Structure-function studies of human ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) is a polypeptide that promotes the survival and/or differentiation of a number of neural cell types. Here we present a structural and functional analysis of the human CNTF molecule. Variant proteins were synthesized byEscherichia coli trnsformmed with mutant cDNA constructs, and purified by SDS-polyacrylamide gel electrophoresis and reverse phase high pressure liquid chromatography. Most variant CNTF proteins lacked neurotrophic activity, but two N-and C-terminal deletions (2–14 and 173–200, respectively) actually displayed a several-fold increase in specific activity. Loss of biological activity was accompanied by changes in the alphahelical nature of CNTF as measured by circular dichroism. These data strengthen the proposed similarity between CNTF and the family of hematopoietic cytokines.     

HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins

Chromatin remodelling complexes containing the nucleosome-dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Our purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. We show that the human homologues of two novel putative histone-fold proteins in Drosophila CHRAC are present in HuCHRAC. The two human histone-fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homologue of Acf1, a subunit of Drosophila ACF. The N-terminus of mouse ACF1 was reported as a heterochromatin-targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. We discuss a possible function for HuCHRAC in heterochromatin dynamics. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC.     

Subunit structures of purified beef mitochondrial cytochromebc 1 complex from liver and heart

The existence of tissue-specific isozymes of cytochromec oxidase has been widely documented. We have now studied if there are differences between subunits of mitochondrialbc ₁ complexes isolated from liver and heart. For this purpose, we have developed a method for the purification of an active ubiquinol-cytochromec oxidoreductase from adult bovine liver that includes solubilization of submitochondrial particles with deoxycholate, ammonium acetate fractionation, resolubilization with dodecyl maltoside, and ion exchange chromatography. The electrophoretic pattern of the liver preparation showed the presence of 11 subunits, with apparent molecular weights identical to the ones reported for the heart complex. Western blot analysis and isoelectric focusing followed by two-dimensional gels ofbc ₁ complexes from liver and heart were compared, and no qualitative differences were observed. In addition, the high-molecular-weight subunits of the purified complexes from both tissues, subunits I, II, V, and VI, were isolated by PAGE in the presence of Coomasie Blue and subjected to limited proteolysis and to chemical digestion with cyanogen bromide and BNPS-skatol, and the peptide patterns were compared. Finally, two of the small-molecular-weight subunits from the liver complex were isolated (subunits VII and X), partially analyzed by amino terminal sequencing, and found to be identical with the reported sequence of their heart counterparts. The data suggest that, in contrast to the case of cytochromec oxidase,bc ₁ complexes from liver and heart do not exhibit tissue-specific differences.     

Isolation and Partial Characterization of Antifungal Metabolites Produced by <Emphasis Type="Italic">Bacillus</Emphasis> sp. IBA 33

Antifungal proteins produced by Bacillus sp. IBA 33 were purified by ammonium sulfate precipitation and DEAE-Sephacel column chromatography. The two purified proteins inhibited the growth of Geotrichum candidum, the sour rot disease agent in lemon. The proteins were stable at 20 (3 months), 40, 60 and 100°C (30 min) and remained active after sterilization at 121°C for 15 min. Their hydrophobic nature was proved and when were developed with ninhydrin they did not show any free amino groups. The infrared spectrum showed vibrational modes corresponding to peptide, ester or ketone links and saturated CH links corresponding to long chain fatty acids. UV scan spectroscopy showed tyrosine and or tryptophan amino acids in their composition. The remarkable thermo-resistance of proteins may be a good feature to be used in the development of a new biocontrol method of Geotrichum candidum.     

Overwintering Is Associated with Reduced Expression of Immune Genes and Higher Susceptibility to Virus Infection in Honey Bees

The eusocial honey bee, Apis mellifera, has evolved remarkable abilities to survive extreme seasonal differences in temperature and availability of resources by dividing the worker caste into two groups that differ in physiology and lifespan: summer and winter bees. Most of the recent major losses of managed honey bee colonies occur during the winter, suggesting that winter bees may have compromised immune function and higher susceptibility to diseases. We tested this hypothesis by comparing the expression of eight immune genes and naturally occurring infection levels of deformed wing virus (DWV), one of the most widespread viruses in A. mellifera populations, between summer and winter bees. Possible interactions between immune response and physiological activity were tested by measuring the expression of vitellogenin and methyl farnesoate epoxidase, a gene coding for the last enzyme involved in juvenile hormone biosynthesis. Our data show that high DWV loads in winter bees correlate with reduced expression of genes involved in the cellular immune response and physiological activity and high expression of humoral immune genes involved in antibacterial defense compared with summer bees. This expression pattern could reflect evolutionary adaptations to resist bacterial pathogens and economize energy during the winter under a pathogen landscape with reduced risk of pathogenic viral infections. The outbreak of Varroa destructor infestation could have overcome these adaptations by promoting the transmission of viruses. Our results suggest that reduced cellular immune function during the winter may have increased honey bee’s susceptibility to DWV. These results contribute to our understanding of honey bee colony losses in temperate regions.     

The ISWI chromatin remodeler organizes the hsrω ncRNA-containing omega speckle nuclear compartments

The complexity in composition and function of the eukaryotic nucleus is achieved through its organization in specialized nuclear compartments. The Drosophila chromatin remodeling ATPase ISWI plays evolutionarily conserved roles in chromatin organization. Interestingly, ISWI genetically interacts with the hsrω gene, encoding multiple non-coding RNAs (ncRNA) essential, among other functions, for the assembly and organization of the omega speckles. The nucleoplasmic omega speckles play important functions in RNA metabolism, in normal and stressed cells, by regulating availability of hnRNPs and some other RNA processing proteins. Chromatin remodelers, as well as nuclear speckles and their associated ncRNAs, are emerging as important components of gene regulatory networks, although their functional connections have remained poorly defined. Here we provide multiple lines of evidence showing that the hsrω ncRNA interacts in vivo and in vitro with ISWI, regulating its ATPase activity. Remarkably, we found that the organization of nucleoplasmic omega speckles depends on ISWI function. Our findings highlight a novel role for chromatin remodelers in organization of nucleoplasmic compartments, providing the first example of interaction between an ATP-dependent chromatin remodeler and a large ncRNA.     

Paced-mating increases the number of adult new born cells in the internal cellular (granular) layer of the accessory olfactory bulb

The continuous production and addition of new neurons during life in the olfactory bulb is well accepted and has been extensively studied in rodents. This process could allow the animals to adapt to a changing environment. Olfactory neurogenesis begins in the subventricular zone where stem cells proliferate and give rise to young undifferentiated neuroblasts that migrate along the rostral migratory stream to the olfactory bulb (OB). Olfaction is crucial for the expression of sexual behavior in rodents. In female rats, the ability to control the rate of sexual interactions (pacing) has important physiological and behavioral consequences. In the present experiment we evaluated if pacing behavior modifies the rate of new cells that reach the main and accessory olfactory bulb. The BrdU marker was injected before and after different behavioral tests which included: females placed in a mating cage (control), females allowed to pace the sexual interaction, females that mated but were not able to control the rate of the sexual interaction and females exposed to a sexually active male. Subjects were sacrificed fifteen days after the behavioral test. We observed a significant increase in the density of BrdU positive cells in the internal cellular layer of the accessory olfactory bulb when females paced the sexual interaction in comparison to the other 3 groups. No differences in the cell density in the main olfactory bulb were found. These results suggest that pacing behavior promotes an increase in density of the new cells in the accessory olfactory bulb.