The effect of reaction media cosolvent water activity, temperature and pH on Novozym 435-caulyzed enantioselective esterification of ketoprofen was systematically explored. Novozym 435 showed high catalytic activity and enantioselectivity in cyclohexane; E value increases markedly by addition of toluene to cyclohexane; the optimum temperature and the initial water activity were found to be 30℃ and 0.09 respectively; pH shows little effect on enzymatic reaction wilson the scope studied.

OBJECTIVETo explore the expression of Toll like receptor 4 (TLR4) on the pulp cells and the change of related signaling molecules under the condition of concomitant lipopolysaccharide (LPS) and transforming growth factor-beta 1 (TGF-beta 1) during the course of pulpitis.

METHODSAfter treated by LPS and TGF-beta 1, the expression of TLR4 on pulp cells was detected by flow cytometry (FCM). The expressions of signaling molecules evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) and nuclear factor-kappaB (NF-kappaB) were detected by real-time polymerase chain reaction (real-time PCR) and Western blot. The secretion of interleukin-6 (IL-6) was detected by enzyme-linked immunosorbent assay (ELISA). Furthermore, the change of corresponding targets in inflamed pulp cells from clinical samples were detected by real-time PCR.

RESULTSAfter treated by LPS and TGF-beta 1 in vitro, there was no change in the expression of TLR4 on pulp cells, but the secretion of proinflammatory cytokines IL-6 increased. LPS and TGF-beta 1 could also increase the expression of signal downstream ECSIT and actived NF-kappaB. Furthermore, the expression of TLR4 mRNA had no increase in inflamed pulp cells from clinical samples, while the expression of TGF-beta 1, ECSIT and IL-6 mRNA increased through real-time PCR.

CONCLUSIONDuring the course of pulpitis, although the expression of TLR4 on pulp cells was inhibited by increased expression of TGF-beta 1, the TLR4 pathway was still activated. This effect could be caused through activation of ECSIT mediated by LPS, which might inhibit the TGF-beta 1 pathway.

Dental Pulp ; Epithelial Cells ; Humans ; Interleukin-6 ; Lipopolysaccharides ; NF-kappa B ; Signal Transduction ; Toll-Like Receptor 4 ; Transforming Growth Factor beta ; Transforming Growth Factor beta1

Objective To compare of the clinical using effect of implantable venous -access port(VAP) among different methods and the postoperative complication.Methods From 2011 to 2014,a total of 359 patients with VAP were included in this study.All patients were divided into two groups according to the surgery approaches methods,307 cases were implanted through internal jugular vein,and other 52 cases were through subclavian vein intubation.Statistics of postoperative flow conditions and complication between the two groups were gathered and com-pared.Results There were 3 cases of postoperative infected,and 13 cases were not free of transfusion,while 2 cases catheter were shed and shifted in the 307 cases of implantation through internal jugular vein,the complication rate was 5.86%;In other group,which in total of 52 cases of implantation through subclavian vein,including 2 cases were post-operative infected,6 cases were not free of transfusion,2 cases are of catheter fracture and5 cases were local hemato-ma,the complication rate was 28.85%.There was difference in rate of catheter leakage,catheter tip dystopy and cath-eter related infection between two groups[the proportion of through internal jugular vein vs that the group through sub-clavian vein:5.86%(18 /307)vs 28.85%(15 /52),χ2 =28.140,P =0.000].Conclusion The study suggests that the two ways of different operation methods of VAP are safe and reliable for long term intermittent venous access.The internal jugular vein implanted group has a slight advantage over the group of subclavian vein implanted,when it comes to develop the patients′living quality.

Objective To investigate the distribution and clonality of the T-cell receptor (TCR) Vβ repertoire in chronic graft versus host disease (cGVHD).Methods The complementarity determining region 3 (CDR3) of the TCRβ gene with 24 variable regions was amplified in peripheral blood mononuclear cells drawn from one cGVHD patient after allogenic bone marrow transplantation (allo-BMT) 35, 39, 43 or 45 months respectively, using RT-PCR, to observe the expression of TCR Vβ repertoire T cells. The PCR products were further analyzed by genescan to evaluate clonality of T cells. Ressults Fourteen or 16 TCR Vβ subfamily T ceils were detected in each sample of cGVHD case. Oligoclonal T cells were identified in TCR Vβ 6, 16, 17, 19 and 21 subfamilies. The stable clonal T cells in all samples were identified in Vβ6, Vβ17 and Vβ21 subfamilies.Conclusion Skewing distribution and stable clonal expansion of T cells can be found in cGVHD cases and it may be related to the initiation of cGVHD.

Chymosin is an important industrial enzyme widely used in cheese manufacture. To improve expression efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799, we designed and synthesized a DNA sequence encoding bovine prochymosin gene (GenBank Accession No. AA30448) by using optimized codons. The synthesized prochymosin gene was amplified by two-step PCR method, and then cloned into the expression vector pKLAC1, resulting in pKLAC1-Prochy. pKLAC1-Prochy was linearized and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YEPD plates containing 1% casein. A recombinant strain chyl with highest activities and multi-copy integration which was detected by using specifical integration primers was chosen and fermented in flask. Prochymosin was expressed in K. lactis successfully. SDS-PAGE analysis revealed that the purified recombinant bovine prochymosin had a molecular mass of 41 kDa. After acid treatment, molecular weight of chymosin is about 36 kDa, the same as native bovine chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 99.67 SU/mL after 96 h cultivation. The activities of chymosin were not prominent increased when galactose was used as carbon source instead of glucose, which proved that the fermentation of recombinant strain does not need galactose inducing. The recombinant K. lactis strain obtained in this study could be further used to produce recombinant chymosin for cheese making.
Animals ; Cattle ; Chymosin ; biosynthesis ; genetics ; Enzyme Precursors ; biosynthesis ; genetics ; Gene Expression Regulation, Fungal ; genetics ; Genetic Vectors ; genetics ; Kluyveromyces ; genetics ; growth & development ; metabolism ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo detect FLG gene mutations in 10 families affected with ichthyosis vulgaris and to explore mutational hot spot of the FLG gene in Chinese Han population.

METHODSPCR and direct sequencing were carried out to identify potential mutations of the FLG gene in above families. One hundred healthy individuals were analyzed as normal controls.

RESULTSThree mutations (3321delA, 5757delCCAG and S2706X) were identified in 7 families. A homozygous mutation 3321delA was also detected in two unrelated patients. No mutations were found in the remaining three families. Neither of the null mutations (5757delCCAG and S2706X) was found in the 100 controls. However, for 3321delA, a heterozygous mutation was also found in two of the controls.

CONCLUSIONThree FLG mutations have been identified in the selected families with ichthyosis vulgaris, and the 3321delA mutation was most prevalent (46.9%). Mutations 5757delCCAG and S2706X were first found in patients with ichthyosis vulgaris. Other candidate genes may underlie the disease in those without a FLG mutation.

Asian Continental Ancestry Group ; Base Sequence ; China ; Female ; Genotype ; Humans ; Ichthyosis Vulgaris ; genetics ; Intermediate Filament Proteins ; genetics ; Male ; Mutation ; Pedigree ; Phenotype

OBJECTIVETo detect the genomic deletion mutation in the NEMO gene of a family with incontinentia pigmenti (IP; MIM 308310).

METHODSA pedigree of IP was investigated. By using long PCR, the Delta4-10 deletion in NEMO gene was tested with specific primers In2/JF3R, and Delta4-10 deletion in pseudogene DeltaNEMO was investigated with primers Rev-2/JF3R. NEMO gene of 80 normal controls was also tested.

RESULTSThe deletion of exons 4-10 in both NEMO gene and the pseudogene DeltaNEMO was detected in all the patients in the family, but was not found in the normal individuals in this IP family and 80 unrelated controls.

CONCLUSIONThe study showed that the family with IP, which showed anticipation, was caused by NEMODelta4-10 deletion in the NEMO gene. Long PCR analysis is proven to be an efficient tool for identification of NEMO rearrangements. It could provide useful information for the genetic counseling of the family involved.

Adolescent ; Child ; Electrophoresis ; Exons ; genetics ; Family ; Female ; Humans ; I-kappa B Kinase ; genetics ; Incontinentia Pigmenti ; genetics ; Infant ; Male ; Pseudogenes ; genetics ; Sequence Deletion

OBJECTIVETo detect mutations of ATP2A2 gene in a pedigree and a sporadic case with Darier disease (DD) and explore the underlying molecular mechanism.

METHODSClinical data of the pedigree and the sporadic case were collected. Genomic DNA was extracted from blood samples of four members from the pedigree (including three patients and one healthy member), the sporadic case and 100 healthy controls. PCR was performed to amplify all coding exons of the ATP2A2 gene. And the products were directly sequenced to detect mutations.

RESULTSA missense mutation c.1484C>T (p.S495L) in exon 12 was detected in all patients of the pedigree. For the sporadic case, a novel splicing mutation c.325-2A>G was detected at the junction between intron 4 and exon 5. The same mutations were not found in the 100 healthy controls.

CONCLUSIONMutations of the ATP2A2 gene may lead to the occurrence of DD in both familial and sporadic cases with DD.

Aged ; Alternative Splicing ; genetics ; Base Sequence ; Child ; DNA Mutational Analysis ; Darier Disease ; genetics ; Family Health ; Female ; Genetic Predisposition to Disease ; genetics ; Humans ; Male ; Mutation, Missense ; Pedigree ; Point Mutation ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics

Objective: This study aims to analyze the relationship among the clinical features, radiologic characteristics and pathological diagnosis in patients with solitary pulmonary nodules, and establish a prediction model for the probability of malignancy. Methods: Clinical data of 372 patients with solitary pulmonary nodules who underwent surgical resection with definite postoperative pathological diagnosis were retrospectively analyzed. In these cases, we collected clinical and radiologic features including gender, age, smoking history, history of tumor, family history of cancer, the location of lesion, ground-glass opacity, maximum diameter, calcification, vessel convergence sign, vacuole sign, pleural indentation, speculation and lobulation. The cases were divided to modeling group (268 cases) and validation group (104 cases). A new prediction model was established by logistic regression analying the data from modeling group. Then the data of validation group was planned to validate the efficiency of the new model, and was compared with three classical models(Mayo model, VA model and LiYun model). With the calculated probability values for each model from validation group, SPSS 22.0 was used to draw the receiver operating characteristic curve, to assess the predictive value of this new model. Results: 112 benign SPNs and 156 malignant SPNs were included in modeling group. Multivariable logistic regression analysis showed that gender, age, history of tumor, ground -glass opacity, maximum diameter, and speculation were independent predictors of malignancy in patients with SPN(P<0.05). We calculated a prediction model for the probability of malignancy as follow: p=e^x/(1+ e^x), x=-4.8029-0.743×gender+ 0.057×age+ 1.306×history of tumor+ 1.305×ground-glass opacity+ 0.051×maximum diameter+ 1.043×speculation. When the data of validation group was added to the four-mathematical prediction model, The area under the curve of our mathematical prediction model was 0.742, which is greater than other models (Mayo 0.696, VA 0.634, LiYun 0.681), while the differences between any two of the four models were not significant (P>0.05). Conclusions Age of patient, gender, history of tumor, ground-glass opacity, maximum diameter and speculation are independent predictors of malignancy in patients with solitary pulmonary nodule. This logistic regression prediction mathematic model is not inferior to those classical models in estimating the prognosis of SPNs.