OBJECTIVE: To observe the changes in protein expression induced by botulinum toxin A (BoNT-A) injection and to characterize the molecular and cellular action of mechanisms of BoNT-A injection on skeletal muscles using proteomic elements as biomarkers. METHODS: BoNT-A was injected into left gastrocnemius muscles of 12 Sprague-Dawley rats (2 months of age) at a dosage of 5 units/kg body weight. For the controls same volume of normal saline was injected to right gastrocnemius muscle of each rat. Muscle samples were obtained at 4 time points (3 rats per time point): 3, 7, 14, and 56 day post-injection. To reveal the alterations in muscle protein, we performed 2-dimensional electrophoresis (2DE) and compared Botox group and normal saline group at each time point. Altered protein spots in 2DE were identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometer (MALDI-TOF MS) proteomics analysis. RESULTS: Compared with normal saline group, 46 protein spots showed changed protein expression. Twelve protein spots demonstrated increased volume and 34 protein spots demonstrated decreased volume. Among spots of decreased volume, 17 spots showed statistically significant differences. Thirty-eight identified proteins were associated with alterations in energy metabolism, muscle contractile function, transcription, translation, cell proliferation, and cellular stress response. CONCLUSION: BoNT-A gives influences on muscle contractile function and energy metabolism directly or indirectly besides neurotoxic effects. Proteomic expression provides better understanding about the effect of BoNT-A on skeletal muscle.
Animals ; Body Weight ; Botulinum Toxins ; Botulinum Toxins, Type A ; Cell Proliferation ; Electrophoresis ; Electrophoresis, Gel, Two-Dimensional ; Energy Metabolism ; Muscle Proteins ; Muscle, Skeletal ; Muscles ; Proteins ; Proteomics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo optimize the performance of Pulsed-Field Gel Electrophoresis (PFGE) for the comparison of inter-laboratory results and information exchange of Borrelia burgdorferi subtyping.

METHODSA panel of 34 strains of B. burgdorferi were used to optimize PFGE for subtyping. In order to optimize the electrophoretic parameters (EPs), all 34 strains of B. burgdorferi were analyzed using four EPs, yielding different Simpson diversity index (D) values and the epidemiological concordance was also evaluated.

RESULTSThe EP of a switch time of 1 s to 25 s for 13 h and 1 s to 10 s for 6 h produced the highest D value and was declared to be optimal for MluI and SmaI PFGE of B. burgdorferi. MluI and SmaI were selected as the first and second restriction enzymes for PFGE subtyping of B. burgdorferi according to discrimination and consistency with epidemiological data.

CONCLUSIONPFGE can be used as a valuable test for routine genospecies identification of B. burgdorferi.

Animals ; Bacterial Proteins ; metabolism ; Bacterial Typing Techniques ; Borrelia burgdorferi ; classification ; genetics ; isolation & purification ; DNA, Bacterial ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Ixodes ; Rats

The object of the research was to extract, purify and identify the type II collagen of Agkistrodon acutus. Type II collagen of A. acutus was extracted by enzyme decomposition method, and purified by ion exchange column chromatography. It was characterized by SDS-PAGE gel electrophoresis, ultraviolet spectrophotometry, infrared absorption spectroscopy and mass spectroscopy. The results showed that the size of C II was about 130 kDa. It absorbed at 223 nm. IR spectrum obtained showed that the triple helical domains of amino-acid sequences were characterized by the repetition of triplets Gly-X-Y. The MS spectrum graphically stated that C II extracted from cow and A. acutus have the similar peptides. The C II of A. acutus was obtained by extraction and purification. Appraisal analysis by SDS-PAGE, UV, IR and MS, C II of A. acutus was consistent with the standard C II of cow. It was proved that the extracted protein was C II.
Agkistrodon ; metabolism ; Animals ; Collagen Type II ; chemistry ; isolation & purification ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Mass Spectrometry ; Reptilian Proteins ; chemistry ; isolation & purification ; metabolism

The amino group PEGylation of rhIFNomega with monomethoxy polyethylene glycol succinimidyl succinate (mPEG-SS, 20 000) was investigated, and the modified mixture was separated and purified by ion exchange chromatography and gel filtration chromatography. Under the optimized purification conditions, the average content ofmono PEG-rhIFNomega in the collect liquid reached 182 microg x mL(-1). The average purified yield of mono PEG-rhIFNomega exceed to 22%, and the purity of mono PEG-rhIFNomega was greater than 98% by SDS-PAGE and RP-HPLC. Relative molecular mass of mono PEG-rhIFNomega was 43 790 detected by MALDI-TOF MS. The apparent molecular mass measured by SDS-PAGE was about 60 810. The purified PEG-rhIFNomega has the characteristics of typical PEGylated protein. Activity reservation rate of mono PEG-rhIFNomega was 15.0%, while the antigenicity decreased by at least 64 folds. In addition, the acid stability, thermal stability and stability in serum and trypsin solution of mono PEG-rhIFNomega were markedly better than those of the rhIFNomega. The pharmacological properties of mono PEG-rhIFNomega were significantly improved. The prepared PEG-rhIFNomega might be developed to a novel safe and long-acting interferon.
Animals ; Antigen-Antibody Reactions ; Chromatography, Gel ; Chromatography, High Pressure Liquid ; Chromatography, Ion Exchange ; Drug Stability ; Electrophoresis, Polyacrylamide Gel ; Interferon Type I ; chemistry ; immunology ; Molecular Weight ; Polyethylene Glycols ; chemistry ; Rabbits ; Recombinant Proteins ; chemistry ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Adiponectin stimulates cholesterol efflux in macrophages and low adiponectin may in part contribute to disturbed reverse cholesterol transport in type 2 diabetes. Monocytes express high levels of annexin A6 that could inhibit cholesterol efflux and it was investigated whether the atheroprotective effects of adiponectin are accompanied by changes in annexin A6 levels. Adiponectin reduces annexin A6 protein whereas mRNA levels are not affected. Adiponectin-mediated activation of peroxisome proliferator-activated receptor alpha (PPARalpha) and AMP-activated protein kinase (AMPK) does not account for reduced annexin A6 expression. Further, fatty acids and lipopolysaccharide that are elevated in obesity do not influence annexin A6 protein levels. Annexin A6 in monocytes from overweight probands or type 2 diabetic patients is significantly elevated compared to monocytes of normal-weight controls. Monocytic annexin A6 positively correlates with body mass index and negatively with systemic adiponectin of the blood donors. Therefore, the current study demonstrates that adiponectin reduces annexin A6 in monocytes and thereby may enhance cholesterol efflux. In agreement with these in vitro finding an increase of monocytic annexin A6 in type 2 diabetes monocytes was observed.
AMP-Activated Protein Kinases/metabolism ; Adiponectin/*pharmacology ; Animals ; Annexin A6/genetics/*metabolism ; Body Mass Index ; CHO Cells ; Case-Control Studies ; Cell Culture Techniques ; Cholesterol/metabolism ; Cricetinae ; Cricetulus ; Diabetes Mellitus, Type 2/*blood ; Down-Regulation/*drug effects ; Electrophoresis, Polyacrylamide Gel ; Humans ; Immunoblotting ; Male ; Middle Aged ; Monocytes/*metabolism ; Obesity/*blood ; PPAR alpha/metabolism ; RNA, Messenger/metabolism ; Recombinant Proteins/pharmacology ; Reverse Transcriptase Polymerase Chain Reaction

Background: Acute gastroenterophathy usually caused by the Rota virus for children under 5 years old. Objectives: To present various types of data on epidemiology of ROTA virus diarrhea in Ho Chi Minh city from 12/2006-11/2007. Material and method: The data were collected from 500 stool specimens of diarrhea diagnosed chilren hosptalised at Thuy Dien Pediatric hospital 1, Ho Chi Minh city from December/2006 to November /2007. Results:There were 322 rotavirus-positive specimens, representing 64.4%. The proportions of monthly distribution of cases with diarrhea due to rotavirus were 90.1%, 54.39%, 85.37%, 74.51%, 72.92%, 41.67%, 26.67%, 58.33%, 79.31%, 52.63%, 69.05% and 57.78%, respectively. The numbers of rotavirus-positive cases in male and female were 216 (65.26%) and 106 (62.72%), respectively. The proportions of Rota virus positive children compared to total number of diarrheal cases with age 0-3, 3-6, 6-12, 12-24, 24-36 and over 36 months were 2.80%, 7.76%, 40.06%, 40.68%, 5.28% and 3.42%, respectively.\r\n', u'The results of typing identification indicated that the phenotypes of 98 among 100 specimens were identified (98%) in which there were sixty-one specimens of G1P8 (61%), one specimen of G2P8 (1%), fourteen specimens of G3P8 (14%), four of specimens of G4P8 (4%), eighteen specimens of GmixedP8 (18%). There were only two specimens of GnontypeableP8 (2%). Conclusion: Further studies should be carried out to clear this issue.\r\n', u'
Rotavirus ; gel type.

AIMTo analyse the alterations of protein expression in the kidney of spontaneously hypertensive rat with losartan.

METHODSThe proteins of the kidney were isolated by two-dimensional gel electrophoresis. The protein spots with significant changes were selected for further identification by LC-MS/MS.

RESULTSThe number of average protein spots of two groups was 570 +/- 48 and 686 +/- 30 respectively. Compared with the SHR, 13 spots had changed significantly after treated with losartan. There were 5 protein spots detected only in SHR group, while 4 up-regulated and 4 down-regulated protein spots were detected in SHR-L group. These differentially expressed proteins were detected by mass spectrometry. 7 spots were identified. There were Heat shock protein (Hsp), Tubulin alpha-1 chain, Transthyretin precursor, Liver regeneration-related protein LRRG03, Ezrin-radixin-moesin binding phosphoprotein 50, Phosphoglycerate kinase 1 and Anionic trypsin I precursor.

CONCLUSIONThe different protein spots expression may play important roles in Losartan's effective protection to hypertension rats renal tissue.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; therapeutic use ; Animals ; Chromatography, Liquid ; Electrophoresis, Gel, Two-Dimensional ; methods ; Hypertension ; drug therapy ; metabolism ; physiopathology ; Kidney ; metabolism ; physiopathology ; Losartan ; pharmacology ; therapeutic use ; Male ; Proteome ; metabolism ; Proteomics ; methods ; Rats ; Rats, Inbred SHR ; Tandem Mass Spectrometry

OBJECTIVETo investigate the effect of Rehmannia glutinosa water extraction (RGL) on adipose metabolic disorder and gene expression of resistin in type 2 diabetes mellitus rats.

METHODThe wistar rats model of 2-DM were induced by high calorie feeding and small dose injection of STZ. Rats were randomly divided into diabetic model group, diabetic model treated with RGL (2.4 g x kg(-1) x d(-1)), RGL (1.2 g x kg(-1) x d(-1)), RGL (0. 6 g x kg(-1) x d(-1)) and normal control group. The levels of FPG, FINS, TG, HDL, LDL, CH and IR were measured, and the mRNA expression of resistin was determined by RT-PCR, the protein expression measured by SDS-PAGD at the end of 8 weeks.

RESULTThe gene expression of resistin in RGL group were lower than that of diabetic model (P < 0.01). The levels of FPG, FINS, TG, LDL, CH, IR in RGL group were lower than that of diabetic model (P < 0.05), and HDL were higher (P < 0.05). CONCLUDE: RGL can improve insulin resistance in the experimental 2-DM rats, can effectively ameliorate adipose metabolic disturbance and decline IR and FINS by increasing the gene expression of resistin.

Animals ; Blood Glucose ; metabolism ; Cholesterol ; blood ; Diabetes Mellitus, Experimental ; blood ; genetics ; pathology ; Diabetes Mellitus, Type 2 ; blood ; genetics ; pathology ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Electrophoresis, Polyacrylamide Gel ; Female ; Gene Expression Regulation ; drug effects ; Hypoglycemic Agents ; isolation & purification ; pharmacology ; Insulin ; blood ; Insulin Resistance ; Male ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Rehmannia ; chemistry ; Resistin ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Triglycerides ; blood

OBJECTIVETo analyze the constituent proteins in donkey hide, the key ingredient for Ejiao, an important traditional Chinese medicine for the blood-related conditions, in hope to eventually decipher the biochemical mechanism behind Ejiao's prominent medicinal efficacy.

METHODTwo methods were employed to extract proteins in donkey skin. One used TriPure isolation reagent to extract the total proteins in donkey skin. Another used 1% sodium dodecyl sulfate (SDS) to heat the sample at 100 degrees C overnight. And then sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and capillary HPLC were used to analyze the component of proteins.

RESULTThere are not only collagen alpha1 (I) and collagen alpha2 (I), but also serum albumin in donkey skin. The content is over 25% in total proteins with the method of TriPure isolation reagent. The content of donkey serum albumin is up to 20% with the method of 1% SDS heating. And two bands, molecular weight are nearly 200 kDa,were found on 7.5% SDS-PAGE. Extracted these proteins to analyze with capillary HPLC, they were found to be the complex products of collagen and serum albumin of donkey.

CONCLUSIONDonkey serum albumin is a main protein component in the hide, which is a clue to expose is the effect of Ejiao on blood.

Animals ; Chromatography, High Pressure Liquid ; Collagen Type I ; analysis ; chemistry ; metabolism ; Collagen Type II ; analysis ; chemistry ; metabolism ; Drug Interactions ; Electrophoresis, Polyacrylamide Gel ; Equidae ; Molecular Weight ; Protein Binding ; Serum Albumin ; analysis ; chemistry ; metabolism ; Skin ; chemistry

BACKGROUND: Polymerase chain reacation (PCR)-based methods have been described for rapid detection and identification of Candida spp. Multiplex PCR assay was developed using internal transcribed spacers and topoisomerase II gene for the accurate identification of Candida species. METHODS: We designed Dual Specificity Oligo (DSO) primers for multiplex PCR. Multiplex PCR was followed by agarose gel electrophoresis to test 8 type strains (C. albicans, C. parapsilosis, C. glabrata, C. tropicalis, C. krusei, C. guilliermondii, C. lusitaniae, C. dubliniensis) and 96 clinical isolates (C. albicans 51 isolates, C. parapsilosis 10 isolates, C. glabrata 10 isolates, C. tropicalis 9 isolates, C. krusei 6 isolates, C. guilliermondii 5 isolates, C. lusitaniae 5 isolates) of Candida spp. RESULTS: With multiplex PCR using DSO primers, the eight Candida type strains each could be easily differentiated and all 96 clinical isolates were identified as the same species as were identified by the conventional method. CONCLUSION: Multiplex PCR followed by electrophoresis can be useful for the simple and rapid identification of Candida species in routine laboratories.
Candida* ; DNA Topoisomerases, Type II ; Electrophoresis ; Electrophoresis, Agar Gel ; Multiplex Polymerase Chain Reaction* ; Sensitivity and Specificity