94 research outputs found
Multiple signaling pathways for Cl(-)-dependent depolarization of mesangial cells: role of Ca2+, PKC, and G proteins
No full text We have previously reported that vasopressin activates chloride channels, leading to depolarization of glomerular mesangial cells via both calcium-dependent and calcium-independent pathways (S.G. Kremer, W.V. Breuer, and K. L. Skorecki, J. Cell. Physiol. 138: 97-105, 1989). However, the calcium-independent pathways were not defined. Using fluorescent probes, we now demonstrate that activation of protein kinase C (PKC) results in cellular depolarization in the absence of a calcium signal. This depolarization is also mediated by an enhanced conductance to chloride. Downregulation of PKC partially attenuated but did not abolish the depolarization response to vasopressin. Depolarization persisted when, in addition, calcium responses were also abolished and prostaglandin production was eliminated, suggesting an additional pathway for depolarization. G protein activation by aluminum fluoride also resulted in cellular depolarization mediated by an enhanced conductance to chloride, which persisted when calcium and PKC-signaling pathways were eliminated. This suggests the presence of a calcium- and PKC-independent pathway for G protein-mediated chloride-dependent depolarization. These findings point to the presence of at least three separate signaling pathways available for the activation of mesangial cell chloride channels, i.e., calcium, PKC, and a G protein. </jats:p
Impact of APOL1 polymorphism and IL-1β priming in the entry and persistence of HIV-1 in human podocytes
No full textBackground
Patients of African ancestry with untreated HIV-1 infection and carrying the G1 or G2 kidney disease risk variants (Vs) at the APOL1 gene have a tenfold higher risk of developing HIV-associated nephropathy (HIVAN) compared to those with the non-risk wild type (WT) G0 variant. However, the mechanistic contribution of the APOL1 allelic state to kidney injury in HIV-1 infection remains to be elucidated.
Results
Non-risk WT APOL1 is associated with lower intracellular levels of HIV-1 in conditionally immortalized human podocytes, while the over expression of G1 or G2 risk Vs significantly increases viral accumulation. The priming of podocytes with exogenous IL-1β facilitates HIV-1 entry, via the up-regulation of DC-SIGN. The over expression of APOL1 G1 and G2 risk Vs in combination with an increase in IL-1β levels causes a greater increase in viral concentration than either condition alone. In turn, HIV-1 and exogenous IL-1β together induce a de novo secretion of endogenous IL-1β and an increase of APOL1 gene expression.
Conclusions
Our findings indicate that the presence of risk Vs of APOL1 is permissive of HIV-1 persistence in human podocytes in synergy with IL-1β, a cytokine that characterizes the inflammatory milieu of acute and chronic phases of HIV-1 infection. The elucidation of these molecular mechanisms explains, at least in part, the higher frequency of HIVAN in populations carrying the risk polymorphic genetic variant of APOL1 gene
Morality in the work of an expert witness
No full textThe author analyses the moral aspects of the work of a court expert witness in the perspective of general professional ethics, ethics of the legal professions, informal professional standards of the profession, quality norms applying to forensic expertises, and finally ethical problems arising out of the domestic and international harmonisation in forensic sciences. The author concludes that the morality of the expert is compounded of the basic principles of the ethics of the legal professions (loyalty, discretion, responsibility, reliability, high quality of services) and the principles of ethics specific to the non-legal specialist in a given area. This generates analogies to business ethics
Etyczne podstawy działania biegłych savdowych
No full textThe author analyses the moral aspects of the work of a court expert witness in the perspective of general
professional ethics, ethics of the legal professions, informal professional standards of the profession,
quality norms applying to forensic expertises, and finally ethical problems arising out of the domestic
and international harmonisation in forensic sciences. The author concludes that the morality of the expert
is compounded of the basic principles of the ethics of the legal professions (loyalty, discretion, responsibility,
reliability, high quality of services) and the principles of ethics specific to the non-legal specialist
in a given area. This generates analogies to business ethics
Phylogenetic applications of whole Y-chromosome sequences and the Near Eastern origin of Ashkenazi Levites
No full textPrevious Y-chromosome studies have demonstrated that Ashkenazi Levites, members of a paternally inherited Jewish priestly caste, display a distinctive founder event within R1a, the most prevalent Y-chromosome haplogroup in Eastern Europe. Here we report the analysis of 16 whole R1 sequences and show that a set of 19 unique nucleotide substitutions defines the Ashkenazi R1a lineage. While our survey of one of these, M582, in 2,834 R1a samples reveals its absence in 922 Eastern Europeans, we show it is present in all sampled R1a Ashkenazi Levites, as well as in 33.8% of other R1a Ashkenazi Jewish males and 5.9% of 303 R1a Near Eastern males, where it shows considerably higher diversity. Moreover, the M582 lineage also occurs at low frequencies in non-Ashkenazi Jewish populations. In contrast to the previously suggested Eastern European origin for Ashkenazi Levites, the current data are indicative of a geographic source of the Levite founder lineage in the Near East and its likely presence among pre-Diaspora Hebrews
Genotyping human ancient mtDNA control and coding region polymorphisms with a multiplexed Single-Base-Extension assay: the singular maternal history of the Tyrolean Iceman
No full textBackground
Progress in the field of human ancient DNA studies has been severely restricted due to the myriad sources of potential contamination, and because of the pronounced difficulty in identifying authentic results. Improving the robustness of human aDNA results is a necessary pre-requisite to vigorously testing hypotheses about human evolution in Europe, including possible admixture with Neanderthals. This study approaches the problem of distinguishing between authentic and contaminating sequences from common European mtDNA haplogroups by applying a multiplexed Single-Base-Extension assay, containing both control and coding region sites, to DNA extracted from the Tyrolean Iceman.
Results
The multiplex assay developed for this study was able to confirm that the Iceman's mtDNA belongs to a new European mtDNA clade with a very limited distribution amongst modern data sets. Controlled contamination experiments show that the correct results are returned by the multiplex assay even in the presence of substantial amounts of exogenous DNA. The overall level of discrimination achieved by targeting both control and coding region polymorphisms in a single reaction provides a methodology capable of dealing with most cases of homoplasy prevalent in European haplogroups.
Conclusion
The new genotyping results for the Iceman confirm the extreme fallibility of human aDNA studies in general, even when authenticated by independent replication. The sensitivity and accuracy of the multiplex Single-Base-Extension methodology forms part of an emerging suite of alternative techniques for the accurate retrieval of ancient DNA sequences from both anatomically modern humans and Neanderthals. The contamination of laboratories remains a pressing concern in aDNA studies, both in the pre and post-PCR environments, and the adoption of a forensic style assessment of a priori risks would significantly improve the credibility of results
Mechanism of Li inhibition of vasopressin-sensitive adenylate cyclase in cultured renal epithelial cells
No full text We investigated the mechanism for lithium-induced inhibition of vasopressin-stimulated adensoine 3',5'-cyclic monophosphate (cAMP) production in the renal epithelial cell line LLC-PK1. In LLC-PK1 membranes lithium caused direct inhibition of hormone-stimulated adenylate cyclase activity by competing with magnesium. Fifty percent inhibition occurred at 20 mM lithium. The maximum transport activity (Vmax) but not the activation constant (Ka) for activation by vasopressin was altered. Activation by GTP and its nonhydrolyzable analogues was also inhibited by lithium. Furthermore, kinetic studies revealed that the lag phase in the activation of adenylate cyclase by 5'-guanylimi-dotriphosphate [Gpp(NH)p] was prolonged from 1 to 3 min, suggesting an effect of lithium on magnesium-dependent activation of the stimulatory GTP binding protein Gs. The function of the corresponding inhibitory GTP-binding protein Gi, as assessed by GTP inhibition of vasopressin-stimulated adenylate cyclase activity in the presence and absence of pertussis toxin pretreatment, was unaffected. Intact LLC-PK1 cells incubated in 10 mM lithium (approximate urinary concentration in lithium-treated patients) attained an intracellular lithium concentration of 17 mM, which led to a 40% reduction in cAMP formation. Magnesium loading of intact cells with the ionophore A23187 reversed the inhibitory effect of lithium. It is concluded that lithium directly inhibits the activation of vasopressin-sensitive adenylate cyclase in renal epithelia by competing with magnesium for activation of Gs. This direct effect on Gs activation accounts for the inhibitory effect of lithium on cAMP production in the intact cell. </jats:p
Radiation inactivation of multimeric enzymes: application to subunit interactions of adenylate cyclase
No full textRadiation inactivation has been applied extensively to determine the molecular weight of soluble enzyme and receptor systems from the slope of a linear ln (activity) vs. dose curve. Complex nonlinear inactivation curves are predicted for multimeric enzyme systems, composed of distinct subunits in equilibrium with multimeric complexes. For the system A1 + A2----A1A2, with an active A1A2 complex (associative model), the ln (activity) vs. dose curve is linear for high dissociation constant, K. If a monomer, A1, has all the enzyme activity (dissociative model), the ln (activity) vs. dose curve has an activation hump at low radiation dose if the inactive subunit, A2, has a higher molecular weight than A1 and has upward concavity when A2 is smaller than A1. In general, a radiation inactivation model for a multistep mechanism for enzyme activation fulfills the characteristics of an associative or dissociative model if the reaction step forming active enzyme is an associative or dissociative reaction. Target theory gives the molecular weight of the active enzyme subunit or complex from the limiting slope of the ln (activity) vs. dose curve at high radiation dose. If energy transfer occurs among subunits in the multimer, the ln (activity) vs. dose curve is linear for a single active component and is concave upward for two or more active components. The use of radiation inactivation as a method to determine enzyme size and multimeric subunit assembly is discussed with specific application to the hormone-sensitive adenylate cyclase system. It is shown that the complex inactivation curves presented in the accompanying paper can be used select the best mechanism out of a series of seven proposed mechanisms for the activation of adenylate cyclase by hormone.</jats:p
Microscopic Hematuria in Adolescents and Young Adults and Risk for End-stage Renal Disease--Reply
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