Regional differences of proliferation activity in the spinal cord ependyma of adult rats

Increased proliferation activity in the central canal ependyma of adult rodent spinal cord was described after injury and is thought to participate in recovery processes. Proliferation activity is scarce under physiological conditions, but still could be of importance, as in vitro studies showed that the spinal cord ependyma is an internal source of neural stem cells. Data from these studies indicate that there are regional differences in the distribution of proliferation activity along the rostro-caudal axis. We analyzed the proliferation activities in the ependyma within the entire extent of intact adult rat spinal cord. To identify proliferating cells we performed immunohistochemistry either for cell cycle S-phase marker BrdU or for the nuclear protein Ki-67. BrdU and Ki-67 positive cells were counted on sections selected from four spinal cord regions — cervical, thoracic, lumbar and sacral/coccygeal. Analysis showed that the number of BrdU positive cells within the ependyma was very low in all subdivisions of the spinal cord. Both BrdU and Ki-67 labeling revealed a significantly higher number of proliferating cells in the ependyma of sacrococcygeal part in comparison to all other spinal cord regions, suggesting that the caudal spinal cord might have potentially higher regeneration capacity compared to more rostral parts.     

Lysine maintenance requirement and efficiency of its utilisation in young pigs as estimated by comparative slaughter technique

An experiment was carried out on weaner pigs (initial BW 10.8 kg) to estimate the maintenance requirement for lysine (Lys) and its marginal efficiency of utilisation using a comparative slaughter technique. Three groups of six pigs each were fed purified diets for 21 days supplying Lys at 19.5, 78 or 195 mg/kg W0.75, which corresponded to 50, 200 or 500% of the assumed maintenance requirement. All other essential amino acids were given at 50% excess. At the end of the experiment, pigs were killed for whole-body nitrogen (N) and amino acid analysis. A representative group of six pigs was analysed at the beginning of the experiment. Based on regression equations, relating Lys or N retention to Lys intake, Lys requirement for zero Lys retention was estimated to be 121 mg/kg W0.75, while Lys requirement corresponding to zero N retention was 41.7 mg/kg W0.75. At N equilibrium, the pigs lost 65 mg of Lys per kg W0.75 daily while at zero Lys retention, the daily N retention was 156 mg/kg W0.75 . The marginal efficiency of lysine utilisation was 0.91. It is concluded that zero lysine retention is a better criterion of lysine maintenance requirement than zero N retention.     

Solving the shugoshin puzzle

Shugoshin proteins form a complex with protein phosphatase 2A (PP2A) that protects centromeric cohesin from separase-mediated cleavage during yeast meiosis I. Recent work shows that this mechanism is conserved from yeast to mammals. Importantly, a model emerges that explains a long-standing puzzle, namely why the shugoshin-PP2A complex mediates protection of centromeric cohesin from separase cleavage specifically during meiosis I, but not during meiosis II or mitosis.     

Pyrrolo-pyrimidones: a novel class of MK2 inhibitors with potent cellular activity

Pyrrolo-pyrimidones of the general structure 1 were synthesized and evaluated for their potential as MK2 inhibitors. Potent derivatives were discovered which inhibit MK2 in the nanomolar range and show potent inhibition of cytokine release from LPS-stimulated monocytes. These derivatives were shown to inhibit phosphorylation of hsp27, a downstream target of MK2 and are modestly selective in a panel of 28 kinases.     

A multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis C virus infection and typing of whole blood

All donor blood samples must be tested pretransfusion to determine the donor blood type. Standard testing protocols require that assays be performed for important bloodborne pathogens such as hepatitis C, syphilis, hepatitis B, and human immunodeficiency virus. We have demonstrated proof of the concept that a protein microarray can type whole blood and detect antibody to significant pathogens simultaneously from the same donor blood sample. The data collected demonstrate the ability of the array to accurately type blood samples while also detecting the presence of antibodies against both human immunodeficiency virus and hepatitis C virus. In conclusion, we have successfully developed a platform capable of typing human whole blood samples, while at the same time testing for the presence of antibodies specific for human immunodeficiency virus/hepatitis C virus. The major benefits of this system are its amenability to expansion with additional assays, for example, rhesus typing and syphilis and/or hepatitis B virus detection, and also the adaptability of the assay to higher-throughput analysis, currently 16 individual samples per slide, but readily expandable to a 96-well format.     

Adaptive Mutations in the V3 Loop of gp120 Enhance Fusogenicity of Human Immunodeficiency Virus Type 1 and Enable Use of a CCR5 Coreceptor That Lacks the Amino-Terminal Sulfated Region

To identify sites in gp120 that interact with the CCR5 coreceptor and to analyze the mechanisms of infection, we selected variants of the CCR5-dependent JRCSF molecular clone of human immunodeficiency virus type 1 (HIV-1) that adapted to replicate in HeLa-CD4 cells that express the mutant coreceptor CCR5(Y14N) or CCR5(G163R), which were previously shown to bind purified gp120-CD4 complexes only weakly. Correspondingly, these mutant CCR5s mediate infections of wild-type virus only at relatively high cell surface concentrations, demonstrating a concentration-dependent assembly requirement for infection. The plots of viral infectivity versus concentration of coreceptors had sigmoidal shapes, implying involvement of multiple coreceptors, with an estimated stoichiometry of four to six CCR5s in the active complexes. All of the adapted viruses had mutations in the V3 loops of their gp120s. The titers of recombinant HIV-1 virions with these V3 mutations were determined in previously described panels of HeLa-CD4 cell clones that express discrete amounts of CCR5(Y14N) or CCR5(G163R). The V3 loop mutations did not alter viral utilization of wild-type CCR5, but they specifically enhanced utilization of the mutant CCR5s by two distinct mechanisms. Several mutant envelope glycoproteins were highly fusogenic in syncytium assays, and these all increased the efficiency of infection of the CCR5(Y14N) or CCR5(G163R) clonal panels without enhancing virus adsorption onto the cells or viral affinity for the coreceptor. In contrast, V3 loop mutation N300Y was selected during virus replication in cells that contained only a trace of CCR5(Y14N) and this mutation increased the apparent affinity of the virus for this coreceptor, as indicated by a shift in the sigmoid-shaped infectivity curve toward lower concentrations. Surprisingly, N300Y increased viral affinity for the second extracellular loop of CCR5(Y14N) rather than for the mutated amino terminus. Indeed, the resulting virus was able to use a mutant CCR5 that lacks 16 amino acids at its amino terminus, a region previously considered essential for CCR5 coreceptor function. Our results demonstrate that the role of CCR5 in infection involves at least two steps that can be strongly and differentially altered by mutations in either CCR5 or the V3 loop of gp120: a concentration-dependent binding step that assembles a critical multivalent virus-coreceptor complex and a postassembly step that likely involves a structural rearrangement of the complex. The postassembly step can severely limit HIV-1 infections and is not an automatic consequence of virus-coreceptor binding, as was previously assumed. These results have important implications for our understanding of the mechanism of HIV-1 infection and the factors that may select for fusogenic gp120 variants during AIDS progression.     

Association of Borrelia garinii and B. valaisiana with Songbirds in Slovakia

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.