Cytokeratin filament modulation in pulmonary microvessel endothelial cells by vasoactive agents and culture confluency.

Recently, bovine pulmonary microvascular endothelial cells (PMV) were shown to contain cytokeratin 8 and 19 intermediate filaments (Patton et al., 1990). In this study, we examine the effect of culture contiguity and vasoactive agents on the content and assembly of cytokeratins in PMV. Immunofluorescent staining of PMV cultures show a progressive increase in cytokeratin filament assembly. In freshly plated PMV, keratin appears as hazy staining (less than 4 hr) and later organizes into keratin 'plaques' (4 days) associated with cell-cell contacts; post confluent (greater than 7 days) PMV cultures contain fully assembled cytokeratin filaments which extend to the cell periphery and approach filaments in apposed cells. Vimentin filaments are also present in freshly plated PMV cultures but unlike cytokeratins, become less filamentous at confluency. This cell density-dependent modulation of cytokeratins is also demonstrated by densitometric analysis of autoradiographs of 35S-methionine labeled keratins in which PMV keratin content is elevated at high cell densities, while vimentin content remains constant. Desmoplakins I and II, components of desmosomes, could not be demonstrated in PMV by immunoblotting. PMV treated with permeability modulating agents (4 x 10(-3) M EGTA, 1 microM cytochalasin B, 1 microM bradykinin, 1 microM A23187, and 1 microM PMA) exhibit border retraction and altered keratin filament staining. From these studies we conclude: 1) cytokeratin 8 and 19 containing intermediate filaments are present in confluent PMV cultures with vimentin but without desmosomes, 2) the state of assembly of PMV cytokeratin and vimentin filaments appears to be oppositely affected by culture contiguity, and 3) treatment of monolayers with vasoactive agents alters the state of assembly of cytokeratin filaments. We speculate that modulation of cytokeratin assembly in PMV may be involved in regulation of pulmonary microvascular structure and function.     

Histopathological and parasitological study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

Background

A nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows was carried out on dairy farms in Sweden. The aim was to investigate the microbial panorama and the occurrence of antimicrobial resistance. Moreover, differences between newly infected cows and chronically infected cows were investigated.

Methods

In total, 583 quarter milk samples were collected from 583 dairy cows at 226 dairy farms from February 2008 to February 2009. The quarter milk samples were bacteriological investigated and scored using the California Mastitis Test. Staphylococci were tested for betalactamase production and presence of resistance was evaluated in all specific udder pathogens. Differences between newly infected cows and chronically infected cows were statistically investigated using logistic regression analysis.

Results

The most common isolates of 590 bacteriological diagnoses were Staphylococcus (S) aureus (19%) and coagulase-negative staphylococci (CNS; 16%) followed by Streptococcus (Str) dysgalactiae (9%), Str. uberis (8%), Escherichia (E.) coli (2.9%), and Streptococcus spp. (1.9%). Samples with no growth or contamination constituted 22% and 18% of the diagnoses, respectively. The distribution of the most commonly isolated bacteria considering only bacteriological positive samples were: S. aureus - 31%, CNS - 27%, Str. dysgalactiae - 15%, Str. uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1%. There was an increased risk of finding S. aureus, Str. uberis or Str. dysgalactiae in milk samples from chronically infected cows compared to findings in milk samples from newly infected cows. Four percent of the S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Overall, resistance to other antimicrobials than penicillin G was uncommon.

Conclusions

Staphylococcus aureus and CNS were the most frequently isolated pathogens and resistance to antimicrobials was rare.     

The phosphoinositol sphingolipids of Saccharomyces cerevisiae are highly localized in the plasma membrane.

To investigate the vital function(s) of the phosphoinositol-containing sphingolipids of Saccharomyces cerevisiae, we measured their intracellular distribution and found these lipids to be highly localized in the plasma membrane. Sphingolipids were assayed in organelles which had been uniformly labeled with [3H]inositol or 32P and by chemical measurements of alkali-stable lipid P, of long chain bases, and of very long chain fatty acids. We have developed an improved method for the preparation of plasma membranes which is based on the procedure of Duran et al. (Proc. Natl. Acad. Sci. USA 72:3952-3955, 1975). On the basis of marker enzyme and DNA assays carried out with a number of preparations, the plasma membranes contained less than 10% vacuolar membranes (alpha-mannosidase) and nuclei (DNA); the contamination by the endoplasmic reticulum (NADPH-cytochrome c reductase) varied from 0 to 20%. The plasma membrane preparations showed a 13-fold increase in the specific activity of vanadate-sensitive ATPase, compared with that in the homogenate, with a yield ranging from 50 to 80%. A comparison of the distribution of the ATPase with that of sphingolipids assayed by a variety of methods showed that 80 to 100% of the sphingolipids are localized in the plasma membrane; the sphingolipids constitute about 30% of the total phospholipid content of the plasma membrane. Minor amounts of sphingolipids that were found in isolated mitochondria and nuclei can be attributed to the presence of small amounts of plasma membrane in these fractions. These results suggest that one or more essential functions of these lipids is in the plasma membrane. Furthermore, sphingolipids may be useful chemical markers of the plasma membrane of S. cerevisiae.     

Type 5 G protein beta subunit (Gbeta5) controls the interaction of regulator of G protein signaling 9 (RGS9) with membrane anchors

The R7 family of regulators of G protein signaling (RGS) proteins, comprising RGS6, RGS7, RGS9, and RGS11, regulate neuronal G protein signaling pathways. All members of the R7 RGS form trimeric complexes with the atypical G protein β subunit, Gβ5, and membrane anchor R7BP or R9AP. Association with Gβ5 and membrane anchors has been shown to be critical for maintaining proteolytic stability of the R7 RGS proteins. However, despite its functional importance, the mechanism of how R7 RGS forms complexes with Gβ5 and membrane anchors remains poorly understood. Here, we used protein-protein interaction, co-localization, and protein stability assays to show that association of RGS9 with membrane anchors requires Gβ5. We further establish that the recruitment of R7BP to the complex requires an intact interface between the N-terminal lobe of RGS9 and protein interaction surface of Gβ5. Site-directed mutational analysis reveals that distinct molecular determinants in the interface between Gβ5 and N-terminal Dishevelled, EGL-10, Pleckstrin/DEP Helical Extension (DEP/DHEY) domains are differentially involved in R7BP binding and proteolytic stabilization. On the basis of these findings, we conclude that Gβ5 contributes to the formation of the binding site to the membrane anchors and thus is playing a central role in the assembly of the proteolytically stable trimeric complex and its correct localization in the cell.     

Analysis of a temperature-sensitive mutant rotavirus indicates that NSP2 octamers are the functional form of the protein

Evidence that NSP2 plays a role in packaging and replication comes from studies on tsE(1400), a rotavirus mutant with a temperature-sensitive (ts) lesion in the NSP2 gene. Cells infected with tsE and maintained at nonpermissive temperature contain few replication-assembly factories (viroplasms) or replication intermediates and produce virus particles that are mostly empty. Sequence analysis has indicated that an A152V mutation in NSP2 is responsible for the ts phenotype of tsE. To gain insight into the effect of the mutation on the octameric structure and biochemical activities of tsE NSP2, the protein was expressed in bacteria and purified to homogeneity. Analytical ultracentrifugation showed that tsE NSP2 formed octamers which, like those formed by wild-type (wt) NSP2, undergo conformational change into more compact structures upon binding of nucleotides. However, exposure to Mg(2+) and the nonpermissive temperature caused disruption of the tsE octamers and yielded the formation of polydisperse NSP2 aggregates, events not observed with wt octamers. Biochemical analysis showed that the RNA-binding, helix-destabilizing and NTPase activities of tsE NSP2 were significantly less at the nonpermissive temperature than at the permissive temperature. In contrast, these activities for wt NSP2 were higher at the nonpermissive temperature. Our results indicate that the octamer is the fully functional form of NSP2 and the form required for productive virus replication. The propensity of tsE NSP2 to form large aggregates provides a possible explanation for the inability of the protein to support packaging and/or replication in the infected cell at the nonpermissive temperature.     

Bioaccumulation of selected metals in the gill,liver and muscle tissue of rednose labeo Labeo rosae from two impoundments on the Olifants River,Limpopo river system,South Africa

Metal concentrations in the gill, muscle and liver tissues of Labeo rosae from two impoundments, Loskop and Flag Boshielo dams on the Olifants River, were evaluated in 2011 to detect patterns in metal associations between tissues and impoundments. Elevated concentrations of Ba, Zn, B, Al, Si and Fe, relative to a pristine site in the catchment, were found in the muscle, liver and gill tissues at both impoundments. Molybdenum concentrations were exceptionally high in all tissues at Loskop Dam and in liver at Flag Boshielo Dam. No definite pattern in the ratio metal concentrations within, or between, fish tissues was identified. The expected trend, liver > gills > muscle, was found at both impoundments, but was less prominent at Loskop Dam. Metal concentrations in muscle of Loskop Dam fish were significantly higher than in those at Flag Boshielo Dam. The inverse was true for liver. The long-term impact of elevated metal concentrations on fish health at both impoundments raises concern.