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

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