Prevalence of bacterial genotypes and outcome of bovine clinical mastitis due to <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> and <Emphasis Type="Italic">Streptococcus uberis</Emphasis>

Background

Streptococcus dysgalactiae and Streptococcus uberis are common causes of clinical mastitis (CM) in dairy cows. In the present study genotype variation of S. dysgalactiae and S. uberis was investigated, as well as the influence of bacterial species, or genotype within species, on the outcome of veterinary-treated CM (VTCM). Isolates of S. dysgalactiae (n = 132) and S. uberis (n = 97) were genotyped using pulsed-field gel electrophoresis. Identical banding patterns were called pulsotypes. Outcome measurements used were cow composite SCC, milk yield, additional registered VTCMs and culling rate during a four-month follow-up period.

Results

In total, 71?S. dysgalactiae pulsotypes were identified. Nineteen of the pulsotypes were isolated from more than one herd; the remaining pulsotypes were only found once each in the material. All S. uberis isolates were of different pulsotypes. During the follow-up period, the SCC of S. dysgalactiae-cows was significantly lower than the SCC of S. uberis-cows (P <0.05). Median SCC of S. dysgalactiae-cows was 71 500 cells/ml and of S. uberis-cows 108 000 cells/ml. No other differences in outcome parameters could be identified between species or genotypes.

Conclusions

Identical S. dysgalactiae genotypes were isolated from more than one herd, suggesting some spread of this pathogen between Swedish dairy herds. The genetic variation among S. uberis isolates was substantial, and we found no evidence of spread of this pathogen between herds. The milk SCC was lower during the follow-up period if S. dysgalactiae rather than S. uberis was isolated from the case, indicating differences in treatment response between bacterial species.

     

Initiation of Hepatitis C Virus Infection Requires the Dynamic Microtubule Network: ROLE OF THE VIRAL NUCLEOCAPSID PROTEIN*

Early events leading to the establishment of hepatitis C virus (HCV) infection are not completely understood. We show that intact and dynamic microtubules play a key role in the initiation of productive HCV infection. Microtubules were required for virus entry into cells, as evidenced using virus pseudotypes presenting HCV envelope proteins on their surface. Studies carried out using the recent infectious HCV model revealed that microtubules also play an essential role in early, postfusion steps of the virus cycle. Moreover, low concentrations of vinblastin and nocodazol, microtubule-affecting drugs, and paclitaxel, which stabilizes microtubules, inhibited infection, suggesting that microtubule dynamic instability and/or treadmilling mechanisms are involved in HCV internalization and early transport. By protein chip and direct core-dependent pull-down assays, followed by mass spectrometry, we identified β- and α-tubulin as cellular partners of the HCV core protein. Surface plasmon resonance analyses confirmed that core directly binds to tubulin with high affinity via amino acids 2-117. The interaction of core with tubulin in vitro promoted its polymerization and enhanced the formation of microtubules. Immune electron microscopy showed that HCV core associates, at least temporarily, with microtubules polymerized in its presence. Studies by confocal microscopy showed a juxtaposition of core with microtubules in HCV-infected cells. In summary, we report that intact and dynamic microtubules are required for virus entry into cells and for early postfusion steps of infection. HCV may exploit a direct interaction of core with tubulin, enhancing microtubule polymerization, to establish efficient infection and promote virus transport and/or assembly in infected cells.HCV⁵ infection is a major cause of chronic liver disease, which frequently progresses to cirrhosis and hepatocellular carcinoma. HCV represents a global public health problem, with 130 million people infected worldwide. There is currently no vaccine directed against HCV and the available antiviral treatments eliminate the virus in 40-80% of patients, depending on the virus genotype (for review, see Ref. 1).HCV has a single-stranded, positive-sense RNA genome of ∼9.6 kilobases encoding a large polyprotein that is processed by both host and viral proteases to produce three structural proteins (core protein and the envelope glycoproteins E1 and E2), p7, and six nonstructural proteins, which are involved in polyprotein processing and replication of the virus genome (for review, see Ref. 2).HCV core is a basic protein, synthesized as the most N-terminal component of the polyprotein, and is followed by the signal sequence of the E1 envelope glycoprotein (3). The polypeptide is cleaved by signal peptidase and signal peptide peptidase, resulting in the release of core from the endoplasmic reticulum membrane and its trafficking to lipid droplets (3-5). Mature core protein forms the viral nucleocapsid (6) and consists of two domains, D1 and D2. D1 lies at the protein N terminus, is composed of about 117 amino acids (aa), and is involved in RNA binding (7). D2 is relatively hydrophobic, has a length of about 55 aa, and targets HCV core to lipid droplets (8).Microtubules (MTs) are ubiquitous cytoskeleton components that play a key role in various cellular processes relating to cell shape and division, motility, and intracellular trafficking (9). MTs are dynamic, polarized polymers composed of α/β-tubulin heterodimers that undergo alternate phases of growth and shrinkage, dependent on so-called “dynamic instability” (10). Active transport by MTs is bidirectional and involves both plus and minus end-directed motors: kinesin and dynein (11, 12).Another mechanism of cytosolic transport on MTs, called “treadmilling” (13, 14) involves polymerization at the plus end and depolymerization at the minus end after severing of MTs by cellular katenin (15).MTs have important functions in the life cycle of most viruses (13, 16, 17). Cytoplasmic transport on MTs provides viruses with the means to reach sites of replication or enables progeny virus to leave the infected cell. Some viruses, such as Ebola virus (18) or reovirus (19), are transported on MTs within membranous compartments, whereas other viruses like herpes simplex virus type 1 (20), murine polyoma virus (21), human cytomegalovirus (22), or adenovirus (23) interact with MT motors or MT-associated proteins to allow their transport along microtubules.Previous studies have established that the cell cytoskeleton is involved in HCV replication, since HCV replication complexes are subjected to intracellular transport and their formation is closely linked to the dynamic organization of endoplasmic reticulum, actin filaments, and the microtubule network (24-26). In addition, intact microtubules are essential for viral morphogenesis and the secretion of progeny virus from infected cells (27). The role of microtubules in HCV cell entry and the initiation of productive HCV infection has not yet been addressed.In this study, we provide evidence that the MT network plays a key role in HCV cell entry and postfusion steps of the virus cycle that lead to the establishment of productive HCV infection. The initial steps of the viral cycle are sensitive to MT-affecting drugs that inhibit MT formation or depolymerize or stabilize microtubules. We also show a unique property of the HCV core protein, its capacity to directly bind to tubulin and to enhance MT polymerization in vitro. Our findings suggest that HCV could exploit the MT network by polymerization-related mechanisms to productively infect its target cell. Thus, microtubules may provide a novel target for therapeutic interventions against HCV infection.     

Freezing at -800 degrees C distorts the DNA composition of bacterial communities in intestinal samples

Terminal-restriction fragment length polymorphism (T-RFLP) was used to evaluate how to store intestinal specimens for bacterial community analysis. Bacterial communities are increasingly often described by means of DNA-based methods and it is common practice to store intestinal or faecal specimens either at -20 degrees C or -80 degrees C. In this study, samples of intestines from five different pigs were stored at -80 degrees C and -20 degrees C, respectively and a thawing and freezing procedure was carried out three times for each intestinal per pig per temperature. The cumulative sum of the T-RFLP peak heights (T-RF intensities) decreased as the temperature decreased. The composition of the bacterial community changed when stored at -80 degrees C compared to the samples stored at -20 degrees C. Thus it is recommended from this study that samples of intestinal content are stored at -20 degrees C before use for bacterial community analysis, instead of the current practice at -80 degrees C.     

Short- and long-term population dynamical consequences of asymmetric climate change in black grouse

Temporal asymmetry in patterns of regional climate change may jeopardize the match between the proximate and ultimate cues of the timing of breeding. The consequences on short- and long-term population dynamics and trends as well as the underlying mechanisms are, however, often unknown. Using long-term data from Finland, we demonstrate that black grouse (Tetrao tetrix) have responded to spring warming by advancing both egg-laying and hatching. However, early summer (the time of hatching) has not advanced, and chicks have to face colder post-hatching conditions. Demonstrating that these conditions are critical to post-hatching survival, we show that chicks are increasingly suffering higher mortality because they hatch too early. Consequently, breeding success and population size has severely declined over the past four decades. Finally, we modelled the impact of this particular climate change scenario on population dynamics and show that the mismatch can further explain the observed collapse of cyclic fluctuations. Because the evolutionary response of grouse is lagging behind the novel selective pressures, seasonally asymmetric climate change is likely to constitute an important determinant of future short- and long-term changes in the dynamics of black grouse populations.     

Hyporesponsiveness to glucocorticoids in mice genetically deficient for the corticosteroid binding globulin

Corticosteroid binding globulin (CBG) is the carrier for glucocorticoids in plasma. The protein is believed to keep the steroids inactive and to regulate the amount of free hormone acting on target tissues (free hormone hypothesis). Here, we generated a mouse model genetically deficient for CBG to test the contribution of the carrier to glucocorticoid action and adrenocortical stress response. The absence of CBG resulted in a lack of corticosterone binding activity in serum and in an approximately 10-fold increase in free corticosterone levels in CBG-null mice, consistent with its role in regulation of circulating free hormone levels. Surprisingly, cbg(-/-) animals did not exhibit features seen in organisms with enhanced glucocorticoid signaling. Rather, the mice exhibited increased activity of the pituitary axis of hormonal control, normal levels of gluconeogenetic enzymes, and fatigue, as well as an aggravated response to septic shock, indicating an inability to appropriately respond to the excess free corticosterone in the absence of CBG. Thus, our data suggest an active role for CBG in bioavailability, local delivery, and/or cellular signal transduction of glucocorticoids that extends beyond a function as a mere cargo transporter.     

Ubiquitin-binding motifs in REV1 protein are required for its role in the tolerance of DNA damage

REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.     

Synthesis and structure–activity relationship of substitutions at the C-1 position of Δ9-tetrahydrocannabinol

A novel series of Δ9-tetrahydrocannabinol (Δ9-THC) analogues were synthesized to determine their potential as cannabinoid receptor modulators. Chemistry focused on conversion of the phenol of Δ9-THC to other functionality through palladium catalyzed reactions with an intermediate triflate 2. Two analogues with sub 100 nM affinity for the CB₁ and CB₂ receptors were identified.     

Demonstration of extensive GABA synthesis in the small population of GAD positive neurons in cerebellar cultures by the use of pharmacological tools

Cultures of dissociated cerebella from 7-day-old mice were maintained in vitro for 1-13 days. GABA biosynthesis and degradation were studied during development in culture and pharmacological agents were used to identify the enzymes involved. The amount of GABA increased, whereas that of glutamate was unchanged during the first 5 days and both decreased thereafter. The presence of aminooxyacetic acid (AOAA, 10 microM) which inhibits transaminases and other pyridoxal phosphate dependent enzymes including GABA-transaminase (GABA-T), in the culture medium caused an increase in the intracellular amount of GABA and a decrease in glutamate. The GABA content was also increased following exposure to the specific GABA-T inhibitor gamma-vinyl GABA. From day 6 in culture (day 4 when cultured in the presence of AOAA) GABA levels in the medium were increased compared to that in medium from 1-day-old cultures. Synthesis of GABA during the first 3 days was demonstrated by the finding that incubation with either [1-(13)C]glucose or [U-(13)C]glutamine led to formation of labeled GABA. Synthesis of GABA after 1 week in culture, when the enzymatic machinery is considered to be at a more differentiated level, was shown by labeling from [U-(13)C]glutamine added on day 7. Altogether the findings show continuous GABA synthesis and degradation throughout the culture period in the cerebellar neurons. At 10 microM AOAA, GABA synthesis from [U-(13)C]glutamine was not affected, indicating that transaminases are not involved in GABA synthesis and thus excluding the putrescine pathway. At a concentration of 5 mM AOAA GABA labeling was, however, abolished, showing that glutamate decarboxylase, which is inhibited at this level of AOAA, is responsible for GABA synthesis in the cerebellar cultures. In conclusion, the present study shows that GABA synthesis is taking place via GAD in a subpopulation of the cerebellar neurons, throughout the culture period.     

Ultrastructure of interstitial cells of Cajal in myenteric plexus of human colon

The role of the interstitial cells of Cajal (ICC) associated with the myenteric plexus (ICC-MP) as regulators of the motility of the colonic external muscle remains unclear. Ultrastructural studies of myenteric interstitial cells are lacking in human colon. We therefore characterized the distinctive ultrastructure of these cells in the myenteric region of the colon by transmission electron microscopy of the region between the main muscle layers in all parts of the colon in unaffected areas of resected specimens from nine adult human patients. ICC-MP were similar in various colonic regions and had myoid features such as scattered caveolae, prominent intermediate filaments, and cytoplasmic dense bodies. We found characteristic dense membrane-associated bands with a patchy basal lamina, invaginating cellular protrusions (peg and socket junctions) between ICC and between ICC and muscle cells, and close contacts (<100 nm) between ICC and nerves. No gap junctions were observed. Fibroblast-like cells (FLC) were abundant showing well-developed secretory organelles, including coated vesicles, but lacked prominent intermediate filaments and caveolae. FLC had a patchy basal lamina, and peg and socket junctions were observed between them. Macrophage-like cells frequently occurred in close apposition with FLC and, more seldomly, with ICC-MP. The ultrastructure of ICC and FLC in the myenteric region of the human colon thus differs characteristically, but significant overlaps in the ultrastructure between ICC and FLC might complicate any interpretation in pathological ultrastructural studies of the human colonic muscle layer. An erratum to this article can be found at