Viability of Cryptosporidium parvum during ensilage of perennial ryegrass

The survival of Cryptosporidium parvum during ensilage of perennial ryegrass was examined in laboratory silos with herbage prepared in one of three different ways; either untreated, inoculated with a strain of Lactobacillus plantarum or by direct acidification with formic acid. The pH values of all silages initially fell below 4.5, but only formic acid-treated silage remained stable at less than pH 4 after 106 d, with the pH of the untreated and inoculant-treated silages rising to above 6. The formic acid-treated silage had a high lactic acid concentration (109 g kg^-1 dry matter (DM)) and low concentrations of propionic and butyric acids after 106 d. However, the untreated and inoculant-treated silages showed an inverse relationship, with low lactic acid concentrations and high concentrations of acetic, propionic and butyric acids. These silages also contained ammonia-N concentrations in excess of 9 g kg^-1 DM. In terms of the viability of Cryptosporidium parvum oocysts very few differences were seen after 14 d of ensilage with ca 50% remaining viable, irrespective of treatment and total numbers had declined from the initial level of 5.9 × 10⁴ to 1 x 10⁴ g^-1 fresh matter. Total oocyst numbers remained approximately the same until the end of the ensiling period, with the percentage of viable oocysts declining to 46, 41 and 32% respectively for formic acid, inoculant and untreated silages. The results are discussed in terms of changes occurring during the silage fermentation, in particular the products which may influence the survival of Cryptosporidium and implications for agricultural practice and the health of silage fed livestock.     

Homogeneous chicken heart mitochondrial creatine kinase purified by dye-ligand and transition-state analog-affinity chromatography

A method for the preparation of homogeneous mitochondrial creatine kinase from chicken heart is presented. The two-column procedure, which can be completed in 2 days, uses Procion red dye and transition-state analog-affinity chromatography. The transition-state analog-affinity chromatographic system utilizes an ADP-hexane-agarose column in conjunction with the transition-state analog complex originally developed by E. J. Milner-White and D. C. Watts (1971, Biochem, J. 122, 727-740) composed of KNO3, MgCl2, creatine, and ADP. The enzyme is a dimer composed of 2 Mr 43,000 subunits. The sequence of the first N-terminal 20 amino acids shows that the enzyme is different from the cytosolic isozymes but similar to human mitochondrial creatine kinase. The enzyme has an extinction coefficient of epsilon 280 nm = 2.22 +/- 0.10 ml X mg-1 X cm-1 and a maximum velocity of 200 IU/ml at pH 7.0. The kinetic constants for the chicken heart mitochondrial isozyme are comparable to values for the canine and human heart isozyme.     

Drug modification of protein thiophosphorylation in permeabilized chromaffin cells

Treatment of permeabilized chromaffin cells with low concentrations of the ATP analog adenosine-5′-O-(3-thiotriphosphate)[³⁵S] results in ³⁵S incorporation into a small number of cellular proteins. Of these proteins, a 47 kilodalton protein is most heavily thiophosphorylated. Permeabilized cells were treated with various drugs known to influence cell functions, more specifically chromaffin granule function, to determine the kinase responsible for thiophosphorylation of the 47 kilodalton protein and if its thiophosphorylation is associated with a specific cell function.

Several drugs which influence the activity of cell kinases were examined for their effect on secretion and thiophosphorylation of the 47 kilodalton protein. There was no qualitative effect of cAMP, cGMP or trifluoperazine on thiophosphorylation of the protein. Both cyclic nucleotides slightly enhanced secretion, while trifluoperazine enhanced only unstimulated catecholamine release. Phorbol 12-myristate 13-acetate had no effect on secretion or ³⁵S incorporation into cell proteins. Only the free calcium concentration of the medium influenced thiophosphorylation of the 47 kilodalton protein, with increased calcium producing increased thiophosphorylation.

Drugs affecting chromaffin vesicle functions were used to assess the relationship between specific functions and thiophosphorylation of the protein. Inhibition of nucleotide translocation with atractyloside or 4,4′diisothiocyanatostilbene-2,2′disulfonic acid or inhibition of the proton translocating ATPase by N-ethylmaleimide inhibited thiophosphorylation of the 47 kilodalton protein, with little effect on secretion. Treatment with rotenone markedly enhanced secretion and thiophosphorylation of the protein. Calcium ionophores had no effect on thiophosphorylation of the protein. Dichloroacetic acid, which inhibits phosphorylation of mitochondrial pyruvate dehydrogenase, had no effect on secretion and a variable effect on thiophosphorylation of the 47 kilodalton protein. The data suggest that thiophosphorylation of the protein may be associated with nucleotide translocation across the vesicle membrane.     

Declines in summer bat activity in central New England 4 years following the initial detection of white-nose syndrome

White-nose syndrome (WNS) was first reported in a hibernating bat population in central New York State in February 2006. Since 2006, WNS has been reported from bat hibernacula across much of eastern United States and adjacent Canada and has been associated with a dramatic decline in the populations of hibernating bats in the northeastern U.S. We are only beginning to discover how these declines are manifest in changes in summer bat abundance and activity at local scales. A 3-year (2004–2006) acoustic survey showed that the forested watershed of the Quabbin Reservoir in central Massachusetts supported an abundant and species-rich summer bat community. In 2010, 4-years following the initial occurrence of WNS, a re-survey of the same habitats and sites found a 72% reduction in bat activity on the watershed. This is the identical rate of decline reported from cave hibernacula surveys (73%). This decline in summer activity levels is most likely a consequence of WNS-caused mortality. The impacts of population losses of this magnitude of a once widespread and abundant taxa are unknown but are presumed to be ecologically significant.     

Alloxan inhibition of a Ca2+- and calmodulin-dependent protein kinase activity in pancreatic islets

Alloxan was found to inhibit a Ca2+- and calmodulin-dependent protein kinase recently identified in pancreatic islets. This effect of alloxan may be specifically related to the inhibitory action of alloxan on insulin secretion from islets since: 1) in islet-cell subcellular fractions, alloxan at micromolar concentrations irreversibly inhibits the Ca2+- and calmodulin-dependent protein kinase activity; 2) pretreatment of intact islets with alloxan at concentrations that inhibit insulin secretion similarly inhibits the protein kinase activity; and 3) alloxan inhibition of both insulin secretion and protein kinase activity in intact islets can be prevented by D-glucose. This inhibition by alloxan appears to be a direct effect on the enzyme since alloxan treatment of either the islet homogenate or the microsomal fraction enriched in protein kinase activity inhibited the kinase activity with similar concentration dependence. These results suggest that alloxan-induced inhibition of a Ca2+- and calmodulin-dependent protein kinase may represent a critical inhibitory site which mediates alloxan-induced inhibition of insulin secretion.     

A general method to quantify quasi-equivalence in icosahedral viruses

A quantitative, atom-based, method is described for comparing protein subunit interfaces in icosahedral virus capsids with quasi-equivalent surface lattices. An integrated, normalized value (between 0 and 1) based on equivalent residue contacts (Q-score) is computed for every pair of subunit interactions and scores that are significantly above zero readily identify interfaces that are quasi-equivalent to each other. The method was applied to all quasi-equivalent capsid structures (T=3, 4, 7 and 13) in the Protein Data Bank and the Q-scores were interpreted in terms of their structural underpinnings. The analysis allowed classification of T=3 structures into three groups with architectures that resemble different polyhedra with icosahedral symmetry. The preference of subunits to form dimers in the T=4 human Hepatitis B virus capsid (HBV) was clearly reflected in high Q-scores of quasi-equivalent dimers. Interesting differences between the classical T=7 capsid and polyoma-like capsids were also identified. Application of the method to the outer-shell of the T=13 Blue tongue virus core (BTVC) highlighted the modest distortion between the interfaces of the general trimers and the strict trimers of VP7 subunits. Furthermore, the method identified the quasi 2-fold symmetry in the inner capsids of the BTV and reovirus cores. The results show that the Q-scores of various quasi-symmetries represent a "fingerprint" for a particular virus capsid architecture allowing particle classification into groups based on their underlying structural and geometric features.     

No intra-locus sexual conflict over reproductive fitness or ageing in field crickets

Differences in the ways in which males and females maximize evolutionary fitness can lead to intra-locus sexual conflict in which genes delivering fitness benefits to one sex are costly when expressed in the other. Trade-offs between current reproductive effort and future reproduction and survival are fundamental to the evolutionary biology of ageing. This leads to the prediction that sex differences in the optimization of age-dependent reproductive effort may generate intra-locus sexual conflict over ageing rates. Here we test for intra-locus sexual conflict over age-dependent reproductive effort and longevity in the black field cricket, Teleogryllus commodus. Using a half-sib breeding design, we show that the most important components of male and female reproductive effort (male calling effort and the number of eggs laid by females) were positively genetically correlated, especially in early adulthood. However, the genetic relationships between longevity and reproductive effort were different for males and females, leading to low genetic covariation between male and female longevity. The apparent absence of intra-locus sexual conflict over ageing suggests that male and female longevity can evolve largely independently of one another.