The culture conditions of Saccharomyces cerevisiae sp.strain by 1.1 b were optimized for the production of D-(-)-mandelate dehydrogenase which is useful for the asymmetric bioreduction of benzoylformate to form D-(-)-mandelate.The optimum medium(per liter)consistes of 60 g peptone,30 g maltose, 0.5 g MgSO_4,0.01 g ZnSO_4,1.0 g KCl.After optimization of the culture medium,the enzyme production in shake flasks is enhanced from 2.56 to 20.21 U/L.The optimum fermentation conditions were determined as follows:medium volume 100 mL(i.e.,40%for a 250-mL shake flask),pH 6.5,inoculum size 10%,temperature 30℃,and cultivation time 25 h.

Objective To determine the changes of interleukin-12(IL-12) and IL-12 mRNA in gastric mucosa of children with helicobacer pylori (Hp) infection,and to study the effects of Hp infection on the expression levels of IL-12 and IL-12 mRNA,and to evaluate its possible roles in the pathogenesis of gastric mucosal inflammation in Hp related gastroduodenal diseases.Methods Biopsy specimens were taken from the antral mucosa on endoscopy in patients with or without Hp infection, which were diagnosed by urease test and Giemsa staining. The expression levels of IL-12 and IL-12 mRNA in gastric mucosa were determined by ELISA and RT-PCR.Results Inflammation of gastric antral mucosa was more severe in Hp-positive mucosa .The expression levels of IL-12 and IL-12 mRNA in Hp-positive mucosa were (66.42?15.15) ng/g and (59.21?15.03)%,which were more than those in (Hp-negative )(22.22?8.79) ng/g and (17.94?7.39)%(P

OBJECTIVETo study the effects of catecholamines on human preadipocyte proliferation and differentiation.

METHODSCatecholamines (epinephrine, isoprenaline and noradrenaline) were added to the culture media of human preadipocytes. The proliferation of cells, the expression of GPDH and lipid droplet accumulation in cytoplasm were observed and recorded. The functions of alpha and beta receptors were examined with adding alpha and beta receptor blockers.

RESULTSEpinephrine and isoprenaline stimulated human preadipocyte proliferation and inhibited GPDH up-regulation during differentiation. The three types of catecholamines inhibited lipid accumulation in cell differentiation. The beta-adrenoceptors played a key role during the process.

CONCLUSIONHuman preadipocytes responded to catecholamine characteristically. The result would be applicable in the study of drugs for obesity.

Adipocytes ; cytology ; drug effects ; Catecholamines ; pharmacology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Epinephrine ; pharmacology ; Humans ; Isoproterenol ; pharmacology ; Norepinephrine ; pharmacology ; Obesity ; drug therapy ; Receptors, Adrenergic, beta ; Up-Regulation

Objective To investigate the expressions of S100B and calcitonin gene related peptide (CGRP) and the pathologic alterations of the hippocampus in kainic acid (KA)-induced epileptic rats. Methods Male SD rats were randomly divided into control group (n=8) and model group (n=40).Animal models of temporal lobe epilepsy were established by intracerebroventricular injection of KA; the same volume of saline was injected into the rats in the control group. Hippocampal tissues within various phases after seizures (6, 12, 24 and 72 h, and 24 h after the success of model making) were performed Nissl staining, Timm staining and immunohistochemical staining. The expressions of S100B and CGRP were observed, and the pathologic alterations of the hippocampal neurons and glial cells were studied.Results All rat models were successfully induced with epileptic seizures. Nissl staining showed that pyknotic neuronal necrosis appeared in the CA3 area of the hippocampus in the model group with cell body atrophy and disappearance of Nissl bodies 1 week after the injection. Timm staining showed that brown particles showed stripped distribution in the CA3 area of the hippocampus and some brown particles in the molecular layer of fascia dentate. Immunohistochemical staining indicated that significant neurons lost and gliosis appeared after seizures with abundant expressions of S100B and CGRP.Conclusion KA-induced epileptic rats express abundant S100B and CGRP and appear such pathological changes as disappearance of Nissl bodies and mossy fiber sprouting, indicating that both S100B and CGRP participate in the onset of epilepsy.

OBJECTIVETo explore the significance and the role of the p53 gene mutation in the exon 4 to 8 in keloid fibroblasts.

METHODSTissue samples from twelve patients with keloid and twelve hyperplastic scar respectively were harvested for in vitro culture of fibroblasts, and normal skin samples from the same patients were employed as the control. Polymerase chain reaction-based single-strained conformational polymorphism (PCR-SSCP) and DNA sequencing were employed to detect p53 gene mutations of the fibroblasts.

RESULTSThe points and frameshift mutations in the exon 4, 5, 6, 7 of p53 gene were identified in 9 of the 12 keloid tissue samples. No p53 gene mutation was detected in all hyperplastic scar and normal skin samples.

CONCLUSIONp53 gene mutation might play an important role in the formation and development of keloids.

Female ; Fibroblasts ; metabolism ; Genes, p53 ; Humans ; Keloid ; genetics ; Male ; Mutation

OBJECTIVETo explore the differential expression of homeobox genes in the normal, wounded human fetal and adult skins and its significance in fetal scarless healing.

METHODSGene chips containing 14 000 human genes were used to investigate homeobox gene expressions of the normal, wounded human fetal and adult skins.

RESULTSThere were significant differences between the expression of homeobox genes, especially for PRX-2, HOXB13, HOXB6 and HOXB7.

CONCLUSIONSThe homeobox gene is in close relation to developmental biology. The different expressions and changes of homeobox genes in the normal, wounded human fetal and adult skin may be a primary cause of different wound healing between fetal and adult skin.

Adolescent ; Adult ; Animals ; Cells, Cultured ; Child ; Female ; Fetus ; Gene Expression ; Gene Expression Profiling ; Genes, Homeobox ; genetics ; Homeodomain Proteins ; genetics ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Oligonucleotide Array Sequence Analysis ; Skin ; Wound Healing ; genetics ; Young Adult

OBJECTIVETo culture the keratinocytes on the acellular pig dermis and establish a composite skin in vitro.

METHODSFull thickness skin taken from neonatal SD rats of approximate 24-hour-old was cultivated in asepsis condition, which was then separated into epidermal and dermal layers with low-temperature enzyme digestion. The basal lamina cells between the two layers were scraped off and the pure keratinocytes were obtained using gradient density centrifugation. In the experimental group, the keratinocytes were cultured on acellular xenodermis(AX) as the three-dimensional frames. In the control, the keratinocytes were cultured without any trophoblast. The air-liquid interface(ALI) culture continued after the primary culture. Routine histological HE staining and assay of Pancytokeratin and Laminin with strepavidin-biotin-peroxidase complex (SABC) method were used to study the morphology of CK and AX.

RESULTSHE staining demonstrated the formation of more than 4 lays of keratinocytes and basement membrane, with slight keratinization of the cells. Pancytokeratin(+) in immunohistochemical results confirmed that the cultured cells on AX were keratinocytes, not other kinds of cells. Laminin(+) indicated that new procreant basement membrane appeared in CK.

CONCLUSIONKeratinocytes cultured on the acellular pig dermis grow well and form basement membrane. This study implies successful construction of composite skin.

Acellular Dermis ; Animals ; Animals, Newborn ; Basement Membrane ; cytology ; Cell Culture Techniques ; Cells, Cultured ; Epidermis ; cytology ; Keratinocytes ; cytology ; Keratins ; analysis ; Laminin ; analysis ; Rats ; Skin ; cytology ; Skin Transplantation ; Staining and Labeling ; methods ; Swine ; Transplantation, Heterologous

OBJECTIVETo investigate effects of low-dose cyclophosphamide and prednisone (CP) metronomic chemotherapy on microvessel density of bone marrow, serum vascular endothelial growth factor (VEGF) and platelet derived growth factor BB (PDGF-BB)in multiple myeloma (MM) patients.

METHODS54 refractory or relapsed MM patients were treated with CP metronomic chemotherapy consisted of oral cyclophosphamide (CTX, 50 mg/d) and prednisone (Pred, 15 mg/d). Bone marrow and peripheral blood of each patient were collected before and 2, 4, 6 months after treatment. Among the 37 assessable patients, 30 cases were responsive with the response rate of 81.08%. Another 17 cases were follow-uped less than 6 months or failure to obtain serum samples or lost to follow-up. Microvessel density of bone marrow was measured by immunohistochemistry and serum VEGF/PDGF-BB expression was analyzed by ELISA in the 37 assessable patients.

RESULTS2, 4, 6 months following CP metronomic chemotherapy, microvessel densities of bone marrow in the responders were 33.1 ± 4.8/HP, 24.8 ± 3.7/HP, 19.7 ± 2.1/HP respectively; the expressions of VEGF were (394 ± 57) ng/L, (268 ± 32) ng/L and (217 ± 20) ng/L respectively; the expressions of PDGF-BB were (304 ± 31) ng/L, (274 ± 31) ng/L and (196 ± 22) ng/L respectively. After CP metronomic chemotherapy, there were significantly lower of microvessel density, VEGF and PDGF-BB levels than pretreatment \[MVD 48.5 ± 5.9/HP, VEGF (517 ± 60) ng/L, PDGF-BB (484 ± 60) ng/L\]in the responders (P < 0.01). While in the non-responders, after treated by CP metronomic chemotherapy for 2 months, microvessel density, the expression of VEGF and the expression of PDGF-BB were 32.5 ± 4.7/HP, 512 ± 39 ng/L and (452 ± 39) ng/L respectively. There were no significant changes of MVD, VEGF and PDGF-BB levels compared with pretreatment \[MVD 33.2 ± 5.6/HP,VEGF (498 ± 55) ng/L, PDGF-BB (488 ± 44) ng/L\] (P > 0.05).

CONCLUSIONSOur findings suggested that continuous low-dose CP metronomic chemotherapy could decrease microvessel density of bone marrow in MM patients. Furthermore, it down-regulated expression of serum VEGF and PDGF-BB to exert its anti-angiogenesis in MM.

Aged ; Aged, 80 and over ; Angiogenesis Inhibitors ; administration & dosage ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Cyclophosphamide ; administration & dosage ; Female ; Humans ; Male ; Microvessels ; drug effects ; Middle Aged ; Multiple Myeloma ; blood ; blood supply ; drug therapy ; Prednisone ; administration & dosage ; Proto-Oncogene Proteins c-sis ; blood ; Vascular Endothelial Growth Factor A ; blood

OBJECTIVETo investigate the effects of transfection of agrin gene on the recovery of muscle function after a free neurovascular muscle transfer.

METHODSThe electrical gene transfection was performed when the gracilis muscle of the SD rat was completed free neurovascular transfer. The experimental group was treated with pCS2+ -agrin, the group with plasmid pCS2+ as the negative control and the group with normal saline as the frank control. The muscle function, expression of neural agrin and the junctional nAChR number was measured after the operation.

RESULTSAt 4, 5 and 10 weeks postoperatively, the pCS2+ -agrin group was significantly better than the control groups in muscle function (P < 0.05 ). The immunohistochemical staining showed an increasing deposition of the agrin protein near the endplate at 1 and 5 weeks after the operation, but decreasing remarkably to the level of control groups at 10 weeks postoperatively. The pCS2+ -agrin group was significantly more than the control groups in junctional nAChR number at every points of the time postoperatively.

CONCLUSIONSTransfection of agrin gene in the transferred muscle may increase the early recovery of muscle function.

Agrin ; genetics ; Animals ; Female ; Genetic Therapy ; Genetic Vectors ; Muscle Proteins ; genetics ; Muscle, Skeletal ; transplantation ; Rats ; Rats, Sprague-Dawley ; Recovery of Function ; Transfection

Human glutamate decarboxylase 65(hGAD65) is an enzyme that catalyzes the transformation of L-glutamic acid into ?-aminobutyric acid.It has been found that Type 1 diabetes mellitus(T1DM)is an autoimmune disease,in which pancreatic islet ?-cells are destroyed due to immune response mediated by autoantigen.hGAD65 is considered as a key autoantigen of the autoimmune response,so anti-hGAD65 antibody(hGAD65-Ab) is the most effective and specific immune marker for T1DM diagnosis,and hGAD65 can be used to detect hGAD65-Ab in serum of T1DM patients.The hGAD65 gene was cloned into pET32a(+),then the recombinant plasmid with hGAD65 was transformed into E.coli BL21(DE3) and expressed by IPTG induction.The fusion protein containing thioredox,hexahistidine and hGAD65(Trx-hGAD65) was mostly insoluble,but the band of soluble Trx-hGAD65 could also be detected by SDS-PAGE,and it was a great improvement compared with the results reported.Trx-hGAD65 was isolated from lysate and purified by immobilized metal ion affinity chromatography(IMAC).After enterokinase digestion and IMAC purification,hGAD65 with high purity was obtained.Detection of thin-layer chromatography(TLC) showed that both Trx-hGAD65 and hGAD65 had enzymatic activity,whereas Trx-hGAD65 had better stability.Furthermore,it was confirmed that Trx-hGAD65 was able to conjugate with hGAD65-Ab in the serum of T1DM patients by ELISA assay.In conclusion,Trx-hGAD65 instead of hGAD65 can be used for T1DM diagnosis,and its application in prophylaxis and therapy of T1DM is expectable.