Structure, function and application of serine carboxypeptidase-like acyltransferases from plants / 生物工程学报

Plant serine carboxypeptidase-like acyltransferases (SCPL-AT) have similar structural characteristics and high homology compared to the serine carboxypeptidase. They can transfer the acyl from acyl glucose esters to many natural products, participate in the acylation modification of plant secondary metabolites, enrich the structural diversity of natural products, and improve the physicochemical properties such as water solubility and stability of compounds. This review summarizes the structural characteristics, catalytic mechanism, functional characterization, and biocatalytic applications of SCPL-AT from plants. This will help to promote the functional characterization of these acyltransferase genes and the biosynthesis of useful plant secondary metabolites by synthetic biotechnology.

From DCPD to NTCP: The long journey towards identifying a functional hepatitis B virus receptor

Hepatitis B virus (HBV) is the prototype of hepatotropic DNA viruses (hepadnaviruses) infecting a wide range of human and non-human hosts. Previous studies with duck hepatitis B virus (DHBV) identified duck carboxypeptidase D (dCPD) as a host specific binding partner for full-length large envelope protein, and p120 as a binding partner for several truncated versions of the large envelope protein. p120 is the P protein of duck glycine decarboxylase (dGLDC) with restricted expression in DHBV infectible tissues. Several lines of evidence suggest the importance of dCPD, and especially p120, in productive DHBV infection, although neither dCPD nor p120 cDNA could confer susceptibility to DHBV infection in any cell line. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a binding partner for the N-terminus of HBV large envelope protein. Importantly, knock down and reconstitution experiments unequivocally demonstrated that NTCP is both necessary and sufficient for in vitro infection by HBV and hepatitis delta virus (HDV), an RNA virus using HBV envelope proteins for its transmission. What remains unclear is whether NTCP is the major HBV receptor in vivo. The fact that some HBV patients are homozygous with an NTCP mutation known to abolish its receptor function suggests the existence of NTCP-independent pathways of HBV entry. Also, NTCP very likely mediates just one step of the HBV entry process, with additional co-factors for productive HBV infection still to be discovered. NTCP offers a novel therapeutic target for the control of chronic HBV infection.

Dimethyl 3, 3', 4, 4'-tetrahydroxy-delta-truxinate isolated from the leaves of Andrographis lineata.Wall. ex. nees suppress adipogenesis in 3T3-L1 preadipocytes for type 2 diabetes

The present investigation elucidates the isolation and characterization of bioactive compound from the ethanolic leaf extract of Andrographis lineata [EtALL] which suppress the differentiation of 3T3-L1 adipocytes. The ethanolic leaf extract was subjected to bioassay guided fractionation in 3T3-L1 cell lines. Five fractions were isolated from the EtALL extract by column chromatography. All the Fractions [I-V] along with EtALL were screened for adipogenesis activity [Oil-Red-O staining].The fraction which showed maximum adipogenesis activity was purified by thin layer chromatography. The bioactive Fraction IV was found to have maximum adipogenic [96.83%] activity and the activity was comparable to Rosiglitazone. The spectroscopic data analysis reveals that, the isolated bioactive compound was Dimethyl 3, 3', 4, 4'-tetrahydroxy-delta-truxinate [DT[delta]T], a combination of truxillic and truxinic acid derivative. DT[delta]T showed insulin mimicking [131.2%], sensitizing [810.02%] and adipogenic activity [80.23%]. Hence our present study concluded that, Dimethyl 3, 3', 4, 4'-tetrahydroxy-delta-truxinate isolated from the ethanolic leaf extract of Andrographis lineata stimulates glucose uptake, potentiates insulin-stimulated glucose in 3T3-L1 adipocytes without increasing adiposity

Expression of the thermostable carboxypeptidase Taq gene in Pichia pastoris GS115 / 生物工程学报

To express recombinant carboxypeptidase from Thermus aquaticus (Cpase Taq) in Pichia pastosis, the open reading frame coding thermostable Cpase Taq was optimized based on the preference of P. pastoris codon usage and synthesized in vitro. The novel gene was cloned into P. pastoris expression vector pHBM905A and the sequence coding 6xHis tag was fused with the ORF of Cpase Taq gene. The recombinant plasmid was named pHBM905A-Cpase Taq and transformed into P. pastoris GS 115. Transformants were induced with 1% methanol for 72 h until the enzyme yield reached 0.1 mg/ml. The enzyme was purified and its enzymatic properties were analyzed. The results showed that the specific enzyme activity reached maximum at 75 °C and pH 7.5, which was about 80 U/mg. It was the first report about the secretory expression of Cpase Taq in P. pastoris GS115. Because of its large-scale preparation, this enzyme may be applied in industrial hydrolysis of peptides into amino acids in the future.

Effect of losartan on the protection of the kidney and PRCP-kallikrein axis of the two-kidney, one-clipped renovascular hypertensive rats / 药学学报

To investigate the effect of losartan on the axis of prolylcarboxypeptidase (PRCP)--kallikrein of the two-kidney, one-clipped (2K1C) hypertensives rats, and explore the novel protection mechanism of losartan on the kidney. Sprague-Dawley (SD) rats were used to develop the 2K1C hypertensive rats. Then, the rats were treated with prazosin (5 mg x kg(-1) x d(-1)) or losartan (5, 15 and 45 mg x kg(-1) x d(-1)) or vehicle, separately. At the same time, the blood pressures were observed. After treated for four weeks, the ratio of right kidney weight and body weight, the change of glomerular morphology, and K+, Na+, creatinine and blood urea nitrogen (BUN) of the serum were used for evaluation of kidney. The expressions of PRCP mRNA in the kidneys were determined by RT-PCR. The protein levels of PRCP, tissue kallikrein, plasma kallikrein, TGF-beta1 in kidney or plasma were measured by Western blotting. Results showed that the changes of body weight and kidney weight ratio, glomerular fibrosis degree and the biochemistrical index of serum induced by hypertension were relieved when the hypertensive rats treated with losartan for four weeks. Meanwhile, treatment of losartan also significantly decreased expression of TGF-beta1 and increased expressions of PRCP, plasma kallikrein and tissue kallikrein. The protective effects of losartan on the kidney of 2K1C hypertensive rats are activation of the axis of PRCP-kallikrein and reducing the expression of TGF-beta1.

Changes of prostaglandin D2,carboxypeptidase A3 and platelet activating factor in guinea pig in anaphylactic shock / 法医学杂志

OBJECTIVE@#To detect the changes of leukotriene E4(LTE4), prostaglandin D2(PGD2), carboxypeptidase A3(CPA3) and platelet activating factor (PAF) in guinea pigs died from anaphylactic shock.@*METHODS@#Guinea pigs were used for establishing anaphylactic shock models. The levels of LTE4, PGD2 and CPA3, and PAF were detected in urine, plasma, and brain tissues with ELISA kit, respectively. The significant biomarkers were selected comparing with control group. The changes of PGD2, CPA3 and PAF in the guinea pigs at time zero, 12 and 24 hours after death were observed and compared respectively. The effect of platelet activating factor acetylhydrolase (PAF-AH) to PAF in guinea pig brain was examined and compared.@*RESULTS@#There were no statistically differences of LTE4 levels in urine observed between experimental group and control group. The levels of CPA3, PGD2 and PAF in the experimental group were significantly higher than that in the control group at 0 h. The levels of PAF at 12 and 24 hours after anaphylactic shock were significantly higher than that in the control group. The levels of PAF decreased significantly after pretreatment with PAF-AH.@*CONCLUSION@#LTE4 in urine cannot be selected as a biomarker to determine the anaphylactic shock. PGD2 and CPA3 in plasma, and PAF in brain tissue may be used as biomarkers to determine the anaphylactic shock. PAF-AH may be potentially useful for clinical treatment of anaphylactic shock.

Measurement of plasma mast cell carboxypeptidase and chymase levels in children with allergic diseases / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To evaluate the roles of plasma mast cell carboxypeptidase and chymase in the diagnosis of allergic diseases by measuring the contents of both in children.</p><p><b>METHODS</b>A total of 59 children with allergic diseases and 53 healthy children were recruited into the study. Plasma levels of mast cell carboxypeptidase and chymase were measured using ELISA.</p><p><b>RESULTS</b>The plasma levels of mast cell carboxypeptidase and chymase in children with allergic children were 1.089 ± 0.752 ng/mL and 0.905(0.375-2.318) ng/mL, respectively, which were significantly higher than those in healthy children [0.593 ± 0.380 ng/mL and 0.454 (0.097-1.077) ng/mL respectively; P<0.05]. There was a significantly positive correlation between plasma mast cell carboxypeptidase and chymase levels in children with allergic diseases (r=0.684, P<0.01).</p><p><b>CONCLUSIONS</b>Plasma levels of mast cell carboxypeptidase and chymase increase in children with allergic diseases, suggesting that mast cell carboxypeptidase and chymase may serve as the indexes for the diagnosis of allergic diseases.</p>

Cloning, expression, purification of spinach carboxyl-terminal processing protease of D1 protein with hydrolysis activity and preparation of polyclonal antibody / 生物工程学报

Carboxyl-terminal processing protease of D1 protein (CtpA) catalyzes carboxyl terminal processing of the D1 protein of photosystem II, which is essential for the assembly of a manganese cluster and consequent light-mediated water oxidation. It is a target for the discovery of wide-spectrum herbicide. We amplified the CtpA gene from spinach cDNA with standard PCR method and constructed it into pET-28a vector to generate a recombinant expression plasmid. Recombinant CtpA fusion protein with His-tag was expressed as soluble protein in Escherichia coli BL21(DE3) after induction with 0.1 mmol/L IPTG at 8 degrees C for 72 h. We purified the CtpA protein with the Ni-NTA affinity chromatography and Superdex 75 gel filtration chromatography respectively, and verified the protein by SDS-PAGE and Western blotting with anti-his antibody. Hydrolysis activity of CtpA was assayed by HPLC method with a synthetic 24-mer oligopeptide corresponding to carboxyl terminal of precursor D1 protein, and gave a total activity of 1.10 nmol/(mg x min). We used the purified CtpA protein as antigen to immune rabbit for the production of polyclonal antibody, and prepared antibody with high specificity and sensitivity. The results obtained in this paper provided the feasibility of high-throughput screening of lead compounds for the protease as inhibitors and mechanism analysis of CtpA enzyme.

E112D polymorphism in the prolylcarboxypeptidase gene is associated with blood pressure response to benazepril in Chinese hypertensive patients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Marked interindividual variation exists in blood pressure response to benazepril, which is considered to have genetic basis. Our objectives were to evaluate whether the E112D polymorphism in the prolylcarboxypeptidase (PRCP) gene has impact on blood pressure response to benazepril.</p><p><b>METHODS</b>Hypertensive patients from Huoqiu County and Yuexi County of Anhui Province received daily treatment with an oral dosage of 10 mg benazepril for 15 days. Genotypes of the E112D polymorphism in the PRCP gene were determined by TaqMan SNP genotyping assay. Multivariate linear and Logistic regressions using generalized estimating equation model were performed in a total of 1092 patients to evaluate the association of PRCP genotypes and blood pressure response to benazepril.</p><p><b>RESULTS</b>Patients carrying ED or DD genotype had a less systolic blood pressure reduction (adjusted beta = -3.7 + or - 1.1, P < 0.001), a less diastolic blood pressure reduction (adjusted beta = -3.1 + or - 0.8, P < 0.001) and a lower percentage of reaching target blood pressure defined as SBP lower than 140 mmHg and DBP lower than 90 mmHg (adjusted OR = 0.6, P = 0.005) than those patients carrying EE genotype. In addition, the results from stratified analysis by county (Huoqiu or Yuexi) were similar to those observed in the pooled population.</p><p><b>CONCLUSIONS</b>Our data suggest that the E112D polymorphism in the PRCP gene may be a useful genetic marker to predict the antihypertensive effect of short-term benazepril treatment in hypertensive patients of Anhui Province, China.</p>

In vivo quality control of photosystem II in cyanobacteria Synechocystis sp. PCC 6803: D1 protein degradation and repair under the influence of light, heat and darkness.

The cells of Synechocystis sp. PCC 6803 were subjected under photoinhibitory irradiation (600 micromolm(-2)s(-1)) at various temperatures (20-40 degrees C) to study in vivo quality control of photosystem II (PSII). The protease biogenesis and its consequences on photosynthetic efficiency (chlorophyll fluorescence ratio Fv/Fm) of the PSII, D1 degradation and repair were monitored during illumination and darkness. The loss in Fv/Fm value and degradation of D1 protein occurred not only under high light exposure, but also continued when the cells were subjected under dark restoration process after high light exposure. No loss in Fv/Fm value or D1 degradation occurred during recovery under growth/low light (30 micromol m(-2) s(-1)). Further, it helped the resynthesis of new D1 protein, essential to sustain quality control of PSII. In vivo triggering of D1 protein required high light exposure to switch-on the protease biosynthesis to maintain protease pool which induced temperature-dependent enzymatic proteolysis of photodamaged D1 protein during photoinhition and dark incubation. Our findings suggested the involvement and overexpression of a membrane-bound FtsH protease during high light exposure which caused degradation of D1 protein, strictly regulated by high temperature (30-40 degrees C). However, lower temperature (20 degrees C) prevented further loss of photoinhibited PSII efficiency in vivo and also retarded temperature-dependent proteolytic process of D1 degradation.

Combined impact of polymorphism of folate metabolism genes; glutamate carboxypeptidase, methylene tetrahydrofolate reductase and methionine synthase reductase on breast cancer susceptibility in Kashmiri women

Folate and methionine play a crucial role in DNA synthesis, repair and the epigenetic profile of cell. Hence, the alterations in the folate metabolism can lead to aberrant proliferation leading to neoplasia. Most of the studies have associated polymorphisms in methylene tetrahydrofolate reductase [MTHFR] and methionine synthase reductase [MTRR] genes with reduced risk of cervical and colorectal cancer. However, the association with breast cancer is still controversial. Further, the ivolvement of Glutamate carboxypeptidase II [GCPII] polymorphism in cancer is not known. In the present study, we analyzed if the individual and combined effects of polymorphisms in folate pathway genes viz., MTHFR 677C> T, MTHFR 1298A> C, MTRR 66A> G and GCP II 1561 C>T, have any role in altering the susceptibility to breast cancer. The DNA of 35 female breast cancer patients and 33 healthy individuals, in the Kashmiri population from India, were analyzed using a PCR-RFLP approach for the above mentioned polymorphisms. Individuals carrying the MTHFR 677CT/TT and GCPII 1561 CT genotype showed a 3.5 [95% CI: 3.1-3.7, P<0.02] and 7.7 [95% CI: 6.7-9.1, P<0.001] fold decreased risk for breast cancer than the wild types [MTHFR 677CC and GCPII 1561 CC]. Subjects with MTRR 66 G-allele showed a 4.5 fold decreased risk [OR: 0.22, 95% CI: 0.20, 0.24, P<0.0005] compared to the wild type [MTRR 66A]. Further, subjects with combined polymorphisms in MTHFR, GCPII and MTRR loci revealed a significant reduction of breast cancer risk. This study indicates [i] a protective role of polymorphisms in MTHFR, GCPII, MTRR against breast cancer in the study subjects, and [ii] combined effect of polymorphisms is more pronounced than single genetic polymorphism, thereby emphasizing the role of gene-gene interaction in the susceptibility to breast cancer

Application of HPLC-ESI-ITMS in the quality control of carboxyterminal sequence confirmation for insulin and insulin chain B / 药学学报

Application of HPLC-ESI-ITMS in the quality control of carboxyterminal sequence confirmation for insulin and insulin chain B was studied. The solution of intact insulin or insulin chain B was added to the solution of carboxypeptidase P (CPP) and carboxypeptidase Y (CPY). Fractions of appropriate volume were removed at some appointed time points, acidified with the same amount of 1% formic acid to stop the digestion, and then briefly vortexed for HPLC-ESI-ITMS analysis. Mobile phase A consisted of 0.02% TFA in 98% ultra-pure water and 2% acetonitrile. Mobile phase B consisted of 0.02% TFA in 98% acetonitrile and 2% ultra-pure water. The solution used for post-column fix consisted of propionic acid and isopropyl alcohol (20 : 80, v/v). Chromatographic separation was carried out on a reversed-phase column (Zorbax Prosphere C18, 300A, 5 microm, 2.1 mm ID x 150 mm length). The molecular weights of the multiply charged ions representing consecutive truncated losses of carboxyterminal amino acids were determined by the use of HPLC-ESI-ITMS. The differences between the consecutive truncated peptides are the experimental weights of the carboxyterminal amino acid residues. The carboxyterminal amino acid residue Ala, which released from chain B of intact insulin, was confirmed in the nanomolar concentration range by analyzing the molecular weight of the truncated peptides. Another one carboxyterminal amino acid Ala was confirmed in the nanomolar concentration range of insulin chain B. In the quality control for recombinant DNA product or natural protein, the confirmation of 1 - 3 carboxyterminal amino acid residues is regarded to be up to standard. One amino acid residue of insulin or insulin chain B could be confirmed accurately in the nanomolar concentration range. The results showed that intact insulin could be directly sequenced in the quality control without separating chain B from chain A. There would be no need to separate chain A from chain B to identify carboxyterminal of intact insulin. Furthermore, the method saved us a lot of trouble from the preparation and purification of insulin chain A and chain B.

Effect and mechanism of recombinant hirudin on fibrinolysis / 药学学报

<p><b>AIM</b>To study the effect of recombinant hirudin (rH) on tPA-induced fibrinolysis and the possible mechanism of its action.</p><p><b>METHODS</b>The effect of rH on thrombin-fibrin complex (Th-Fn) was detected by 99mTc labeled rH. In the in vitro clot lysis, tPA as plasminogen activator, and recalcified plasma as plasminogen resource were used to study the influence of rH on fibrinolysis by detecting TAFIa, D-Dimer and FXIII.</p><p><b>RESULTS</b>In a canine model of femoral artery thrombosis, a clear radioactivity strip was imaged in 30 - 60 min on a part image, and the femoral vein thrombosis developed at 30 min. rH efficiently inhibited clot regeneration. Addition of TM could inhibit clot lysis obviously, and CPI could shorten the delay of clot lysis which due to TAFIa. There was a dose-dependent relationship with TM concentration and TAFI activation. FXIII activation was inhibited by low concentration of rH ( < or = 0.2 u x mL(-1)), and the level of fibrinolysis product, D-Dimer, increased.</p><p><b>CONCLUSION</b>rH could inhibit the thrombin binding to fibrin. rH inhibited the activation of TAFI and FXIII by combining with thrombin which resulted in enhancement of thrombolysis.</p>

Cloning, expression and sequence analysis and tissue distribution of angiotensin-converting enzyme 2 (ACE2) gene in adult mice / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>o clone angiotensin-converting enzyme 2(ACE2) gene, to analyze its amino acids and nucleotides sequence and to investigate tissue distribution of ACE2 in adult mice.</p><p><b>METHODS</b>The full-length ACE2 encoding sequence was amplified from the RNA of mice kidney tissue by RT-PCR technique, cloned into plasmid pGEM-T easy, then subcloned into plasmid pcDNA3.1+. After identification of DNA sequence, the recombinant plasmid pmACE2 was transfected into Cos7 cells with lipofectin reagent. The transient expression of ACE2 molecule was detected by SDS-PAGE. Sequence analysis was conducted with CLUSTALX program. Tissue distribution of ACE2 in mice was detected by RT-PCR.</p><p><b>RESULTS</b>A fragment about 2.6 kb was amplified and the recombinant plasmid pmACE2 was confirmed by two-enzyme digesting and DNA sequencing. The cloned DNA sequence was consistent with that previously reported, except for 3 variations: A701G, T1102C and T1330C. SDS-PAGE proved that expression of a soluble, truncated products form of ACE2 was a glycoprotein of approximately 80 kD in Cos7 cells. The predicted mice ACE2 sequence contained an N-terminal signal sequence (amino acid residues 1-18), a single HHEMGHIQ zinc-binding domain (amino acid residues 373-380) and C-terminal membrane anchor (amino acid residues 738-765). Mice ACE2 showed 84 % identity with that of human, and 90 % identity with that of rat. Expression of ACE2 was the greatest in lungs, hearts and kidneys, and moderate levels were also detected in testes and livers.</p><p><b>CONCLUSION</b>Mice ACE2 gene has been cloned and successfully expressed in vitro. The tissue-specific expression of ACE2 in different species is not identical.</p>

Estudo da regulação da expressão do gene da carboxipeptidase M de camundongo / Expression regulation studies of the carboxypeptidase M gene in mouse

A caracterização da estrutura molecular da carboxipeptidase M humana originou os estudos da enzima em camundongos. Utilizando análise computacional a estrutura do gene em camundongos foi determinada comparando a seqüência de humanos no banco de dados genômico de camundongos. As duas seqüências apresentaram 82 por cento de similaridade entre elas. Foram determinados todos os fatores envolvidos na caracterização do RNAm como tamanho de exons e introns, região de catálise, região hidrofóbica, peptídeo sinal, sítios de glicosilação e glutâmicos catalíticos. A análise computacional proporcionou também a observação de um EST o qual também se tomou objeto de estudos. Após serem definidos todos os fatores pertencentes ao gene iniciamos os experimentos com a demonstração da atividade luciferase para os promotores da CPM e do EST. A análise foi feita em três diferentes tipos celulares para evidenciar a existência de atividade promotora e sua diferente regulação nessas linhagens. O EST foi clonado e transfectado em células para dosagem fluorimetrica de sua atividade carboxipeptidase básica. A técnica de PCR em tempo real foi utilizada para obtenção dos resultados de expressão gênica relativa tanto da carboxipeptidase M como do EST em camundongos nas fases embrionária, neonatal e adulta. Com base nos dados obtidos pudemos concluir a estrutura da carboxipeptidase M e do EST encontrado, sua regulação transcricional e padrão de expressão nos diversos RNAs extraídos de tecidos de camundongos.

preliminary characterization of digestive proteease in the mid-gut of the third larval instar of gasterophilus intestinalis

Protinases contained in the mid-gut of the early third instar of Gasterophilus intestinals have been tentatively identified by midgut hydrolysis of synthetic substrates. Trypsin was identified by maximal hydrolysis of benzoyl-DL-arginine-p-nitroanilide [BApNA] at pH 8 and chymotrypsin by maximal hydrolysis of benzoyl-L-tyrosine ethyl ester [BTEE] at pH 9. Carboxypeptidase A and B were identified by their maximal hydrolysis of hippuryl-DL-phenyllactic acid and hippuryl-L-arginine at pH 9 and 8 respectively. Aminopeptidase was identified by maximal hydrolysis of leucine-p-nitroanilide at pH 9. The mid-gut also showed activity of aspartic proteinase and identified it as cathepsin D.A drug [Banmith 12.5% pyrantel tartarate] used for the routine control of helminthes parasites of horses and donkies in Egypt was used in vitro to investigate its effect on the optimal activity of studied enzymes. It was found that the drug has no effect on trypsin and carboxypeptidase A while it decreases the activity of chymotrypsin, aminopeptidase and acidic proteinase and was also found to increase the activity of carboxypeptidase B greatly

Thrombin-activatable fibrinolysis inhibitor and prothrombin fragment 1+2 in type II diabetics: possible relation to albuminuria and macrovascular complications

Forty type II diabetic patients [diabetic group] and 40 healthy age- and sex-matched persons [control group] were enrolled. The study involved assessment of body mass index [MBI], glycemic state [fasting and postprandial blood glucose, glycated hemoglobin [HbAlc]], low- density lipoprotein cholesterol [LDL], albuminuria by urinary albumin excretion rate [UAE], plasma PF 1+2 and TAFI, also, reverse transcriptase-polymerase chain reaction [RT-PCR] for TAFI mRNA in adipocytes and cell cultures. Plasma PF 1+2 and TAFI showed significant positive correlations with CVD risk factors including BMI, LDL and albuminuria. Also, they showed significant positive correlation with history of arterial and venous [only for TAFI] CVD. Mean values for these markers were statistically higher in the diabetic group compared to the control group. Furthermore, their values were statistically higher in patients with history of CVD compared to those without prior history. In conclusion, plasma PF 1+2 and TAFI reflected a prothrombotic and hypofibrinolysis state that may explain the increased CVD among diabetics. TAFI gene expression in adipocytes would explain the increased risk of CVD among obese diabetics

Cloning and expression of extracellular domain of prostate specific membrane antigen in Escherichia coli and preparation of polyclonal antibody / 生物工程学报

Human Prostate Specific Membrane Antigen(PSMA) cDNA was amplified using total RNA extracted from prostate carcinoma tissue by RT-PCR. The cDNA fragment of extracellular domain of PSMA(edPSMA) gene was amplified by PCR and cloned into expression vector pMAL-c2x. Sequence analysis of both PSMA and edPSMA revealed identity to the GenBank reported. The edPSMA was expressed in E. coli as part of a fusion protein with MBP as the induction of IPTG. Western blot analysis showed the recombinant protein could react with PSMA monocloned antibodies 4G5. MBP-edPSMA fusion protein were purified by amylose resin affinity chromatography and showed to be homogeneity in SDS-PAGE(120 kD). BALB/C mice were immunized with the purified protein for the preparation of polyclonal antibody. The polyclonal antibody, which had a title of 1:12,800, were indicated the specificity to prostate tissue.

Heterologous expression of syntaxin 6 in Saccharomyces cerevisiae

The molecular mechanisms of vesicular protein transport in eukaryotic cells are highly conserved. Members of the syntaxin family play a pivotal role in the membrane fusion process. We have expressed rat syntaxin 6 and its cytoplasmic domain in wild-type and pep12 mutant strains of Saccharomyces cerevisiae to elucidate the role of the syntaxin 6-dependent vesicular trafficking step in yeast. Immunofluorescence microscopy revealed a punctate, Golgi-like staining pattern for syntaxin 6, which only partially overlapped with Pep12p in wild-type yeast cells. In contrast to Pep12p, syntaxin 6 was not mislocalized to the vacuole upon expression from 2 micron vectors, which might be attributed to conserved sorting and retention signals. Syntaxin 6 was not capable of complementing the sorting and maturation defects of the vacuolar hydrolase CPY in pep12 null mutants. No dominant negative effects of either syntaxin 6 or syntaxin 6DC overexpression on CPY sorting and maturation were observed in wild-type yeast cells. We conclude that syntaxin 6 and Pep12p do not act at the same vesicular trafficking step(s) in yeast and higher eukaryotes

Serum butyrylcholinesterase of non-human primate shows amine sensitive aryl acyl amidase and metallopeptidase activities and characteristics similar to those of the human serum enzyme.

Butyrylcholinesterase (BChE) was purified from monkey serum and the catalytic activities were examined. The enzyme has a molecular mass of approximately equal to 74 kDa as seen by SDS-gel electrophoresis. Monkey serum BChE also exhibits an amine sensitive aryl acylamidase (AAA) and a metallocarboxypeptidase activity. The tyramine activation of the aryl acylamidase activity and the metal chelator inhibition of the peptidase activity were characteristics similar to those of the human enzyme. Studies on 65Zn2+ binding and zinc chelate Sepharose chromatography showed that monkey serum BChE and human serum BChE have similar characteristics. Limited alpha chymotrypsin digestion of monkey serum BChE followed by Sephadex gel chromatography cleaved the enzyme into a 36 kDa fragment exhibiting peptidase activity. However the 20 kDa fragment corresponding to cholinesterase and aryl acylamidase activity was not detectable possibly due to the unstable nature of the fragment. Immunological studies showed that a polyclonal antibody against human serum BChE cross reacted with monkey serum BChE. The identical nature of the catalytic activities of human serum BChE and monkey serum BChE supports the postulate that all three catalytic activities co-exist in the same enzyme. This is the first time that purification and characterisation of the monkey serum BChE which has the highest sequence identity and immunological identity with that of human serum BChE, is being reported.