The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.
Animals ; Antigens, Protozoan/*chemistry/*isolation & purification ; Blotting, Western ; Chromatography, High Pressure Liquid ; Electrophoresis, Polyacrylamide Gel/methods ; Humans ; Leishmania major/growth & development/*immunology ; Protein Isoforms/chemistry/isolation & purification

OBJECTIVETo study the epidemic status of human immunodeficiency virus type 1 (HIV-1) subtypes and sequence variations in Henan province and to explore their epidemic characteristics and transmission sources and routes.

METHODSHIV-1 env gene was amplified by nested PRC from uncultured peripheral blood mononuclear cells (PBMCs) obtained from 1157 HIV-1 carriers confirmed in Henan from 2005 to 2006. The C2-V3 region (about 350450 bp) of HIV-1 env was sequenced.

RESULTSOf 1157 samples, there were 4 HIV-1 strains including subtype B', C and recombinant subtype BC and AE, accounting for 96.456% (1116/1157), 0.346% (4/1157), 2.593% (30/1157) and 0.605% (7/1157), respectively. In comparison with the sequences of the international strains of RL42, C.95in21068, 07BC.CN.97.C54A and 01AE.TH.90.CM240, the genetic divergence was (8.971 +/- 3.182)%, (5.109 +/- 0.112)%, (3.568 +/- 0.254)% and (4.775 +/- 0.128)%, respectively. The phylogenetic tree analysis showed that 1116 Henan B' strain was closely related to those of Thailand B' and was far different from other international subtypes. The major transmit route in subtype B' strain was mainly found among former blood donators, while sexual transmission was the major route for BC spreading. For AE, the major transmission was sex and blood transfusion, and sex was defined as the major route for C.

CONCLUSIONThere are four HIV-1 strains including subtype B', C and recombinant subtype BC and AE identified in Henan province currently, and the dominant subtype B' is closely related to HIV-1 strains of Thailand B'. While, for non-B' subtype, there exists the aggregating phenomenon in some local areas. Therefore, it is necessary to attach importance and to strengthen the HIV test and surveillance on migrant populations.

Adolescent ; Adult ; Base Sequence ; China ; DNA, Viral ; Female ; HIV-1 ; classification ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Protein Isoforms ; Sequence Analysis, Protein ; Viral Proteins

OBJECTIVETo identify and clone the gene encoding human M96 gene and study its expression spectrum in several blood cell lines.

METHODSAccording to the sequence of human EST which was highly homologous to the mouse M96 gene, primers used for library screening were synthesized, then the human adult testis and fetal brain cDNA library were screened. The gene was analyzed by making use of BLAST and CLUSTAL W, and its expression spectrum was studied by multiple-cell lines Northern blot analysis. The expression change of M961 in cell differentiation was observed by use of K562 cell line induced by hemin.

RESULTSTwo cDNA clones encoding human M96 gene were isolated, identified and named as M961, and M962. They were found to be isoforms of each other. Northern, blot showed that M961 gene was expressed highly in CEM, Hel, Dami and K562 cell lines. However, during K562 cell line differentiation, process the expression of M961 elevated only slightly.

CONCLUSIONSM961 gene was expressed highly in pluripotent cell lines with erythrocytic and megakaryocytic potentials.

Alternative Splicing ; Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; DNA, Neoplasm ; genetics ; isolation & purification ; Hemin ; pharmacology ; Humans ; K562 Cells ; Molecular Sequence Data ; Protein Isoforms ; genetics ; isolation & purification ; Protein Splicing ; Zinc Fingers ; genetics

The organic anion transporters (OATs) are expressed in various tissues, primarily in the kidney and liver, but they are also expressed in the placenta, small intestine, and the choroid plexus, which are all epithelial tissues that transport xenobiotics. Six isoforms of OATs are currently known. Considering the variety of organic anionic compounds, other OATs isoforms can be assumed. In this connection, we have searched for a new isoform in the expressed sequence tag (EST) database. We found the new candidate clone AK052752 in the mouse kidney cDNA library and we named it mouse organic anion transporter like protein 1 (mOATLP1). The mOATLP1 cDNA consisted of 2221 base pairs that encoded a 552 amino acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of mOATLP1 showed 37 to 63% identity to other members of the OAT family. According to the tissue distribution based on Northern blot analysis, 2.7 kb and 2.9 kb mOATLP1 transcripts (approximate sizes) were observed in the kidney and liver. An 85-kDa band (approximate) was detected using Western blot analysis of mouse kidney performed with a synthesized oligopeptide-induced mOATLP1 antibody. Immunohistochemical results showed mOATLP1 was stained in the blood vessels, glomeruli (the parietal epithelial cells and podocytes), distal convoluted tubules, connecting tubules, and inner medullary collecting tubules. mOATLP1 appears to be a novel candidate for an organic anion transporter isoform identified in the kidney.
Tissue Distribution ; Sequence Homology, Amino Acid ; Rabbits ; Protein Structure, Tertiary ; Protein Isoforms/isolation & purification ; Phylogeny ; Organic Anion Transporters/*isolation & purification/*metabolism ; Oligopeptides/immunology ; Multigene Family ; Molecular Sequence Data ; Mice ; Kidney/*metabolism ; Immunohistochemistry ; Cloning, Molecular ; Blotting, Western ; Animals ; Amino Acid Sequence

Organic ion transporters are expressed in various tissues that transport endogenous and exogenous compounds including their metabolites. There are organic anion transporter (OAT), organic cation transporter (OCT), organic anion transporter like protein (OATLP) and organic cation transporter like (OCTL). Considering the variety of charged organic ionic compounds, the existence of numerous isoforms of organic ion transporters can be assumed. In the present study, we have searched for a new isoform in the expressed sequence tag (EST) database using human organic anion transporter 4 (hOAT4) amino acid sequence as a "query". We found a candidate clone (BC021449) from the mouse kidney cDNA library. This clone was identified as an ortholog of ORCTL3 or OCTL-1. The mOCTL1 cDNA consists of 2016 base pairs encoding 551 amino acid residues with 12 putative transmembrane domains. The deduced amino acid sequence of mOCTL1 showed 35 to 40% identity to those of the other members of the OATs and OCTs. According to the tissue distribution, examined by Northern blot analysis, about a 2.4-kb transcript of mOCTL1 was observed in the kidney. About a 90-kDa band was detected when Western blot analysis in the mouse kidney was done by using antibody against synthesized oligopeptide of mOCTL1. The immunohistochemical result showed that mOCTL1 was stained at the glomerulus (the parietal epithelial cells and podocytes), pars recta of proximal tubule, distal convoluted tubules, connecting tubules and collecting tubules. From these results, we conclude that mOCTL1 may be a candidate for an organic ion transporter isoform in the mouse kidney.
Amino Acid Sequence ; Animals ; Blotting, Western ; Gene Library ; Humans ; Immunohistochemistry ; Kidney/*metabolism ; Mice ; Molecular Sequence Data ; Organ Specificity ; Organic Cation Transport Proteins/genetics/*isolation & purification ; Organic Cation Transporter 1/genetics/*isolation & purification ; Protein Isoforms/isolation & purification

Plant betaine aldehyde dehydrogenase (BADH) is a physiologically important enzyme in response to salt or drought stress. In this study, two BADH genes (PeBADH1 and PeBADH2) were cloned from Populus euphratica. Both PeBADH1 and PeBADH2 genes encode the proteins of 503 amino acid residues, with a calculated molecular mass of 54.93 kDa and 54.90 kDa, respectively. Reverse transcription PCR showed the divergence of expression pattern between the PeBADH1 and PeBADH2 genes in P. euphratica. The recombinant PeBADH1 and PeBADH2 proteins were overexpressed in E. coli, and purified by Ni-affinity chromatography. The PeBADH2 protein had 1.5-fold higher enzymatic activity towards the substrate aldehyde than PeBADH1 protein. The PeBADH1 protein revealed higher thermal stability than PeBADH2 protein. These results indicated obvious functional divergence between the PeBADH1 and PeBADH2 genes.
Amino Acid Sequence ; Betaine-Aldehyde Dehydrogenase ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Plant ; physiology ; Molecular Sequence Data ; Plant Proteins ; biosynthesis ; chemistry ; genetics ; Populus ; genetics ; Protein Isoforms ; chemistry ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Substrate Specificity

OBJECTIVETo find the molecular mechanism of decreasing blood fat effect of Darning capsule on hyperlipemic rat, we study the expression of connexin43 in the myocardium before and after using the capsule.

METHODForty Wistar rats were randomly divided into 5 group: control group, hyperlipemia model group, Daming capsule group of high dose, middle dose and low dose (200, 100, 50 mg kg(-1) d(-1)). Each group had 8 rats. Hyperlipemic rat model was made firstly, the blood was obtained via vena caudalis and the indexes of TC, TG, LDL, HDL and NEFA in the serum were measured. The myocardial total RNA was extracted by Trizol method. To compare the expression of connexin43 in the following groups: hyperlipemia, normal and drug, we used the technique of RT-PCR, immunostaining and microconfoul.

RESULTThe concentrations of TC, TG, LDL and NEFA in hyperlipemic serum were increased (P <0. 05), while that of HDL was decreased (P <0. 05). After treating with Daming capsule, the concentration of the preceding four indexes were decreased and the concentrations of HDL was increased up to nearly normal level. No significant difference was found in the ECG of the three groups. As compared with the normal group, the mRNA expressions of connexin43 in hyperlipemia group was weakened (P <0.05), while that of the drug group was enhanced(P <0.05). The same result in immunostaining was observed.

CONCLUSIONHyperlipemic rat model was successfully established and Daming capsule has the effect of lowering blood lipid. Furthermore, the molecular mechanism of Darning capsule is related with the change of Cx43 closely.

Animals ; Capsules ; Cholesterol ; blood ; Connexin 43 ; biosynthesis ; genetics ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Fatty Acids, Nonesterified ; blood ; Hyperlipidemias ; blood ; metabolism ; Hypolipidemic Agents ; administration & dosage ; pharmacology ; Male ; Myocardium ; metabolism ; Plants, Medicinal ; chemistry ; Protein Isoforms ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; Random Allocation ; Rats ; Rats, Wistar ; Triglycerides ; blood