Comparison of milk produced by cows cloned by nuclear transfer with milk from non-cloned cows

Cloning technologies, including embryo splitting and nuclear transfer, were introduced into dairy cattle breeding in the early 1980s. With the recent worldwide attention on the cloning of sheep ("Dolly") and cows ("Gene"), the potential food safety concerns for food products derived from cloned animals needs to be addressed. There has been no study of the composition of milk produced by cloned cows. In this preliminary study, we evaluated the composition of milk from 15 lactating non-embryonic cell cloned cows and six non-cloned lactating cows over a single season. The cloned cows came from five unique genetic lines and three distinct breeds. Milk samples were analyzed for total solids, fat, fatty acid profile, lactose, protein and compared to non-cloned and literature values. Gross chemical composition of milk from cloned cows was similar to that of the non-cloned cows and literature values. Our results lead us to conclude that there are no obvious differences in milk composition produced from cloned cows compared to non-cloned cows.     

Cyclooxygenase-2 deficiency leads to intestinal barrier dysfunction and increased mortality during polymicrobial sepsis

Sepsis remains the leading cause of death in critically ill patients, despite modern advances in critical care. Intestinal barrier dysfunction may lead to secondary bacterial translocation and the development of the multiple organ dysfunction syndrome during sepsis. Cyclooxygenase (COX)-2 is highly upregulated in the intestine during sepsis, and we hypothesized that it may be critical in the maintenance of intestinal epithelial barrier function during peritonitis-induced polymicrobial sepsis. COX-2(-/-) and COX-2(+/+) BALB/c mice underwent cecal ligation and puncture (CLP) or sham surgery. Mice chimeric for COX-2 were derived by bone marrow transplantation and underwent CLP. C2BBe1 cells, an intestinal epithelial cell line, were treated with the COX-2 inhibitor NS-398, PGD(2), or vehicle and stimulated with cytokines. COX-2(-/-) mice developed exaggerated bacteremia and increased mortality compared with COX-2(+/+) mice following CLP. Mice chimeric for COX-2 exhibited the recipient phenotype, suggesting that epithelial COX-2 expression in the ileum attenuates bacteremia following CLP. Absence of COX-2 significantly increased epithelial permeability of the ileum and reduced expression of the tight junction proteins zonula occludens-1, occludin, and claudin-1 in the ileum following CLP. Furthermore, PGD(2) attenuated cytokine-induced hyperpermeability and zonula occludens-1 downregulation in NS-398-treated C2BBe1 cells. Our findings reveal that absence of COX-2 is associated with enhanced intestinal epithelial permeability and leads to exaggerated bacterial translocation and increased mortality during peritonitis-induced sepsis. Taken together, our results suggest that epithelial expression of COX-2 in the ileum is a critical modulator of tight junction protein expression and intestinal barrier function during sepsis.     

Tandem action of glycosyltransferases in the maturation of vancomycin and teicoplanin aglycones: novel glycopeptides

The glycopeptides vancomycin and teicoplanin are clinically important antibiotics. The carbohydrate portions of these molecules affect biological activity, and there is great interest in developing efficient strategies to make carbohydrate derivatives. To this end, genes encoding four glycosyltransferases, GtfB, C, D, E, were subcloned from Amycolatopsis orientalis strains that produce chloroeremomycin (GtfB, C) or vancomycin (GtfD, E) into Escherichia coli. After expression and purification, each glycosyltransferase (Gtf) was characterized for activity either with the aglycones (GtfB, E) or the glucosylated derivatives (GtfC, D) of vancomycin and teicoplanin. GtfB efficiently glucosylates vancomycin aglycone using UDP-glucose as the glycosyl donor to form desvancosaminyl-vancomycin (vancomycin pseudoaglycone), with k(cat) of 17 min(-1), but has very low glucosylation activity, < or = 0.3 min(-1), for an alternate substrate, teicoplanin aglycone. In contrast, GtfE is much more efficient at glucosylating both its natural substrate, vancomycin aglycone (k(cat) = 60 min(-1)), and an unnatural substrate, teicoplanin aglycone (k(cat) = 20 min(-1)). To test the addition of the 4-epi-vancosamine moiety by GtfC and GtfD, synthesis of UDP-beta-L-4-epi-vancosamine was undertaken. This NDP-sugar served as a substrate for both GtfC and GtfD in the presence of vancomycin pseudoaglycone (GtfC and GtfD) or the glucosylated teicoplanin scaffold, 7 (GtfD). The GtfC product was the 4-epi-vancosaminyl form of vancomycin. Remarkably, GtfD was able to utilize both an unnatural acceptor, 7, and an unnatural nucleotide sugar donor, UDP-4-epi-vancosamine, to synthesize a novel hybrid teicoplanin/vancomycin glycopeptide. These results establish the enzymatic activity of these four Gtfs, begin to probe substrate specificity, and illustrate how they can be utilized to make variant sugar forms of both the vancomycin and the teicoplanin class of glycopeptide antibiotics.     

Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer’s disease, and suggest that misregulated EV traffic may be an underlying defect.     

Catalytic reductive dehalogenation of hexachloroethane by molecular variants of cytochrome P450cam (CYP101).

CYP101 (cytochrome P450cam) catalyses the oxidation of camphor but has also been shown to catalyse the reductive dehalogenation of hexachloroethane and pentachloroethane. This reaction has potential applications in the biodegradation of these environmental contaminants. The hexachloroethane dehalogenation activity of CYP101 has been investigated by mutagenesis. The effects of active-site polarity and volume were probed by combinations of active-site mutations. Increasing the active-site hydrophobicity by the Y96A and Y96F mutations strengthened hexachloroethane binding but decreased the rate of reaction. Increasing the polarity with the F87Y mutation drastically weakened hexachloroethane binding but did not affect the rate of reaction. The Y96H mutation had little effect at pH 7.4, but weakened hexachloroethane binding while increasing the rate of dehalogenation by up to 40% at pH 6.5, suggesting that the imidazole side-chain was partially protonated at pH 6.5 but not at pH 7.4. Substitutions by bulkier side-chains at F87, T101 and V247 weakened hexachloroethane binding but increased the dehalogenation rate. The effect of the individual mutations was additive in multiple mutants, and the most active mutant for hexachloroethane reductive dehalogenation at pH 7.4 was F87W-V247L (80 min-1 or 2.5 x the activity of the wild-type). The results suggested that the CYP101 active site shows good match with hexachloroethane, the Y96 side-chain plays an important role in both hexachloroethane binding and dehalogenation, and hexachloroethane binding and dehalogenation places conflicting demands on active-site polarity and compromises were necessary to achieve reasonable values for both.     

The role of superior temporal cortex in auditory timing

Recently, there has been upsurge of interest in the neural mechanisms of time perception. A central question is whether the representation of time is distributed over brain regions as a function of stimulus modality, task and length of the duration used or whether it is centralized in a single specific and supramodal network. The answers seem to be converging on the former, and many areas not primarily considered as temporal processing areas remain to be investigated in the temporal domain. Here we asked whether the superior temporal gyrus, an auditory modality specific area, is involved in processing of auditory timing. Repetitive transcranial magnetic stimulation was applied over left and right superior temporal gyri while participants performed either a temporal or a frequency discrimination task of single tones. A significant decrease in performance accuracy was observed after stimulation of the right superior temporal gyrus, in addition to an increase in response uncertainty as measured by the Just Noticeable Difference. The results are specific to auditory temporal processing and performance on the frequency task was not affected. Our results further support the idea of distributed temporal processing and speak in favor of the existence of modality specific temporal regions in the human brain.     

Phosphorylation of high-mobility-group proteins by the calcium-phospholipid-dependent protein kinase and the cyclic AMP-dependent protein kinase

Purified lamb thymus high-mobility-group (HMG) proteins 1, 2, and 17 have been investigated as potential substrates for the Ca2+-phospholipid-dependent protein kinase and the cAMP-dependent protein kinase. HMG proteins 1, 2, and 17 are phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the reactions are totally Ca2+ and lipid dependent and are not inhibited by the inhibitor protein of the cAMP-dependent protein kinase. HMG 17 is phosphorylated predominantly in a single seryl residue, Ser 24 in the sequence Gln-Arg-Arg-Ser 24-Ala-Arg-Leu-Ser 28-Ala-Lys, with the second seryl moiety, Ser 28, modified to a markedly lesser degree. HMGs 1 and 2 are also phosphorylated in only seryl residues but with each there are multiple phosphorylation sites. HMG 17, but not HMG 1 or 2, is also phosphorylated by the cAMP-dependent protein kinase with the site phosphorylated being the minor of the two phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the Km for phosphorylation by the cAMP-dependent enzyme is 50-fold higher than that by the Ca2+-phospholipid-dependent enzyme. HMG 17 is an equally effective substrate for the Ca2+-phospholipid-dependent protein kinase either as the pure protein or bound to nucleosomes. Preliminary evidence has indicated that lamb thymus HMG 14 is also a substrate for the Ca2+-phospholipid-dependent enzyme. It is phosphorylated with a Km similar to that of HMG 17 (4-6 microM), and a comparison of tryptic peptides suggests that it is phosphorylated in a site that is homologous with Ser 24 of HMG 17 and distinct from the sites phosphorylated by the cAMP-dependent protein kinase.     

The roles of geological history and colonization abilities in genetic differentiation between mammalian populations in the Philippine archipelago

Aim To test hypotheses that: (1) late Pleistocene low sea‐level shorelines (rather than current shorelines) define patterns of genetic variation among mammals on oceanic Philippine islands; (2) species‐specific ecological attributes, especially forest fidelity and vagility, determine the extent to which common genetic patterns are exhibited among a set of species; (3) populations show reduced within‐population variation on small, isolated oceanic islands; (4) populations tend to be most highly differentiated on small, isolated islands; and (5) to assess the extent to which patterns of genetic differentiation among multiple species are determined by interactions of ecological traits and geological/geographic conditions. Location The Philippine Islands, a large group of oceanic islands in Southeast (SE) Asia with unusually high levels of endemism among mammals. Methods Starch‐gel electrophoresis of protein allozymes of six species of small fruit bats (Chiroptera, Pteropodidae) and one rodent (Rodentia, Muridae). Results Genetic distances between populations within all species are not correlated with distances between present‐day shorelines, but are positively correlated with distances between shorelines during the last Pleistocene period of low sea level; relatively little intraspecific variation was found within these ‘Pleistocene islands’. Island area and isolation of oceanic populations have only slight effects on standing genetic variation within populations, but populations on some isolated islands have heightened levels of genetic differentiation, and reduced levels of gene flow, relative to other islands. Species associated with disturbed habitat (all of which fly readily across open habitats) show more genetic variation within populations than species associated with primary rain forest (all of which avoid flying out from beneath forest canopy). Species associated with disturbed habitats, which tend to be widely distributed in SE Asia, also show higher rates of gene flow and less differentiation between populations than species associated with rain forest, which tend to be Philippine endemic species. One rain forest bat has levels of gene flow and heterozygosity similar to the forest‐living rodent in our study. Main conclusions The maximum limits of Philippine islands that were reached during Pleistocene periods of low sea level define areas of relative genetic homogeneity, whereas even narrow sea channels between adjacent but permanently isolated oceanic islands are associated with most genetic variation within the species. Moreover, the distance between ‘Pleistocene islands’ is correlated with the extent of genetic distances within species. The structure of genetic variation is strongly influenced by the ecology of the species, predominantly as a result of their varying levels of vagility and ability to tolerate open (non‐forested) habitat. Readily available information on ecology (habitat association and vagility) and geological circumstances (presence or absence of Pleistocene land‐bridges between islands, and distance between oceanic islands during periods of low sea level) are combined to produce a simple predictive model of likely patterns of genetic differentiation (and hence speciation) among these mammals, and probably among other organisms, in oceanic archipelagos.