Molecular characterization of seipin and its mutants: implications for seipin in triacylglycerol synthesis

The human lipodystrophy gene product Berardinelli-Seip congenital lipodystrophy 2/seipin has been implicated in adipocyte differentiation, lipid droplet (LD) formation, and motor neuron development. However, the molecular function of seipin and its disease-causing mutants remains to be elucidated. Here, we characterize seipin and its mis-sense mutants: N88S/S90L (both linked to motoneuron disorders) and A212P (linked to lipodystrophy) in cultured mammalian cells. Knocking down seipin significantly increases oleate incorporation into triacylglycerol (TAG) and the steady state level of TAG, and induces the proliferation and clustering of small LDs. By contrast, overexpression of seipin reduces TAG synthesis, leading to decreased formation of LDs. Expression of the A212P mutant, however, had little effect on LD biogenesis. Surprisingly, expression of N88S or S90L causes the formation of many small LDs reminiscent of seipin deficient cells. This dominant-negative effect may be due to the N88S/S90L-induced formation of inclusions where wild-type seipin can be trapped. Importantly, coexpression of wild-type seipin and the N88S or S90L mutant can significantly reduce the formation of inclusions. Finally, we demonstrate that seipin can interact with itself and its mutant forms. Our results provide important insights into the biochemical characteristics of seipin and its mis-sense mutants, and suggest that seipin may function to inhibit lipogenesis.     

B lymphocyte inhibition of anti-tumor response depends on expansion of Treg but is independent of B-cell IL-10 secretion

The mechanisms by which B lymphocytes inhibit anti-tumor immunity remain poorly understood. Murine EMT-6 mammary tumors grow readily in immune competent mice (BALB/c), but poorly in B-cell-deficient μ^?/? BALB/c mice (BCDM). T regulatory cell (Treg) expansion and function were impaired in BCDM compared with BALB/c. In this study, we compared tumor growth, Treg cell proliferation, tumor lymphocyte infiltration and cytolytic T cell activity in BALB/c, BCDM and BCDM partially reconstituted with B cells by adoptive transfer (BCDM+B). Partial reconstitution of BCDM with adoptively transferred B cells restored EMT-6 tumor growth, which was independent of IL-10 secretion by B cells. Instead, high frequencies of intratumoral B cells were associated with increased recruitment and proliferation of Treg cells within the tumor microenvironment. The B-cell-dependent accumulation of Treg within the tumor microenvironment was associated with reduced tumor infiltration by CD49+ NK and CD8+ T cells and reduced cytotoxic T cell activity against EMT-6 targets. Our studies indicate that tumor-dependent immunosuppression of T-cell-mediated anti-tumor immunity is coordinated within the tumor microenvironment by B-cell-dependent cross talk with Treg cells, which does not require production of IL-10 by B cells.     

The effect of the lunar cycle on fecal cortisol metabolite levels and foraging ecology of nocturnally and diurnally active spiny mice

We studied stress hormones and foraging of nocturnal Acomys cahirinus and diurnal A. russatus in field populations as well as in two field enclosures populated by both species and two field enclosures with individuals of A. russatus alone. When alone, A. russatus individuals become also nocturnally active. We asked whether nocturnally active A. russatus will respond to moon phase and whether this response will be obtained also in diurnally active individuals. We studied giving-up densities (GUDs) in artificial foraging patches and fecal cortisol metabolite levels. Both species exhibited elevated fecal cortisol metabolite levels and foraged to higher GUDs in full moon nights; thus A. russatus retains physiological response and behavioral patterns that correlate with full moon conditions, as can be expected in nocturnal rodents, in spite of its diurnal activity. The endocrinological and behavioral response of this diurnal species to moon phase reflects its evolutionary heritage.     

Frequency and Pathophysiology of Acute Liver Failure in Ornithine Transcarbamylase Deficiency (OTCD)

BackgroundAcute liver failure (ALF) has been reported in ornithine transcarbamylase deficiency (OTCD) and other urea cycle disorders (UCD). The frequency of ALF in OTCD is not well-defined and the pathogenesis is not known.AimTo evaluate the prevalence of ALF in OTCD, we analyzed the Swiss patient cohort. Laboratory data from 37 individuals, 27 females and 10 males, diagnosed between 12/1991 and 03/2015, were reviewed for evidence of ALF. In parallel, we performed cell culture studies using human primary hepatocytes from a single patient treated with ammonium chloride in order to investigate the inhibitory potential of ammonia on hepatic protein synthesis.ResultsMore than 50% of Swiss patients with OTCD had liver involvement with ALF at least once in the course of disease. Elevated levels of ammonia often correlated with (laboratory) coagulopathy as reflected by increased values for international normalized ratio (INR) and low levels of hepatic coagulation factors which did not respond to vitamin K. In contrast, liver transaminases remained normal in several cases despite massive hyperammonemia and liver involvement as assessed by pathological INR values. In our in vitro studies, treatment of human primary hepatocytes with ammonium chloride for 48 hours resulted in a reduction of albumin synthesis and secretion by approximately 40%.ConclusionIn conclusion, ALF is a common complication of OTCD, which may not always lead to severe symptoms and may therefore be underdiagnosed. Cell culture experiments suggest an ammonia-induced inhibition of hepatic protein synthesis, thus providing a possible pathophysiological explanation for hyperammonemia-associated ALF.     

Enhanced Erythrocyte Adhesiveness/Aggregation in Obesity Corresponds to Low‐Grade Inflammation

Objective: Previous studies have suggested that obesity enhances the inflammatory response, producing macromolecules involved in the induction and/or maintenance of increased erythrocyte aggregation. The objectives of this study were to evaluate the correlation between inflammation markers, erythrocyte adhesiveness/aggregation, and the degree of obesity and to assess phosphatidylserine expression on erythrocyte surface membrane of obese vs. nonobese individuals. Research Methods and Procedures: Erythrocyte adhesiveness/aggregation in the peripheral venous blood was evaluated by using a new biomarker, phosphatidylserine expression was assessed by means of flow cytometry, and markers of inflammation were measured in 65 subjects: 30 obese [body mass index (BMI) = 41 ± 7.7 kg/m²] and 35 nonobese (BMI = 24 ± 2.7 kg/m²) individuals. Pearson correlations and Student's t test were performed. Results: A highly significant difference was noted in the degree of erythrocyte adhesiveness/aggregation and markers of inflammation between the study groups. BMI correlated with erythrocyte adhesiveness/aggregation (r = 0.42, p = 0.001), erythrocyte sedimentation rate (r = 0.42, p = 0.001), high‐sensitive C‐reactive protein (r = 0.55, p < 10^?4), fibrinogen (r = 0.37, p = 0.004), and white blood cell count (r = 0.45, p < 10^?4). The degree of erythrocyte adhesiveness/aggregation correlated with erythrocyte sedimentation rate (r = 0.5, p < 10^?4), high‐sensitive C‐reactive protein (r = 0.56, p < 10^?4), fibrinogen (r = 0.54, p < 10^?4), and white blood cell count (r = 0.32, p = 0.01). Discussion: Our results suggest that obesity‐related erythrocyte adhesiveness/aggregation is probably mediated through increased concentrations of adhesive macromolecules in the circulation and not necessarily through hyperlipidemia or phosphatidylserine exposure on erythrocyte's membrane.     

Variation in phosphorus and sulfur content shapes the genetic architecture and phenotypic associations within the wheat grain ionome

Dissection of the genetic basis of wheat ionome is crucial for understanding the physiological and biochemical processes underlying mineral accumulation in seeds, as well as for efficient crop breeding. Most of the elements essential for plants are metals stored in seeds as chelate complexes with phytic acid or sulfur‐containing compounds. We assume that the involvement of phosphorus and sulfur in metal chelation is the reason for strong phenotypic correlations within ionome. Adjustment of element concentrations for the effect of variation in phosphorus and sulfur seed content resulted in drastic change of phenotypic correlations between the elements. The genetic architecture of wheat grain ionome was characterized by quantitative trait loci (QTL) analysis using a cross between durum and wild emmer wheat. QTL analysis of the adjusted traits and two‐trait analysis of the initial traits paired with either P or S considerably improved QTL detection power and accuracy, resulting in the identification of 105 QTLs and 617 QTL effects for 11 elements. Candidate gene search revealed some potential functional associations between QTLs and corresponding genes within their intervals. Thus, we have shown that accounting for variation in P and S is crucial for understanding of the physiological and genetic regulation of mineral composition of wheat grain ionome and can be implemented for other plants.     

Lignocellulose-degrading enzyme production by white-rot <Emphasis Type="Italic">Basidiomycetes</Emphasis> isolated from the forests of Georgia

The production of lignocellulolytic enzymes by eleven basidiomycetes species isolated from two ecosystems of Georgia was investigated for the first time under submerged (SF) and solid-state fermentation (SSF) of lignocellulosic by-products. Notable intergeneric and intrageneric differences were revealed with regard to the extent of hydrolase and oxidase activity. Several fungi produced laccase along with hydrolases in parallel with growth during the trophophase, showing that the synthesis of this enzyme is not connected with secondary metabolism. The lignocellulosic substrate type had the greatest impact on enzyme secretion. Some of the substrates significantly stimulated lignocellulolytic enzyme synthesis without supplementation of the culture medium with specific inducers. Exceptionally high carboxymethyl cellulase (CMCase, 122 U ml⁻¹) and xylanase (195 U ml⁻¹) activities were revealed in SF of mandarin peelings by Pseudotremella gibbosa IBB 22 and of residue after ethanol production (REP) by Fomes fomentarius IBB 38, respectively. The SSF of REP by T. pubescens IBB 11 ensured the highest level of laccase activity (24,690 U l⁻¹), whereas the SSF of wheat bran and SF of mandarin peels provided the highest manganese peroxidase activity (570–620 U l⁻¹) of Trichaptum biforme IBB 117. Moreover, the variation of lignocellulosic growth substrate provides an opportunity to obtain enzyme preparations containing different ratios of individual enzymes.     

RAG-3D: a search tool for RNA 3D substructures

To address many challenges in RNA structure/function prediction, the characterization of RNA''s modular architectural units is required. Using the RNA-As-Graphs (RAG) database, we have previously explored the existence of secondary structure (2D) submotifs within larger RNA structures. Here we present RAG-3D—a dataset of RNA tertiary (3D) structures and substructures plus a web-based search tool—designed to exploit graph representations of RNAs for the goal of searching for similar 3D structural fragments. The objects in RAG-3D consist of 3D structures translated into 3D graphs, cataloged based on the connectivity between their secondary structure elements. Each graph is additionally described in terms of its subgraph building blocks. The RAG-3D search tool then compares a query RNA 3D structure to those in the database to obtain structurally similar structures and substructures. This comparison reveals conserved 3D RNA features and thus may suggest functional connections. Though RNA search programs based on similarity in sequence, 2D, and/or 3D structural elements are available, our graph-based search tool may be advantageous for illuminating similarities that are not obvious; using motifs rather than sequence space also reduces search times considerably. Ultimately, such substructuring could be useful for RNA 3D structure prediction, structure/function inference and inverse folding.     

Evidence for anextra-cellular function for protein kinase A

In addition to itsintra-cellular functions, cAMP-dependent protein kinase (PKA) may well have anextra-cellular regulatory role in blood. This suggestion is based on the following experimental findings: (a) Physiological stimulation of blood platelets brings about a specific release of PKA, together with its co-substrates ATP and Mg⁺⁺; (b) In human serum, an endogenous phosphorylation of one protein (p75, Mr 75 kDa) occurs; this phosphorylation is enhanced by addition of cAMP and blocked by the Walsh-Krebs specific PKA inhibitor; (c) No endogenous phosphorylation of p75 occurs in human plasma devoid of platelets, but the selective labeling of p75 can be reproduced by adding to plasma the pure catalytic subunit of PKA; (d) p75 was shown to be vitronectin (V), a multifunctional protein implicated in processes associated with platelet activation, and thus a protein whose function may require modulation for control; (e) The phosphorylation of vitronectin occurs at one site (Ser³⁷⁸) which, at physiological pH, is buried in its two-chain form (V₆₅₊₁₀) but becomes exposed in the presence of glycosaminoglycans (GAGs) e.g. heparin or heparan sulfate. Such a transconformation may be used for targeting the PKA phosphorylation to vitronectin molecules bound to GAGs, for example in the extracellular matrix or on cell surfaces; (f) From the biochemical point of view (Km values and physiological concentrations) the phosphorylation of vitronectin can take place at the locus of a hemostatic event; (g) The phosphorylation of Ser³⁷⁸ in vitronectin alters its function, since it significantly reduces its ability to bind the inhibitor-1 of plasminogen activator(s) (PAI-1). Physiologically, this functional modulation may be involved in unleashing PAI-1, allowing its translocation to control the inhibitory function of PAI-1 and, through it, regulating the conversion of plasminogen to active plasmin.Dedicated to Edmond H. Fischer and Edwin G. Krebs, with gratitude for teaching us the right measure of thoroughness and vision in research.