Reciprocal modulation of regulatory action of prolactin and somatotropin on DNA synthesis in granulosa cells of cows

A comparative analysis of the individual and joint influence of bovine prolactin (BPRL) and bovine somatotropin (BST) on DNA synthesis in cultured bovine granulosa cells from follicles of 3-5 mm in diameter was conducted, and a possibility for regulation of BPRL binding to receptors on the cells by BST was examined. Despite a unidirectional growth-promoting action of BPRL and BST on granulosa cells, their joint mitogenic effect on the cells was not additive at low concentrations of BPRL. The addition of BST to the culture medium did not alter the biphasic character of dependence of DNA synthesis in the cells on BPRL concentration, but increased the maximum effective concentration of the latter. When culturing granulosa cells with BST, the absence of its influence on the level of BPRL specific binding to the cells was shown. This fact suggests that BST modulating action on the mitogenic effect of BPRL is not a result of a change in the number of free receptors for PRL on granulosa cells. The results obtained are discussed in relation to the notion of similarity of receptor functional properties and intracellular signal ways for PRL and ST.     

Subcellular Distribution of Distinct Nucleolin Subfractions Recognized by Two Monoclonal Antibodies

Monoclonal antibodies binding to different domains of nucleolin have been used to localize nucleolin in tissue culture cells ofXenopus laevis.The monoclonal antibody b6-6E7 binds to an epitope in the N-terminal domain, which contains arrays of phosphorylation consensus sites. This monoclonal antibody binds to nucleolin of oocytes and of eggs with high affinity. In contrast, the monoclonal antibody Nu-1H6 binds poorly to the modified forms of nucleolin arising during meiosis and mitosis. In interphase cells, monoclonal antibody b6-6E7 preferentially stains the periphery of the nucleoli, where most of the rRNA accumulates. Staining by monoclonal antibody Nu-1H6 complements this pattern by staining mainly the center of the nucleoli. The epitope of monoclonal antibody Nu-1H6 is within the central domain of nucleolin, which contains the first two RNA binding domains. RNase treatment of cells results in loss of nucleolin from nucleoli. In mitotic cells, both monoclonal antibodies decorate the surface of condensing chromosomes in prophase. The periphery of the condensed chromosomes in metaphase and anaphase is preferentially stained by monoclonal antibody b6-6E7.     

B30.2-like domain proteins: update and new insights into a rapidly expanding family of proteins

The B30.2 domain is a conserved region of around 170 amino acids associated with several different protein domains, including the immunoglobulin folds of butyrophilin and the RING finger domain of ret finger protein. We recently reported several novel members of this family as well as previously undescribed protein families possessing the B30.2 domain. Many proteins have subsequently been found to possess this domain, including pyrin/marenostrin and the midline 1 (MID1) protein. Mutations in the B30.2 domain of pyrin/marenostrin are implicated in familial Mediterranean fever, and partial loss of the B30.2 domain of MID1 is responsible for Opitz G/BBB syndrome, characterized by developmental midline defects. In this study, we scrutinized the available sequence data bases for the identification of novel B30.2 domain proteins using highly sensitive database-searching tools. In addition, we discuss the chromosomal localization of genes in the B30.2 family, since the encoded proteins are likely to be involved in other forms of periodic fever, autoimmune, and genetic diseases.      

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Evolution of the Selfing Syndrome in Arabis alpina (Brassicaceae)

Introduction

The transition from cross-fertilisation (outcrossing) to self-fertilisation (selfing) frequently coincides with changes towards a floral morphology that optimises self-pollination, the selfing syndrome. Population genetic studies have reported the existence of both outcrossing and selfing populations in Arabis alpina (Brassicaceae), which is an emerging model species for studying the molecular basis of perenniality and local adaptation. It is unknown whether its selfing populations have evolved a selfing syndrome.

Methods

Using macro-photography, microscopy and automated cell counting, we compared floral syndromes (size, herkogamy, pollen and ovule numbers) between three outcrossing populations from the Apuan Alps and three selfing populations from the Western and Central Alps (Maritime Alps and Dolomites). In addition, we genotyped the plants for 12 microsatellite loci to confirm previous measures of diversity and inbreeding coefficients based on allozymes, and performed Bayesian clustering.

Results and Discussion

Plants from the three selfing populations had markedly smaller flowers, less herkogamy and lower pollen production than plants from the three outcrossing populations, whereas pistil length and ovule number have remained constant. Compared to allozymes, microsatellite variation was higher, but revealed similar patterns of low diversity and high Fis in selfing populations. Bayesian clustering revealed two clusters. The first cluster contained the three outcrossing populations from the Apuan Alps, the second contained the three selfing populations from the Maritime Alps and Dolomites.

Conclusion

We conclude that in comparison to three outcrossing populations, three populations with high selfing rates are characterised by a flower morphology that is closer to the selfing syndrome. The presence of outcrossing and selfing floral syndromes within a single species will facilitate unravelling the genetic basis of the selfing syndrome, and addressing which selective forces drive its evolution.     

Exploring Genomic,Geographic and Virulence Interactions among Epidemic and Non-Epidemic St. Louis Encephalitis Virus (Flavivirus) Strains

St. Louis encephalitis virus (SLEV) is a re-emerging arbovirus in South America. In 2005, an encephalitis outbreak caused by SLEV was reported in Argentina. The reason for the outbreak remains unknown, but may have been related to virological factors, changes in vectors populations, avian amplifying hosts, and/or environmental conditions. The main goal of this study was to characterize the complete genome of epidemic and non-epidemic SLEV strains from Argentina. Seventeen amino acid changes were detected; ten were non-conservative and located in proteins E, NS1, NS3 and NS5. Phylogenetic analysis showed two major clades based on geography: the North America and northern Central America (NAnCA) clade and the South America and southern Central America (SAsCA) clade. Interestingly, the presence of SAsCA genotype V SLEV strains in the NAnCA clade was reported in California, Florida and Texas, overlapping with known bird migration flyways. This work represents the first step in understanding the molecular mechanisms underlying virulence and biological variation among SLEV strains.     

EpCAM expression in the prostate cancer makes the difference in the response to growth factors

Introduction

Epithelial cell adhesion molecule (EpCAM) is expressed in tumors with an epithelial cell of origin, in a heterogeneous manner. Prostate cancer stem-like cells highly express EpCAM. However, little is known about how EpCAM is involved in the ability of cells to adapt to micro-environmental changes in available growth factors, which is one of the essential biological phenotypes of cancer stem-like cells (CSCs).

Methods

EpCAM-high and EpCAM-low subpopulations of cells were established from the prostate cancer cell line PC-3. Signal transductions in response to serum starvation, and on the exposure to EGF ligand or the specific inhibitor were analyzed in terms. Furthermore, we analyzed the expression level of amino acid transporters which contribute to the activation of mTOR signal between the two subgroups.

Results

EpCAM-high and EpCAM-low PC-3 subpopulations showed markedly different responses to serum starvation. EpCAM expression was positively correlated with activation of the mTOR and epithelial growth factor receptor (EGFR) signaling pathways. Furthermore, AMP-activated protein kinase (AMPK) was gradually de-activated in EpCAM-low PC-3 cells in the absence of serum.

Conclusions

EpCAM regulates the AMPK signaling pathway, essential for the response to growth factors characterized by EGF. LAT1, the amino acid transporter stabilized at the cellular membrane by EpCAM, is likely to be responsible for the difference in the susceptibility to EGF between EpCAM-high and EpCAM-low PC-3 cells.     

High-Melting Lipid Mixtures and the Origin of Detergent-Resistant Membranes Studied with Temperature-Solubilization Diagrams

The origin of resistance to detergent solubilization in certain membranes, or membrane components, is not clearly understood. We have studied the solubilization by Triton X-100 of binary mixtures composed of egg sphingomyelin (SM) and either ceramide, diacylglycerol, or cholesterol. Solubilization has been assayed in the 4–50°C range, and the results are summarized in a novel, to our knowledge, form of plots, that we have called temperature-solubilization diagrams. Despite using a large detergent excess (lipid/detergent 1:20 mol ratio) and extended solubilization times (24–48 h) certain mixtures were not amenable to Triton X-100 solubilization at one or more temperatures. DSC of all the lipid mixtures, and of all the lipid + detergent mixtures revealed that detergent resistance was associated with the presence of gel domains at the assay temperature. Once the system melted down, solubilization could occur. In general adding high-melting lipids limited the solubilization, whereas the addition of low-melting lipids promoted it. Lipidomic analysis of Madin-Darby canine kidney cell membranes and of the corresponding detergent-resistant fraction indicated a large enrichment of the nonsolubilized components in saturated diacylglycerol and ceramide. SM-cholesterol mixtures were special in that detergent solubilization was accompanied, for certain temperatures and compositions, by an independent phenomenon of reassembly of the partially solubilized lipid bilayers. The temperature at which lysis and reassembly prevailed was ∼25°C, thus for some SM-cholesterol mixtures solubilization occurred both above and below 25°C, but not at that temperature. These observations can be at the origin of the detergent resistance effects observed with cell membranes, and they also mean that cholesterol-containing detergent-resistant membrane remnants cannot correspond to structures existing in the native membrane before detergent addition.     

Migration Stimulating Factor (MSF) promotes fibroblast migration by inhibiting AKT

The protein kinase AKT is activated strongly by many motogenic growth factors, yet has recently been shown capable of inhibiting migration in several cell types. Here we report that treatment with Migration Stimulating Factor (MSF), a truncated form of fibronectin that promotes the migration of many cell types, inhibits AKT activity in human fibroblasts and endothelial cells. In fibroblasts, treatment with either MSF or the AKT inhibitor, Akti-1/2, stimulated migration into 3D collagen gels to a similar extent and the effects of Akti-1/2 on migration could be blocked by the expression of an inhibitor-resistant mutant, AKT1 W80A. These data indicate that MSF promotes fibroblast migration, at least in part, by inhibiting the activity of AKT.     

Polymorphism and signatures of selection in the multimammate rat DQB gene

In order to test if DQB is a good candidate marker to investigate the relationship between major histocompatibility complex genes and pathogens in natural populations of Mastomys natalensis, we assessed the polymorphism and evolutionary history of this gene. Twenty-four individuals were genotyped for exon 2 of DQB using capillary electrophoresis single-strand conformation polymorphism, cloning, and sequencing. We found 21 different alleles. Four individuals show three alleles implying a duplication event in the history of this gene. Each distinct sequence translates to give a distinct amino acid sequence and there are strong signals of positive selection on peptide binding sites. Signals of recombination were found in the sequences suggesting that recombination has played a role in generating allelic diversity. Although trans-taxon polymorphism is present at the interspecific level in DQB exon 2 sequences of Mus species, we did not find any evidence of allele sharing among Muridae genera. This indicates a temporal limit of DQB allele sharing in Muridae of less than 8 Mya. In conclusion, although DQB seems to be a good marker to investigate pathogen-driven selection, the polymorphism of gene copy number may restrict its utility in natural populations.