A single functional testis and long deferent duct papillae: the peculiar male reproductive tract of the classically polyandrous, sex-role reversed Black Coucal (Centropus grillii)

In many birds, the male reproductive tract is asymmetric with the left testis being larger than the right one. Coucals (Centropodidae) represent an exception as the asymmetry is typically reversed. Here, we describe the functional morphology of the reproductive tract of the African Black Coucal (Centropus grillii), a bird species with reversed sex-roles and a classical polyandrous mating system. In this species, the left testis has been reduced to a tiny, disk-like vestige that in almost all cases examined was not visible macroscopically. The vestigial left testis apparently does not participate in sperm production but has retained a vestigial left excurrent duct system. By contrast, the right deferent duct was engorged with sperm, suggesting a sperm storage function. Both deferent ducts opened on the tip of spectacularly long, erectile deferent duct papillae into the urodaeum of the male cloaca. Behavioural observations suggest that they may function as “pseudophalli”. Testis mass represented 0.49% of body mass, less than half the size of other classical polyandrous bird species. Assuming moderate to high levels of sperm competition this represents a paradox. Heavy demands on the energy budget while caring for altricial young may have prevented males from maintaining two testes and large bilateral sperm storage devices. Reduced testis mass may be compensated for by frequent transfer of small amounts of sperm. Female Black Coucals have not evolved any corresponding cloacal specializations, but like all other birds have sperm storage tubules.     

A role for tumor protein TPD52 phosphorylation in endo-membrane trafficking during cytokinesis

Tumor protein D52 is expressed at high levels in exocrine cells containing large secretory granules where it regulates Ca²⁺-dependent protein secretion; however, D52 expression is also highly induced in multiple cancers. The present study investigated a role for the Ca²⁺-dependent phosphorylation of D52 at the single major phospho-acceptor site serine 136 on cell division. Ectopic expression of wild type D52 (D52wt) and the phosphomutants serine 136/alanine (S136A) or serine 136/glutamate (S136/E) resulted in significant multinucleation of cells. D52wt and S136/E each resulted in a greater than 2-fold increase in multinucleated cells compared to plasmid-transfected controls whereas the S136/A phospho-null mutant caused a 9-fold increase in multinucleation at 48 h post-transfection. Electron microscopy revealed D52 expression induced a marked accumulation of vesicles along the mid-line between nuclei where the final stages of cell abscission normally occurs. Supporting this, D52wt strongly colocalized on vesicular structures containing the endosomal regulatory protein vesicle associated membrane protein 8 (VAMP 8) and this colocalization significantly increased with elevations in cellular Ca²⁺. As VAMP 8 is known to be necessary for the endo-membrane fusion reactions that mediate the final stages of cytokinesis, these data indicate that D52 expression and phosphorylation at serine 136 play an important role in supporting the Ca²⁺-dependent membrane trafficking events necessary for cytokinesis in rapidly proliferating cancer cells.     

Correlated light and electron microscopy illuminates the role of mitochondrial inner membrane remodeling during apoptosis

In addition to their role in providing ATP for cellular functions via oxidative phosphorylation, mitochondria also play a critical role in initiating and/or regulating apoptosis through the release of proteins such as cytochrome c from intermembrane and intracristal compartments. The mechanism by which these proteins are able to cross the outer mitochondrial membrane has been a subject of controversy. This paper will review some recent results that demonstrate that inner mitochondrial membrane remodeling does occur during apoptosis in HeLa cells but does not appear to be a requirement for release of cytochrome c from intracristal compartments. Inner membrane remodeling does appear to be related to fragmentation of the mitochondrial matrix, and the form of the remodeling suggests a topological mechanism for inner membrane fission and fusion.     

Molecular monitoring of inactivation efficiencies of bacteria during pulsed electric field treatment of clinical wastewater

Aims: The applicability of an alternative wastewater disinfection concept based on the pulsed electric field (PEF) treatment is tested with molecular biology techniques using clinical wastewaters. Methods and Results: Hospital wastewater was treated with the PEF technology. The inactivation efficiencies of bacteria were successfully monitored with real‐time polymerase chain reaction (PCR). As the differentiation between living and dead bacterial cells is important for the determination of the disinfection efficiency, propidium monoazide (PMA) was applied. PMA selectively penetrates cells with compromised membranes and intercalates into the DNA inhibiting a subsequent PCR amplification. The rates of reduction were examined for specific pathogens and wastewater populations using PCR‐denaturing gradient gel electrophoresis. The results showed that the main part of the bacterial population could be inactivated efficiently with the PEF treatment. Moreover, it was demonstrated that naturally occurring nuclease activities were not affected by the PEF treatment in contrast to a thermal treatment. Conclusions: The results indicated that the PEF treatment is an appropriate alternative disinfection concept for the treatment of clinical wastewaters and surpass the disadvantages of other disinfection methods. Significance and Impact of the Study: With the use of propidium monoazide for live–dead distinction, a new concept could be developed for the evaluation of disinfection methods.     

Influenza C virus and bovine coronavirus esterase reveal a similar catalytic mechanism: new insights for drug discovery

Both, the influenza C (INF-C) virus haemagglutinin esterase fusion and bovine coronavirus (BCoV) haemagglutinin esterase surface glycoproteins exhibit a lectin binding capability and a receptor-destroying 9-O-acetyl esterase activity that recognise 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2))-containing glycans. Here we report nuclear magnetic resonance and molecular modelling studies on the 9-O-acetyl esterase showing that the alpha-configured Neu5,9Ac(2) is strictly preferred by the INF-C and BCoV esterases. Interestingly, we have discovered that the INF-C esterase function releases acetate independently of the chemical nature of the aglycon moiety, whereas subtle differences in substrate recognition were found for BCoV esterase. Analysis of the apo and complexed X-ray crystal structure of INF-C esterase revealed that binding of 9-O-acetylated N-acetylneuraminic acids is a dynamic process that involves conformational rearrangement of serine-57 in the esterase active site. This study provides valuable insights towards the design of drugs to combat INF-C virus and coronavirus infections causing outbreaks of upper respiratory infections and severe diarrhea in calves, respectively.     

Molecular epidemiology of Bacillus anthracis: determining the correct origin

We analyzed and compared strains of Bacillus anthracis isolated from husbandry and industrial anthrax cases in Switzerland between 1952 and 1981 with published data using multiple-locus variable-number tandem repeat analysis. Strains isolated from autochthonous cases of anthrax in cattle belong to genotype B2, together with strains from continental Europe, while human B. anthracis strains clustered with genotype A4. These strains could be traced back to outbreaks of human anthrax that occurred between 1978 and 1981 in a factory processing cashmere wool from the Indian subcontinent. We interpret the worldwide occurrence of B. anthracis strains of cluster A4 to be due to the extensive global trade of untreated cashmere wool during the last century.     

Following nature's roadmap: folding factors from plasma cells led to improvements in antibody secretion in S. cerevisiae

Therapeutic protein production in yeast is a reality in industry with an untapped potential to expand to more complex proteins, such as full‐length antibodies. Despite numerous engineering approaches, cellular limitations are preventing the use of Saccharomyces cerevisiae as the titers of recombinant antibodies are currently not competitive. Instead of a host specific approach, the possibility of adopting the features from native producers of antibodies, plasma cells, to improve antibody production in yeast. A subset of mammalian folding factors upregulated in plasma cells for expression in yeast and screened for beneficial effects on antibody secretion using a high‐throughput ELISA platform was selected. Co‐expression of the mammalian chaperone BiP, the co‐chaperone GRP170, or the peptidyl‐prolyl isomerase FKBP2, with the antibody improved specific product yields up to two‐fold. By comparing strains expressing FKBP2 or the yeast PPIase Cpr5p, the authors demonstrate that speeding up peptidyl‐prolyl isomerization by upregulation of catalyzing enzymes is a key factor to improve antibody titers in yeast. The findings show that following the route of plasma cells can improve product titers and contribute to developing an alternative yeast‐based antibody factory.     

TNF-alpha enhances intracellular glucocorticoid availability.

For understanding the mechanism(s) relating inflammation to corticosteroid action, the effect of tumour necrosis factor-alpha (TNF-alpha) on 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), the enzyme regulating access of 11beta-hydroxycorticosteroids to receptors, was studied in LLC-PK(1) cells. We observed (i) NAD-dependent enzyme activity and mRNA for 11beta-HSD2, but not 11beta-HSD1, (ii) increasing 11beta-HSD2 activity with increasing degree of differentiation and (iii) a concentration-dependent down-regulation by TNF-alpha, phorbol myristate acetate (PMA) or glucose of activity and mRNA of 11beta-HSD2. The decrease of activity and mRNA by glucose and PMA, but not that by TNF-alpha, was abrogated by the protein kinase C inhibitor GF-109203X. The effect of TNF-alpha on 11beta-HSD2 was reversed by inhibiting the mitogen-activated protein kinases ERK with PD-098050 and p38 by SB-202190, or by activating protein kinase A with forskolin. Overexpression of MEK1, an ERK activator, down-regulated the 11beta-HSD2 activity. In conclusion, TNF-alpha decreases 11beta-HSD2 activity and thereby enhances glucocorticoid access to glucocorticoid receptors to modulate the inflammatory response.     

Isoindolinone ureas: a novel class of KDR kinase inhibitors

A series of substituted isoindolinone ureas was prepared and evaluated for enzymatic and cellular inhibition of KDR kinase activity. Several of these analogs, such as 14c, are potent inhibitors of KDR both enzymatically (< 50 nM) and cellularly < or = 100 nM). A 3D KDR/CDK2/MAP kinase overlay model with several structurally related tyrosine kinase inhibitors was used to predict the binding interactions of the isoindolinone ureas with the KDR active site.