-domain structure is a partially folded intermediate that is kinetically stable at lower temperatures and that mature fully folded OmpA is a large pore.     

Local genetic structure in a clonal dioecious angiosperm

We used seven microsatellite loci to characterize genetic structure and clonal architecture at three different spatial scales (from meters to centimetres) of a Cymodocea nodosa population. C. nodosa exhibits both sexual reproduction and vegetative propagation by rhizome elongation. Seeds remain buried in the sediment nearby the mother plant in a dormant stage until germination. Seed dispersal potential is therefore expected to be extremely restricted. High clonal diversity (up to 67% of distinct genotypes) and a highly intermingled configuration of genets at different spatial scales were found. No significant differences in genetic structure were found among the three spatial scales, indicating that genetic diversity is evenly distributed along the meadow. Autocorrelation analyses of kinship estimates confirmed the absence of spatial clumping of genets at small spatial scale and the expectations of a very restricted seed dispersal (observed dispersal range 1-21 m) in this species.     

Codon bias and plasticity in immunoglobulins

Immunoglobulin genes experience Darwinian evolution twice. In addition to the germline evolution all genes experience, immunoglobulins are subjected, upon exposure to antigen, to somatic hypermutation. This is accompanied by selection for high affinity to the eliciting antigen and frequently results in a significant increase in the specificity of the responding population. The hypermutation mechanism displays a strong sequence specificity. Thus arises the opportunity to manipulate codon bias in a site-specific manner so as to direct hypermutation to those parts of the gene that encode the antigen-binding portions of the molecule and away from those that encode the structurally conserved regions. This segregation of mutability would clearly be advantageous; it would enhance the generation of potentially useful variants while keeping mutational loss to acceptably low levels. But it is not clear that the advantage gained would be large enough to produce a measurable effect within the background stochasticity of the evolutionary process. I have performed a pair of statistical tests to determine whether site- specific codon bias in human immunoglobulin genes is correlated with the sequence specificity of the somatic mutation mechanism. The sequence specificity of the mutator was determined by analysis of a database of published immunoglobulin intron sequences that had experienced somatic mutation but not selection. The site-specific codon bias was determined by analysis of published sequences of human germline immunoglobulin V genes. Both tests strongly suggest that evolution has acted to enhance the plasticity of immunoglobulin genes under somatic hypermutation.      

Soluble Adenylyl Cyclase Controls Mitochondria-dependent Apoptosis in Coronary Endothelial Cells

The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC- and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells.Increasing evidence suggests that apoptosis of endothelial cells (EC)³ may be responsible for acute and chronic vascular diseases, e.g. through atherogenesis (1), endothelial dysfunction (2), or thrombosis (3). Within several signaling mechanisms, a cAMP-dependent signaling pathway plays a substantial role in mediating apoptotic cell death induced by various stress factors. Elevation of the cellular cAMP either by forskolin-induced stimulation of the G-protein-responsive transmembrane adenylyl cyclase (tmAC) or by treatment with cAMP analogs has been shown to lead to both induction and suppression of apoptosis in different cell types (4–7). This discrepancy may be due to differences in cell types and experimental models. Alternatively, a lack of specificity of tmAC-induced signals, especially directed to distant intracellular targets like mitochondria, may be a cause of the discrepancy. Indeed, the classical model of cAMP signaling requires the diffusion of cAMP from plasma membrane-localized tmAC to targets localized throughout the cell. Diffusion of cAMP throughout the cytosol makes it difficult to selectively activate distally localized targets without also activating more proximal targets. Therefore, such diffusion of cAMP would likely diminish specificity, selectivity, and signal strength. This model is further complicated by the presence of phosphodiesterases, which degrade cAMP, thus preventing its diffusion.In addition to tmAC, a second source of cAMP, soluble adenylyl cyclase (sAC), was demonstrated for mammalian cells (8, 9). Cytosolic localization of sAC provides both specificity and selectivity by permitting generation of cAMP proximal to intracellular targets. Furthermore, this model for cAMP action incorporates phosphodiesterases, which would act to limit diffusion and prevent nonspecific effector activation.Whether sAC participates in apoptosis was unknown. A previous report demonstrated that sAC is co-localized with mitochondria (10). Because mitochondria play a fundamental role in apoptosis (11), we hypothesized that sAC may influence the development of apoptosis by modulating the mitochondrial pathway of apoptosis. Therefore, we aimed to examine the role of sAC in apoptotic cell death, especially its role in the modulation of the mitochondria-dependent pathway of apoptosis. For this purpose, apoptosis was induced in rat coronary EC by simulated in vitro ischemia or by acidosis. By applying pharmacological inhibition of sAC or small interfering RNA (siRNA)-mediated sAC knockdown, we found that sAC activity is required for the induction of apoptosis by ischemia or acidosis. Additionally, translocation of sAC to mitochondria and the sAC-dependent release of cytochrome c suggest that this cyclase specifically regulates the mitochondrial pathway of apoptosis.     

Intracellular receptors for 5alpha-dihydrostestosterone in the epididymis of adult rats. Comparison with the androgenic receptors in the ventral prostate and the androgen binding protein (ABP) in the testicular and epididymal fluid

Epididymal cytosol fractions of adult short-time castrated rats contained at least two different androgen protein complexes by experiments $\text{in vivo}$ (Complex I and II).Complex I is probably located intracellularly in the epididymal cells. It was specific for 5α-dihydrotestosterone (DHT) and appeared to be very similar to the cytoplasmic DHT-receptor complexes in rat ventral prostate. By ultracentrifugation on sucrose gradients, it sedimented as heavy aggregates 8–10 S complexes and 3–4 S complexes, which dissociated into 3–4 S complexes at high ionic strength. Complex I was eluted in the void volume from columns of Sephadex G-200.Complex II was also specific for DHT and showed physical properties similar to those of the androgen binding protein (ABP) in the testicular fluid. It was eluted between immunoglobulin G (IgG) (53 Å) and albumin (36 Å) by gel filtration on Sephadex G-200. The sedimentation coefficient was 4.5–5 S (mean 4.6 S_W, 20) at both high and low ionic strength.Complex I and the cytosol receptors for DHT in the rat ventral prostate were both destroyed by heating at 50° C for 30 min, addition of 1 mM p-chloro-mercuri-phenyl-sulphonate (PCMPS) and charcoal absorption (1 mg/mg protein) overnight, whereas complex II was not influenced by similar treatment.Hemi-castration for 4 weeks caused complex II to disappear completely from the castrated side, confirming the intraluminal localization of this complex. Complex I was not influenced by such treatment, indicating that this protein is located within the epididymal cells. The similarity between complex I and the cytoplasmic DHT-receptor complexes in the ventral prostate also suggests that complex I represents the cytoplasmic receptors for DHT in the epididymis.     

Characterisation of <Emphasis Type="Italic">mycobacteria</Emphasis> isolated from slaughter cattle in pastoral regions of Uganda

Background  

Bovine tuberculosis is a zoonotic problem in pastoral cattle and communities in Uganda. Tuberculin tests in pastoral cattle had shown a high herd but low animal prevalence, with a high proportion of avian reactors. No work had been done to identify the mycobacterial species involved. The objective of the study was to isolate and characterise Mycobacterial species causing tuberculous lesions in slaughtered animals. Lesioned organs compatible with bovine tuberculosis in slaughtered cattle from pastoral areas in Uganda were collected and cultured to isolate mycobacteria. AccuProbe culture identification kits for the Mycobacterium tuberculosis complex, M. avium complex and M. avium were used to identify the isolates. Spoligotyping and Insertion Sequence (IS) 1311 and IS1245 Restriction Fragment Length Polymorphism analysis (RFLP) were used to further characterise the isolates.