Factors affecting trimethylarsine and dimethylselenide formation byCandida humicola

Selenite, selenate, and tellurate inhibited the conversion of arsenate to trimethylarsine byCandida humicola. Trimethylarsine disappeared from the gas phase when incubated withC. humicola in the presence of selenium or tellurium salts. The fungus generated dimethyl-selenide from selenite and selenate and an unidentified gas from tellurate. Sulfate but not arsenate, tellurate, or phosphate inhibited the conversion of selenate to dimethylselenide. Arsenate-grown cells generated trimethylarsine from arsenate, and selenate-grown cells formed dimethylselenide from selenate with almost no lag phase. Cells grown in media with selenate or with no additions only formed the alkylarsine from arsenate after a lag phase, and those grown in solutions with arsenate or no additions produced dimethylselenide slowly from selenate.     

Pigmented Nevi—Induced Changes in the Junctional Component

The pigmented nevus represents a potentially more dynamic lesion than has been indicated by most published studies. New nevus cell clusters frequently appear in the epidermis over the residual portion of a nevus that remains after partial surgical excision. Even in relatively inactive nevi in adults, new junctional nevus cells may be induced by surgical trauma. This stimulated growth usually regresses by the time one year or more has elapsed. The growth of nevus cells is probably comparable to that induced in other cells by traumatic injury. There is no evidence to suggest that it is related to the development of melanoma in pigmented nevi.     

In vitro human keratinocyte migration rates are associated with SNPs in the KRT1 interval

Efforts to develop effective therapeutic treatments for promoting fast wound healing after injury to the epidermis are hindered by a lack of understanding of the factors involved. Re-epithelialization is an essential step of wound healing involving the migration of epidermal keratinocytes over the wound site. Here, we examine genetic variants in the keratin-1 (KRT1) locus for association with migration rates of human epidermal keratinocytes (HEK) isolated from different individuals. Although the role of intermediate filament genes, including KRT1, in wound activated keratinocytes is well established, this is the first study to examine if genetic variants in humans contribute to differences in the migration rates of these cells. Using an in vitro scratch wound assay we observe quantifiable variation in HEK migration rates in two independent sets of samples; 24 samples in the first set and 17 samples in the second set. We analyze genetic variants in the KRT1 interval and identify SNPs significantly associated with HEK migration rates in both samples sets. Additionally, we show in the first set of samples that the average migration rate of HEK cells homozygous for one common haplotype pattern in the KRT1 interval is significantly faster than that of HEK cells homozygous for a second common haplotype pattern. Our study demonstrates that genetic variants in the KRT1 interval contribute to quantifiable differences in the migration rates of keratinocytes isolated from different individuals. Furthermore we show that in vitro cell assays can successfully be used to deconstruct complex traits into simple biological model systems for genetic association studies.     

R-type Ca(2+)-channel-evoked CICR regulates glucose-induced somatostatin secretion

Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing beta-cells and glucagon-secreting alpha-cells, the islets contain somatostatin-releasing delta-cells. Somatostatin is a powerful inhibitor of insulin and glucagon secretion. It is normally secreted in response to glucose and there is evidence suggesting its release becomes perturbed in diabetes. Little is known about the control of somatostatin release. Closure of ATP-regulated K(+)-channels (K(ATP)-channels) and a depolarization-evoked increase in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (>or=10 mM) is unaffected by the K(ATP)-channel activator diazoxide and proceeds normally in K(ATP)-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca(2+)-induced Ca(2+)-release (CICR). This constitutes a novel mechanism for K(ATP)-channel-independent metabolic control of pancreatic hormone secretion.     

Integrin-linked kinase regulates senescence in an Rb-dependent manner in cancer cell lines

Anti-integrin-linked kinase (ILK) therapies result in aberrant mitosis including altered mitotic spindle organization, centrosome declustering and mitotic arrest. In contrast to cells that expressed the retinoblastoma tumor suppressor protein Rb, we have shown that in retinoblastoma cell lines that do not express Rb, anti-ILK therapies induced aberrant mitosis that led to the accumulation of temporarily viable multinucleated cells. The present work was undertaken to: 1) determine the ultimate fate of cells that had survived anti-ILK therapies and 2) determine whether or not Rb expression altered the outcome of these cells. Our data indicate that ILK, a chemotherapy drug target is expressed in both well-differentiated, Rb-negative and relatively undifferentiated, Rb-positive retinoblastoma tissue. We show that small molecule targeting of ILK in Rb-positive and Rb-deficient cancer cells results in increased centrosomal declustering, aberrant mitotic spindle formation and multinucleation. However, anti-ILK therapies in vitro have different outcomes in retinoblastoma and glioblastoma cell lines that depend on Rb expression. TUNEL labeling and propidium iodide FACS analysis indicate that Rb-positive cells exposed to anti-ILK therapies are more susceptible to apoptosis and senescence than their Rb-deficient counterparts wherein aberrant mitosis induced by anti-ILK therapies exhibit mitotic arrest instead. These studies are the first to show a role for ILK in chemotherapy-induced senescence in Rb-positive cancer lines. Taken together these results indicate that the oncosuppressive outcomes for anti-ILK therapies in vitro, depend on the expression of the tumor suppressor Rb, a known G₁ checkpoint and senescence regulator.     

Massive conformation change in the prion protein: Using dual-basin structure-based models to find misfolding pathways

We employ all-atom structure-based models with a force field with multiple energetic basins for the C-terminal (residues 166-226) of the mammalian prion protein. One basin represents the known alpha-helical (αH) structure while the other represents the same residues in a left-handed beta-helical (LHBH) conformation. The LHBH structure has been proposed to help describe one class of in vitro grown fibrils, as well as possibly self-templating the conversion of normal cellular prion protein to the infectious form. Yet, it is unclear how the protein may make this global rearrangement. Our results demonstrate that the conformation changes are not strongly limited by large-scale geometry modification and that there may exist an overall preference for the LHBH conformation. Furthermore, our model presents novel intermediate trapping conformations with twisted LHBH structure.     

Relaxin stimulates leukocyte adhesion and migration through a relaxin receptor LGR7-dependent mechanism

Leukocytes are critical effectors of inflammation and tumor biology. Chemokine-like factors produced by such inflammatory sites are key mediators of tumor growth that activate leukocytic recruitment and tumor infiltration and suppress immune surveillance. Here we report that the endocrine peptide hormone, relaxin, is a regulator of leukocyte biology with properties important in recruitment to sites of inflammation. This study uses the human monocytic cell line THP-1 and normal human peripheral blood mononuclear cells to define a novel role for relaxin in regulation of leukocyte adhesion and migration. Our studies indicate that relaxin promotes adenylate cyclase activation, substrate adhesion, and migratory capacity of mononuclear leukocytes through a relaxin receptor LGR7-dependent mechanism. Relaxin-stimulated cAMP accumulation was observed to occur primarily in non-adherent cells. Relaxin stimulation results in increased substrate adhesion and increased migratory activity of leukocytes. In addition, relaxin-stimulated substrate adhesion resulted in enhanced chemotaxis to monocyte chemoattractant protein-1. These responses in THP-1 and peripheral blood mononuclear cells are relaxin dose-dependent and proportional to cAMP accumulation. We further demonstrate that LGR7 is critical for mediating these biological responses by use of RNA interference lentiviral short hairpin constructs. In summary, we provide evidence that relaxin is a novel leukocyte stimulatory agent with properties affecting adhesion and chemomigration.     

Genes within the Idd5 and Idd9/11 diabetes susceptibility loci affect the pathogenic activity of B cells in nonobese diabetic mice

Autoreactive T cells clearly mediate the pancreatic beta cell destruction causing type 1 diabetes (T1D). However, studies in NOD mice indicate that B cells also contribute to pathogenesis because their ablation by introduction of an Igmunull mutation elicits T1D resistance. T1D susceptibility is restored in NOD.Igmunull mice that are irradiated and reconstituted with syngeneic bone marrow plus NOD B cells, but not syngeneic bone marrow alone. Thus, we hypothesized some non-MHC T1D susceptibility (Idd) genes contribute to disease by allowing development of pathogenic B cells. Supporting this hypothesis was the finding that unlike those from NOD donors, engraftment with B cells from H2g7 MHC-matched, but T1D-resistant, nonobese-resistant (NOR) mice failed to restore full disease susceptibility in NOD.Igmunull recipients. T1D resistance in NOR mice is mainly encoded within the Idd13, Idd5.2, and Idd9/11 loci. B cells from NOD congenic stocks containing Idd9/11 or Idd5.1/5.2-resistance loci, respectively, derived from the NOR or C57BL/10 strains were characterized by suppressed diabetogenic activity. Immature autoreactive B cells in NOD mice have an impaired ability to be rendered anergic upon Ag engagement. Interestingly, both Idd5.1/5.2 and Idd9/11-resistance loci were found to normalize this B cell tolerogenic process, which may represent a mechanism contributing to the inhibition of T1D.