OBJECTIVE: To establish a reliable, exact and practical method to prepare DNA samples for sensitivity-test purposes. METHODS: The micromanipulation method was employed to prepare exact quantity DNA samples used to study the sensitivity of Profiler Plus Kit-ABI PRISM 310 system. RESULTS: We succeed in establishing a micromanipulation method to prepare groups of DNA samples, which contain 1-11 cells in turn, and also succeed in using them to study the sensitivity of Profiler Plus Kit-ABI310 system. CONCLUSION The method we have established is proved to be a reliable, exact and practical way to prepare DNA samples for sensitivity-test purposes.
DNA/isolation & purification* ; DNA Fingerprinting/methods* ; Microscopy ; Polymerase Chain Reaction ; Sensitivity and Specificity ; Specimen Handling/methods* ; Tandem Repeat Sequences

AIMTo develop a near-infrared diffuse reflectance analysis (NIRDRA) method for rapid noninvasive quantitative determination of ambroxol hydrochloride in half-finished product particles and non-blister-packed, blistered tablets.

METHODSAll spectra were measured with a Fourier transform spectrometer equipped with a PbS and a InGaAs detector, an external integrating sphere, a rotating sample cup, and a fibre-optic probe for reflectance measurements. All samples were scanned from 12,000 cm-1 to 4,000 cm-1, and each sample spectrum was obtained as an automatic mean of 64 scans. No spectrum pre-processing method was used, and spectral regions, 4,602-4,247, 12,000-7,498 and 6,102-5,446, 12,000-5,446 cm-1 were selected to develope mathematical models by partial least square method for half-finished product particles and non-blister-packed, blistered tablets samples, respectively.

RESULTSThe optimal rank and mean square error determined for half-finished product particles and non-blister-packed, blistered tablets samples by cross validation method all was 6 and 0.306, 0.972 and 1.492, respectively, the average recovery was 100%, 100% and 102% respectively; and the RSD was 1.17%, 1.70% and 1.78% respectively.

CONCLUSIONResults showed that the NIRDRA method was rapid, simple, noninvasive and sensitive, and it can be applied to assay the content of ambroxol hydrochloride in half-finished product particles non-blister-packed and blistered tablets.

Ambroxol ; administration & dosage ; analysis ; Expectorants ; administration & dosage ; analysis ; Quality Control ; Spectroscopy, Near-Infrared ; methods ; Tablets

Objective To investigate the association between cutaneous adverse drug reactions (CADRs) caused by antiepileptic drugs and HLA-B*1502 allele. Methods In 31 epileptic patients presented to the Epilepsy Clinic of the Second Affiliated Hospital of Guangzhou Medical College between January 2007 and May 2008, 13 had CADR to carbanazepine (CBZ) including 6 with Stevens-Johnson syndrome (SJS) and 7 with mild maculopapular exanthona (MPE);15 were CBZ-tolerant, and 3 had lamotrigine (LTG)-indueed MPE. All the patients underwent examinations using polymerase chain reaction with sequence specific palmers to analyze HLA -B*1502 allele frequencies, with 30 healthy subjects without a history of using CBZ or LTG as the control. Results HLA-B*IS02 allele frequency was 100% (6/6) in patients with CBZ-SJS, 57% (4/7) in patients with CBZ-induced MPE, and 33% (1/3) in patients with LTG-induced MPE. The frequency was 7% (1/15) in CBZ-tolerant patients and 10% (3/30) in the control subjects. Compared with the CBZ-tolerant patients and the control subjects, the patients with CBZ-induced SJS and MPE had significantly increased HLA -B*1502 allele frequency (P<0.05). Conclusions HLA-B*1502 allele is associated with CADRs to CBZ in epileptic patients.

Recombinant adeno-associated viruses (rAAV) vectors have been shown to mediate long-term transgene expression in mice and nonhuman primates. We have adapted viral vector system based on two rAAV vectors, namely rAAV1 and rAAV2. We have generated rAAV vectors expressing the envelope glycoprotein (E1 and E2) derived from Chinese HCV patient (genotype 1b) and used these to immunize BALB/c mice. We detected the total antibody titer by IFA and neutralizing antibody (nAb) using in vitro HCV neutralizing assays based on HCV pseudotyped particles. Furthermore, IFN-gamma ELISpot assay was used to assess the T cellular response against HCV at 12 weeks after rAAV1-E1E2 immunization. We also analyzed HCV envelope glycoprotein expression in muscle of rAAV1-E1E2 immunized mice. Our data showed: (i) rAAV1 directed long-term expression of HCV genes in mice; (ii) immunized intramuscularly with a single dose of rAAV elicited durable and effective immune responses in mice; and (iii) Moreover, rAAV1-E1E2 induced higher total antibody and nAb titers than rAAV2-E1E2 did. These data suggest that rAAV1 vectors could stimulate robust, durable, and effective immune responses against HCV.
Animals ; Antibodies, Viral ; blood ; Dependovirus ; genetics ; metabolism ; Female ; Genetic Vectors ; genetics ; metabolism ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; virology ; Humans ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; administration & dosage ; genetics ; immunology ; Viral Envelope Proteins ; administration & dosage ; genetics ; immunology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

Human parathyroid hormone (hPTH) was highly expressed in Escherichia coli by inserted the synthesized whole hPTH cDNA into the vectors pBV220 and pET22b. After expression and disruption, the purified product was acquired through cation exchange chromatography and reverse phase chromatography. From the results of N-terminal sequencing and MALDI-TOF-MS analysis the recombiant prtein was indentified as intact hPTH. In in vitro Bioassays the recombinant hPTH stimulated adenylate cyclase as the standard did. In ovariectomized rats the recombinant hPTH markedly increased the femoral bone mass and bone mineral density.
Amino Acid Sequence ; Animals ; Base Sequence ; Bone Density ; drug effects ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Molecular Sequence Data ; Ovariectomy ; Parathyroid Hormone ; chemistry ; genetics ; metabolism ; pharmacology ; Rats ; Rats, Wistar ; Sequence Alignment ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo explore the P75NTR expression in the mouse testis and its relationship with nestin.

METHODSWe observed the location of the expressions of P75NTR and nestin in the testis of the nestin-GFP transgenic mouse on postnatal day (PND) 5, 14 and 30 using immunofluorescence, and detected the expression levels of P75NTR in the testicular tissue of mice in different age groups by real-time quantitative PCR (RTqPCR) and flow cytometry. Then we cultured the P75NTR positive cells in neural stem cell culture medium and observed their neuronal differentiation capacity by orientation differentiation.

RESULTSImmunofluorescence showed the expressions of P75NTR and nestin in the Leydig cells of the mouse testis. RTqPCR and flow cytometry exhibited the peak of the P75NTR expression on PND 14. The positive rates of P75NTR were (2.88 +/- 0.52), (9.54 +/- 1.81) and (2.63 +/- 0.43)% on PND 5, 14 and 30, respectively. The P75NTR positive cells obtained also expressed nestin and P75NTR and had the capacity of neuronal differentiation.

CONCLUSIONP75NTR and nestin are co-expressed in the Leydig cells of the mouse testis, and the P75NTR positive cells have the ability of neural differentiation, which is presumably attributed to neural crest cells.

Animals ; Intermediate Filament Proteins ; genetics ; metabolism ; Leydig Cells ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Nerve Tissue Proteins ; genetics ; metabolism ; Nestin ; Receptor, Nerve Growth Factor ; genetics ; metabolism ; Testis ; cytology ; metabolism

OBJECTIVETo add DXS7133, GATA198A10, DXS9896 and DXS6797 to the panel of forensically validated X chromosome markers, and apply the multiplex amplification system to a population study and forensic analysis on the Hans of Chengdu.

METHODSThe PCR products were detected by the polyacrylamide gel electrophoresis and silver staining method. Hardy-Weinberg equilibrium of females was tested and every forensically interested value was calculated.

RESULTSSequencing revealed that their common sequence motifs were tetranucleotide repeats. Population genetic data were obtained by analyzing 120 unrelated females and 100 males from Chengdu Han ethnic group. In this population, DXS7133, GATA198A10, DXS9896 and DXS6797 exhibited 6, 6, 11, 8 distinguishable alleles respectively. Chi-square test demonstrated that genotype frequencies in females did not depart from Hardy-Weinberg equilibrium. Power of discrimination for female samples for the four loci were 0.7962, 0.8021, 0.9675, and 0.9444. The parentage testing in 32 cases revealed a typical X-linked inheritance and no mutations.

CONCLUSIONDXS7133, GATA198A10, DXS9896 and DXS6797, which are highly polymorphic in Chengdu Han population, are appropriate for individual identification and paternity testing involving a female child.

Asian Continental Ancestry Group ; genetics ; Chi-Square Distribution ; China ; Chromosomes, Human, X ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Male ; Microsatellite Repeats ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic ; genetics

OBJECTIVETo solve the problems in the accuracy and standardization of short tandem repeats-polymerase chain reaction (STR-PCR) typing, the authors adopted the molecular clone technology in producing the standard allelic ladders of D1S1676, D2S2735, D11S1977 and D22S444 loci and applied them in a population study on the Hans in Chengdu, China.

METHODSPCR was used to produce several different allelic fragments of these loci. PCR products were eluted from the gel and re-amplified by PCR. The purified allelic fragments were then blunt-end subcloned individually into the pGEMR-T plasmid vectors and the recombinant were transfected into competent E.coli DH5alpha TM cells. The results of sequencing confirmed that the size and the construction of the inserts were correct. The recombinant plasmids DNA with the inserts were then used as template for re-amplification to generate the four loci standard ladders.

RESULTSThe authors succeeded in producing large quantity of standard allelic ladder of these four loci, with which the genetic polymorphisms of these loci in Chengdu Han population of China were studied.

CONCLUSIONThis method is of high value for forensic DNA typing to construct standard ladders. D1S1676, D2S2735 loci are robust for forensic analysis in Chinese Han population, whereas the value of D11S1977 and D22S444 loci is limited.

Alleles ; Asian Continental Ancestry Group ; genetics ; China ; Genetics, Population ; methods ; Humans ; Microsatellite Repeats ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic

OBJECTIVE: To explore the protective effect of HSP72 on the acute injury of cardiomyocyte induced by oxidative stress. METHODS: Cardiomyocytes of neonatal rats treated with heat shock (42 degrees C, 30 min, recovery for 6 h) to induce the expression of HSP72 and HSP72 antisense oligonucleotide was transformed to block the expression of HSP72. 0.5 mmol/L (final concentration) H2O2 was added into the culture medium to mimic oxidative stress, and to induce the acute injury of neonatal cardiomyocytes. The release of LDH and the total protein synthesis were applied to evaluate the injury of cardiomyocyte of neonatal rats. RESULTS: Oxidative stress could significantly increase the release of LDH, and inhibit the total protein synthesis. By inducing the expression of HSPs, heat shock pretreatment significantly reduced the release of LDH and relieved the oxidative stress-mediated inhibition of total protein synthesis. Moreover, HSP7-2 anti-sense oligonucleotide could remarkably block the protective effect of heat shock pretreatment on the cellular injuries induced by H2O2. CONCLUSION HSP72 plays a most important role in the acute injury of cardiomyocyte mediated by oxidative stress.
Animals ; Animals, Newborn ; Cells, Cultured ; HSP72 Heat-Shock Proteins ; pharmacology ; L-Lactate Dehydrogenase ; metabolism ; Myocytes, Cardiac ; pathology ; Oligonucleotides, Antisense ; pharmacology ; Oxidative Stress ; Protein Biosynthesis ; Random Allocation ; Rats ; Rats, Wistar

Objective To analyze the characteristics of cognitive function impairment in PD patients without dementia and their influencing factors, and provide evidence for early recognition and treatment of cognitive deficits in PD patients. Methods Fifty-six PD patients without dementia,admitted to our hospital from January 2010 to October 2011 were assessed with mini-mental state examination (MMSE) and Montreal cognitive assessment (MoCA) for cognitive function. Logistic stepwise regression was employed to analyze the influencing factors of cognitive function impairment.Results Using MMSE scores as the standard for recognition, PD patients with mild cognitive impairment (MCI) accounted for 7.14％ (6/56); however,using MoCA scores,they accounted for 71.43％(40/56). Both of the MMSE and MoCA scores were positively correlative with the education degree of the patients (r=0.483,P=-0.007; r=0.503,P=0.000).In the cognitive domain of MMSE,the scores of visuospatial function,and abilities of delayed recall,calculation,attention and repetition had significant decrement; and MMSE indicated that abilities of delayed recall, immediate memory, calculation and attention,and visuospatial function were main cognitive disturbances of PD.In the cognitive domain of MoCA,the scores of visuospatial and executive functions,and abilities of delayed recall,abstraction and repetition had significant decrement; MoCA indicated that visuospatial and executive functions,abilities of delayed recall,denomination,attention,abstraction and repetition were main cognitive disturbances of PD. Logistic regression analysis showed that education degree and clinical types were the main influencing factors of cognitive function impairment in PD patients. Conclusion Cognitive impairment is very common in patients with PD,and the main cognitive deficits involve visuospatial and executive functions,abilities of delayed recall,calculation,attention,abstraction and repetition; education degree and clinical types are the influencing factors of cognitive impairment of PD.