Presence of Various Carbohydrate Moieties Including β-Galactose and N-Acetylglucosamine Residues on Solubilized Porcine Brain Neurokinin-1/Substance P Receptors

Neurokinin-1 (NK-1)/substance P (SP) receptors were solubilized using 10 mM 3-[( cholamidopropyl)-dimethylammonio]-1- propanesulfate from porcine striatal membranes (solubilization yield, 80%). In solubilized preparations, [3H]SP apparently bound to a single class of high-affinity sites (KD = 0.82 +/- 0.13 nM) as in membrane homogenates. The ligand selectivity pattern observed in both membrane and solubilized receptor preparations indicated that [Sar9,Met(O2)11]SP = SP much greater than senktide = [Nle10]neurokinin A. This suggests the selective labeling of the NK-1 receptor class in both assays. Solubilized receptors were retained on agarose-coupled lectins that bind N-acetylglucosamine-galactose and beta-galactose (Ricinus communis I and Ricinus communis II), mannose (concanavalin A and lentil), and N-acetylglucosamine (wheat germ agglutinin) but not on lectins binding fucose (Lotus A) and N-acetylgalactosamine (Doli-chos biflorus A). Thus, it appears that porcine brain NK-1/SP receptors are enriched with various carbohydrate moieties, beta-galactose and N-acetylglucosamine-galactose residues being especially abundant. This situation is rather different from that in various other members of the rhodopsin seven-transmembrane receptor superfamily.     

From Principal Component to Direct Coupling Analysis of Coevolution in Proteins: Low-Eigenvalue Modes are Needed for Structure Prediction

Various approaches have explored the covariation of residues in multiple-sequence alignments of homologous proteins to extract functional and structural information. Among those are principal component analysis (PCA), which identifies the most correlated groups of residues, and direct coupling analysis (DCA), a global inference method based on the maximum entropy principle, which aims at predicting residue-residue contacts. In this paper, inspired by the statistical physics of disordered systems, we introduce the Hopfield-Potts model to naturally interpolate between these two approaches. The Hopfield-Potts model allows us to identify relevant ‘patterns’ of residues from the knowledge of the eigenmodes and eigenvalues of the residue-residue correlation matrix. We show how the computation of such statistical patterns makes it possible to accurately predict residue-residue contacts with a much smaller number of parameters than DCA. This dimensional reduction allows us to avoid overfitting and to extract contact information from multiple-sequence alignments of reduced size. In addition, we show that low-eigenvalue correlation modes, discarded by PCA, are important to recover structural information: the corresponding patterns are highly localized, that is, they are concentrated in few sites, which we find to be in close contact in the three-dimensional protein fold.     

Recognition of phlorizin by the carriers of sucrose and hexose in broad bean leaves

Phlorizin (1-[2-(β- d -glucopyranosyloxy)-4, 6-dihydroxyphenyl]-3-(4-hydroxyphenyl)-1-propanone) is a well-known non-transported inhibitor of glucose uptake in animal cells. The effects of this compound were studied on the transmembrane potential difference (PD) of broad bean ( Vicia faba L. cv. Aguadulce) mesophyll cells, and on the uptake of amino acids and sugars by the leaf tissues. Phlorizin (5 m M ) induced a marginal depolarization (7 to 10 mV) of the normal PD (-140 mV), and a slight decrease in the uptake of glycine and serine. By contrast, phlorizin induced a stronger inhibition of the uptake of glucose and 3–O-methylglucose, and more particularly of sucrose uptake and phloem loading. In tissues aged for 12 h, 5 m M phlorizin inhibited the absorption of sucrose from a 1 m M solution by 70%. Kinetic experiments demonstrated that phlorizin acted as a competitive inhibitor for the sucrose carrier and for the hexose carrier. Efflux experiments showed that the counter-exchange of sucrose and of 3–O-methylglucose was also phlorizin-sensitive. Overall, the data show that phlorizin is recognized by the hexose carrier and, more efficiently, by the sucrose carrier of the material investigated, but that it is not transported across the membrane.     

Mitotic CDK Promotes Replisome Disassembly,Fork Breakage,and Complex DNA Rearrangements

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Specific MALDI imaging and profiling for biomarker hunting and validation: fragment of the 11S proteasome activator complex, Reg alpha fragment, is a new potential ovary cancer biomarker

MALDI imaging mass spectrometry represents a new analytical tool to directly provide the spatial distribution and relative abundance of proteins in tissue. Twenty-five ovary carcinomas (stages III and IV) and 23 benign ovaries were directly analyzed using MALDI-TOF MS. The biomarker with the major prevalence (80%) has been fully identified using MALDI MS and nanoESI MS and MS/MS after separation by RP-HPLC and trypsin enzymatic digestion. This marker with an m/z of 9744 corresponds to 84 amino acid residues from the 11S proteasome activator complex, named PA28 or Reg-alpha. Validation of this marker has been performed using MALDI imaging, classical immunocytochemistry with an antibody raised against the C-terminal part of the protein, specific MALDI imaging, and Western blot analysis. The validation, using immunocytochemistry, confirmed the epithelial localization of this fragment with nucleus localization in benign epithelial cells and a cytoplasmic localization in carcinoma cells. This indicates that this antibody could be used to discriminate the borderline tumor cases. At this point, a multicentric study needs to be conducted in order to clearly establish the potential of this biomarker. Taken together these studies reflect that direct tissue analysis and specific MALDI imaging strategies facilitate biomarker hunting and validation which can be named pathological proteomics.     

Point of Care Strategy for Rapid Diagnosis of Novel A/H1N1 Influenza Virus

Background

Within months of the emergence of the novel A/H1N1 pandemic influenza virus (nA/H1N1v), systematic screening for the surveillance of the pandemic was abandoned in France and in some other countries. At the end of June 2009, we implemented, for the public hospitals of Marseille, a Point Of Care (POC) strategy for rapid diagnosis of the novel A/H1N1 influenza virus, in order to maintain local surveillance and to evaluate locally the kinetics of the pandemic.

Methodology/Principal Findings

Two POC laboratories, located in strategic places, were organized to receive and test samples 24 h/24. POC strategy consisted of receiving and processing naso-pharyngeal specimens in preparation for the rapid influenza diagnostic test (RIDT) and real-time RT-PCR assay (rtRT-PCR). This strategy had the theoretical capacity of processing up to 36 samples per 24 h. When the flow of samples was too high, the rtRT-PCR test was abandoned in the POC laboratories and transferred to the core virology laboratory. Confirmatory diagnosis was performed in the core virology laboratory twice a day using two distinct rtRT-PCR techniques that detect either influenza A virus or nA/N1N1v. Over a period of three months, 1974 samples were received in the POC laboratories, of which 111 were positive for nA/H1N1v. Specificity and sensitivity of RIDT were 100%, and 57.7% respectively. Positive results obtained using RIDT were transmitted to clinical practitioners in less than 2 hours. POC processed rtRT-PCR results were available within 7 hours, and rtRT-PCR confirmation within 24 hours.

Conclusions/Significance

The POC strategy is of benefit, in all cases (with or without rtRT-PCR assay), because it provides continuous reception/processing of samples and reduction of the time to provide consolidated results to the clinical practitioners. We believe that implementation of the POC strategy for the largest number of suspect cases may improve the quality of patient care and our knowledge of the epidemiology of the pandemic.     

Sphingosine impairs mitochondrial function by opening permeability transition pore

Growing evidence suggest that, in the heart, sphingosine participates to contractile dysfunction by altering calcium transients and mitochondria function. However, mechanisms underlying sphingosine-induced cardiac mitochondria dysfunction are poorly understood. Here, we studied the effects of sphingosine on isolated cardiac mitochondria of either wild-type or Bcl-2 overexpressing transgenic mice. Sphingosine induced reductions in ADP-coupled respiration, membrane potential, mitochondrial cytochrome c content and ATP production, which were partially prevented by cyclosporine A and mitochondrial Bcl-2 overexpression. These data suggest that sphingosine promotes mitochondrial permeability transition pore opening, which may result in uncoupled respiration and participate in cardiac contractile dysfunction.     

Phytochemical analysis and toxicological evaluation of the ethanolic Leaves extract of Hypoestes rosea on the morphology and biochemical indices of the Kidneys of albino Wistar Rats

BackgroundHypoestes rosea (family: Acanthacea), has been harnessed and utilized for treatment of several ailments. However, there is the paucity of available data on nephrotoxicity associated with this herb. Here, we investigated the phytochemical profile and toxicological effect of H. rosea on Wistar Rats.MethodsTwenty rats (weight range: 75–100 g) were assigned into five study groups, viz; (a) control (without treatment) (b) treatment group 1, orally administered with 50 mg/kg (c) treatment group 2, orally administered with 100 mg/kg (d) treatment group 3, orally administered with 250 mg/kg, and (e) treatment group 4, orally administered with 300 mg/kg of H. rosea, respectively for 28 days of four rats per group. The rats were made unconscious by using oral administration of chloroform. Cardiac punctures were made, and blood samples collected into 10 ml labeled plain container, allowed to clot and spun to harvest serum for determination of sodium, potassium, chloride, bicarbonate, urea and creatinine using colorimetric, back-titrimetric, Urease-Berthelot and Jaffe’s reaction methods respectively. Kidneys of rats were harvested, weighed and immediately fixed in 10% neutral buffered formalin for histological analysis.ResultMean serum sodium (p = 0.049), potassium (p = 0.007), and urea (p < 0.001) levels were significantly higher among the treatment groups compared to controls. Histopathological findings of kidney sections revealed mild glomerular infiltration in treatment groups 2–4. Additionally, sclerosis was observed in groups 3–4. Phytochemical analysis of H. rosea revealed presence of alkaloids, flavonoids, saponins, tannins, terpenoids, steroids and reducing sugars.ConclusionFrom the findings in this study, H. rosea leaf extract causes significant damage to the kidneys of Wistar rats at higher doses. Of which, the damages were dose-dependent in direct proportionality manner. To better determine the safe dosage and ideal duration of consumption, there is the need for further studies on H. rosea.