Chimaeric protein A/protein G and protein G/alkaline phosphatase as reporter molecules.

The IgG binding domains of staphylococcal protein A and streptococcal protein G were expressed as a chimaera using the pGEX vector which has been advocated because its fusion proteins tend to be soluble and easily isolated on immobilised glutathione. This chimaera was soluble and abundant (yield = 18 mg/l of bacterial culture) and was tested by double diffusion in agarose and by ELISA. It was found to bind IgG of all species that either parent could bind. It was superior to protein A or protein G in binding mouse Ig. A chimaera of protein G and alkaline phosphatase was also constructed and found to be soluble and abundant (yield = 20 mg/l of bacterial culture). This protein could be used as a secondary reagent in ELISA at 5 micrograms/ml for human, rabbit and mouse and at 25 micrograms/ml for sheep.     

Immunogenicity of genetically engineered glutathione S-transferase fusion proteins containing a T-cell epitope from diphtheria toxin.

Glutathione S-transferase (GST) has been shown to induce a marginal antibody response in experimental animals as well as partial protection against a number of parasitic worms, including Schistosoma and Fasciola species. The objective of our study was to increase the immunogenicity of GST by adding heterologous T-cell epitopes at the carboxy terminus of the protein. We generated recombinant GST proteins by attaching one or three tandem repeats of a T-cell epitope of CRM197, a nontoxic variant of diphtheria toxin. This T-cell epitope encoding the region of amino acids 366 to 383 of CRM197, when contained in a GST fusion protein and/or after purification as a recombinant peptide, retained the ability to induce a CRM197-specific T-cell response. The fusion protein containing a single T-cell epitope induced a strong T-cell proliferative response to GST and also enhanced anti-GST antibody production in mice. The addition of three repeats of the epitope did not augment the responses when compared with the responses of GST itself. The results suggest that the addition of a single T-cell epitope to a larger protein like GST increases the immunogenicity of the protein.     

肠毒素大肠杆菌耐热肠毒素与谷胱甘肽巯基转移酶融合蛋白的制备和耐热肠毒素抗体测定

目的：弥补ST的弱抗原性，实现ST抗体测定。方法：采用生物工程技术，融合表达GST和带有前导序列的突变耐热肠毒素proSTm，制备ST融合蛋白，测定ST抗体。结果：测到了抗ST的血清IgG和黏膜IgA。结论：融合蛋白GST／proSTm能有效提高ST的抗原活性，可直接用于ST抗体测定。     

Characterization of glutathione S-transferase from dust mite, Der p 8 and its immunoglobulin E cross-reactivity with cockroach glutathione S-transferase

BACKGROUND: Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems. OBJECTIVE: To assess the allergenicity of native and recombinant mite glutathione S-transferase (GST) (Der p 8) and study the IgE cross-reactivity between Der p 8 and cockroach GST. METHODS: Der p 8 cDNA encoding a new isoform was isolated and expressed in yeast. Native Der p 8 was affinity purified from mite extract. IgE reactivity to native and recombinant Der p 8 was assessed by ELISA using sera from allergic subjects from Taiwan, Singapore and Malaysia. IgE cross-reactivity between Der p 8 and cockroach GST was examined by IgE inhibition assays. RESULTS: Our Der p 8 cDNA encoded a basic isoform (pI=8.5) containing six polymorphic residues located at positions 46, 106, 149, 160, 167 and 184. At least 8 isoforms of native Der p 8 were detected by two-dimensionalgel and immunoblot analyses. Sera from Taiwanese asthmatics showed 96% and 84% IgE reactivity to native Der p 8 and recombinant Der p 8, respectively. Native Der p 8 showed 75% and 65% IgE reactivity with sera from Malaysia and Singapore, respectively. CONCLUSIONS: A high frequency of sensitization to mite GST among allergic subjects was observed but the titres of IgE reactivity were low. The IgE cross-reactivity between mite and cockroach GST suggests that GST is a panallergen.     

HPV antibody detection by ELISA with capsid protein L1 fused to glutathione S-transferase

An alternative enzyme linked immunosorbent assay (ELISA) system was developed to analyze antibodies to human papillomavirus capsid antigens. The assay uses glutathione crosslinked to casein to capture the major capsid protein L1 from human papillomavirus (HPV) types 6b, 16 and 18 fused to glutathione S-transferase (GST) as antigen. The method allows efficient one-step purification of L1 fusion protein from crude bacterial lysates on ELISA plates coated with glutathione casein. The GST-L1 capture ELISA detected HPV 16 antibodies with high type specificity. Comparison with the current "gold-standard" for L1-serology that uses virus-like particles (VLP) as antigen demonstrated similar assay sensitivity. Pairwise comparison of the absorbance values of 105 human sera obtained in the two ELISA formats for HPV 16 showed a R(2) value of linear regression of 0.68. Conformity of the two ELISAs in classification of sera as HPV 16 L1 antibody-positive or -negative was verified with Cohen's kappa test, yielding a value of 0.62. These data indicate that the GST-L1 capture ELISA is similar in performance to the VLP ELISA. The ease of antigen production and purification in the GST-based ELISA will be advantageous to screen large sample numbers in vaccine trials or epidemiological studies examining immune responses to many HPV types in parallel.     

A generic capture ELISA for recombinant proteins fused to glutathione S-transferase: validation for HPV serology

An enzyme-linked immunosorbent assay (ELISA) system has been developed that uses glutathione crosslinked to casein as capture protein to bind recombinant protein antigens fused to N-terminal glutathione S-transferase (GST). The method allows simple and efficient immobilization and one-step purification of overexpressed recombinant antigens from crude lysates on ELISA plates coated with glutathione casein. Several antigens can be tested in parallel under the same conditions without the need to biochemically purify or renature the proteins. An additional undecapeptide epitope fused to the C-terminus of each antigen permits the detection and quantification of any full-length protein antigen bound to the ELISA plate with one single monoclonal antibody. The ELISA system was applied with four antigens to detect antibodies against E6 and E7 proteins of human papillomavirus types 16 and 18. Antibody reactivities of 164 sera from patients with cervical carcinoma and healthy individuals were in good agreement with those determined using a previously established capture ELISA with biochemically purified and renatured proteins as antigens although the GST capture ELISA was more sensitive with no loss of specificity. The GST capture ELISA could be adapted to provide standardized antibody assays for many protein antigens.     

Immunodominant B-cell epitope in the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis cross-reacting with glutathione S-transferase

The TB1-5 76C monoclonal antibody raised against a synthetic 60-mer peptide in the N-terminal part of the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis has previously been shown to react with a linear epitope in the KRRITPKD region, residues 131-138 in Mce1A, and to cross-react with Mce1F. Six additional monoclonal antibodies raised against the same peptide were also shown to cross-react with Mce1F. Four of them reacted with a linear epitope in the same area, indicating that this area is immunodominant but showed distinct differrences in fine specificity. Two monoclonal antibodies did not react with synthetic peptides from this region on the solid phase in enzyme-linked immunosorbent assay, indicating greater influence of conformation on reactivity. None of the monoclonal antibodies reacted with 14-mer synthetic peptides from the corresponding area in Mce2A, Mce3A, Mce4A, M. avium, M. smegmatis or M. leprae. The reaction pattern of the monoclonal antibodies was analysed in relation to our model of the Mce1A molecule (AK Das et al. Biochem Biophys Res Commun 2003;302:442-7).The epitope is located on the surface of Mce1A, at the distal beta-strand-loop region in the beta-domain supporting its potential role in promoting uptake of M. tuberculosis in host cells. Monoclonal antibody TB1-5 19C cross-reacted with glutathione S-transferase of Schistosoma japonicum containing a PKE triplet. Monoclonal antibody TB1-5 76C gave a major band at about 44 kDa in Western blotting of M. tuberculosis sonicate, whereas polyclonal rabbit anti-Mce1A peptide antibodies reacting with the extended TTPKNPTKRRITPKDVI area of Mce1A showed a distinct band above the 160 kDa molecular mass standard.     

A novel type of glutathione S-transferase in Onchocerca volvulus.

Onchocerca volvulus is a pathogenic human filarial parasite which, like other helminth parasites, is capable of evading the host's immune responses by a variety of defense mechanisms which are likely to include the detoxification and repair mechanisms of the enzyme glutathione S-transferase (GST). In this study, we show that one of the previously described GSTs from O. volvulus appears to possess the characteristics of a secreted enzyme. When the complete O. volvulus GST1 (OvGST1) sequence presented here is compared with those of other GSTs, 50 additional residues at the N terminus are observed, the first 25 showing characteristics of a signal peptide. This is consistent with the N-terminal sequence data on the native mature enzyme which begins at amino acid 26, based on the deduced protein sequence from the cDNA. The native protein, without the signal peptide sequence, possesses a 24-amino-acid extension not present in other GSTs. The deduced amino acid sequence of the OvGST1 cDNA clone was shown to possess four potential N-glycosylation sites. Digestion of O. volvulus homogenate with endoglycosidase, followed by detection of OvGST1 with specific antibody, indicated that the enzyme possesses at least two N-linked oligosaccharide chains. Gel filtration of the Escherichia coli-produced recombinant OvGST1 showed that it is enzymatically active as a nonglycosylated dimer. OvGST1 is found in the media surrounding adult worms maintained in culture, indicating that, in vitro, this enzyme is released from the worm. The strongest immunostaining for OvGST1 was observed in the outer cellular covering of the adult worm body, the syncytial hypodermis, especially in the interchordal hypodermis, where the peripheral membrane forms a series of lamellae which run into the outer zone of the hypodermal cytoplasm.     

Identification and localization of glutathione S-transferase as a potential target enzyme in Brugia species

Brugia filarial nematodes are pathogenic lymphatic-dwelling parasites that, like other helminths, may modify the host's defense mechanisms by a major detoxification process involving glutathione-binding proteins such as glutathione S-transferases (GSTs). In the present study, soluble extracts of third-stage larvae, adult male and female worms, microfilariae of either B. pahangi or B. malayi or the adult worm excretory–secretory products of B. malayi were used to determine GST activity. These extracts and affinity-purified fractions of B. pahangi adult worms had a specific enzymatic activity when 1-chloro-2,4-dinitrobenzene was used as a substrate. The observance of this enzyme in all life cycle stages of Brugia spp. demonstrates its ubiquitous nature. Lavage of intraperitoneally infected jirds, but not that of uninfected jirds, also showed increased enzymatic activity, suggesting that GST is secreted in vivo. Soluble proteins of both Brugia spp. were strongly recognized by antibodies in sera from rabbits immunized with affinity-purified native GST of Onchocerca volvulus. Immunohistochemical studies localized these proteins in adult worms, demonstrating cross-reactivity between the GST of these two filarial nematodes. The effect of this enzyme on the motility and viability of adult worms, microfilariae, and larvae was tested in vitro using a battery of known GST inhibitors. Of all those tested, ethacrynic acid, N-ethylmalemide, 4-nitropyridine-oxide, or 1-chloro-2,4-dinitrobenzene at micromolar concentrations reduced the viability and motility of microfilariae, third-stage larvae, and adult worms. These results suggest that Brugia GSTs are major metabolic enzymes and may play an important role in the parasite's survival. Received: 25 October 1999 / Accepted: 21 April 2000     

Recombinant cholera toxin B subunit and gene fusion proteins for oral vaccination

The B subunit portion of cholera toxin (CTB) is a safe and effective oral immunizing agent in humans, affording protection against both cholera and diarrhoea caused by enterotoxigenic Escherichia coli producing heat-labile toxin (LT) (Clemens et al., 1986; 1988). CTB may also be used as a carrier of various "foreign" antigens suitable for oral administration. To facilitate large-scale production of CTB for vaccine development purposes, we have constructed recombinant overexpression systems for CTB proteins in which the CTB gene is under the control of strong foreign (non-cholera) promoters and in which it is also possible to fuse oligonucleotides to the CTB gene and thereby achieve overexpression of hybrid proteins (Sanchez and Holmgren, 1989; Sanchez et al., 1988). We here expand these findings by describing overexpression of CTB by a constitutive tacP promoter as well as by the T7 RNA-polymerase promoter, and also by describing gene fusions leading to overexpression of several hybrid proteins between heat-stable E. coli enterotoxin (STa)-related peptides to either the amino or carboxy ends of CTB. Each of the hybrid proteins, when tested as immunogens in rabbits, stimulated significant anti-STa as well as anti-CTB antibody formation, although the anti-STa antibody levels attained (c.a. 1-15 micrograms/ml specific anti-STa immunoglobulin) were too low to give more than partial neutralization of STa intestinal challenge in baby mice. The hybrid proteins also had a near-native conformation, as apparent from their oligomeric nature and their strong reactivity with both a neutralizing antibody against the B subunit and a neutralizing monoclonal antibody (mAb) against STa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophoretic and immunological analysis of human glutathione S-transferase isozymes

Several electrophoretically distinct glutathione S-transferase isozymes from different tissues have been purified and characterized. The data confirm the suggestion that GST-1, GST-2 and GST-3 are the products of separate genetic loci.
An apparently muscle-specific isozyme termed GST-4 has been identified and shown to differ structurally from GST-1, GST-2 and GST-3. It is likely that GST-4 is the product of an additional gene locus.
Two isozymes termed GST-5 and GST-6 were purified from brain. GST-5 has a different isoelectric point, but shares many structural features with GST-1. GST-5 may be a brain-specific post-translationally modified product of the GST-1 gene. GST-6 is an acidic isozyme found in many tissues. The data indicate that GST-6 is composed of two dissimilar subunits that do not cross-react with antiserum directed against GST-1, GST-2 or GST-3. These observations therefore suggest that GST-6 may have an independent genetic origin.     

Expression of A and B subunits of Shiga-like toxin II as fusions with glutathione S-transferase and their potential for use in seroepidemiology.

We used the plasmid vector pGEX-2T for the expression of recombinant subunits of Shiga-like toxin II (SLT-II). The 5' terminus of the genes that code for either the SLT-IIA or SLT-IIB subunits was genetically fused to the 3' terminus of the gene coding for the enzyme glutathione S-transferase, which serves as a carrier in this expression system. The subunit genes were constructed synthetically by polymerase chain reaction, with appropriate restriction sites to permit in-frame downstream insertion of the genes. The resulting plasmids containing the A and B subunit genes were designated pFG1 and pFG2, respectively. Induction of Escherichia coli laboratory strains harboring pFG1 with isopropyl-beta-D-thiogalactopyranoside (IPTG) yielding only small quantities of SLT-IIA fusion proteins. Since IPTG induction was lethal for cells harboring pFG2, we constructed the recombinant plasmid pFG4, which contained a subgenic fragment of slt-IIB but without the 5' signal sequence. With this construct we were able to express very large quantities of a 33.5-kDa fusion protein, which was purified by affinity chromatography on immobilized glutathione and used as an antigen in immunoblot analysis. Rabbit serum against native SLT-II, as well as all of 12 serum samples with high neutralizing activity against SLT-II, reacted with SLT-IIB purified from an E. coli pFG4 expression system, whereas only 3 of 208 human serum samples with low neutralization titers and none of 54 serum samples with no SLT-II-neutralizing capability reacted. Failure of specific reactivity with the SLT-IIB fusion protein in the majority of human serum samples with low neutralizing activity suggests that serum factors other than immunoglobulins may be responsible for neutralizing activity in these cases. The immunoblot assay with recombinant SLT-IIB as the antigen can be recommended for use in a diagnostic setting as a simple and reliable approach to detect specific human serum antibodies to SLT-II.     

P-糖蛋白、多药耐药相关蛋白和谷胱苷肽S-转移酶在神经母细胞瘤中的表达及意义

目的　探讨P 糖蛋白、多药耐药相关蛋白 (MRP)和谷胱苷肽S 转移酶 (GST π)在小儿神经母细胞瘤 (NB)中的表达及临床意义。方法　应用免疫组织化学SP法检测 70例NB组织中P 糖蛋白、MRP和GST π的表达。结果　P 糖蛋白、MRP和GST π的阳性率分别为 61 4% ( 43 / 70 )、3 8 6%( 2 7/ 70 )和 5 1 4% ( 3 6/ 70 )。P 糖蛋白与MRP、P 糖蛋白与GST π、MRP与GST π及P 糖蛋白、MRP与GST π共表达阳性率分别为 3 2 9% ( 2 3 / 70 )、3 5 7% ( 2 5 / 70 )、2 7 1% ( 19/ 70 )和 2 4 3 % ( 17/ 70 )。P 糖蛋白、GST π与MRP均具有相关性 (r =0 3 87,P =0 0 0 1;r=0 3 0 0 ,P =0 0 12 ) ,P 糖蛋白与GST π无相关性 (r=0 169,P =0 161)。年龄大于 1岁的NBP 糖蛋白和MRP的表达阳性率 ( 72 3 % ,48 9% )均高于小于 1岁者 ( 3 9 1% ,17 4% ,P <0 0 5 )。分化性NBP 糖蛋白、MRP和GST π表达 ( 93 3 %、86 7%和80 0 % )均明显高于无分化的NB( 5 2 7%、2 5 5 %和 43 6% ,P <0 0 5 )。有转移的NBMRP表达( 61 9% )高于无转移者 ( 2 8 6% ,P <0 0 5 )。P 糖蛋白、MRP和GST π表达与性别和分期无关。MRP阳性者中位生存期和 2年累计生存率 ( 2 0个月 ,48 9% )均较阴性者 ( 2 4个月 ,77 9% )下降 (P <0 0 5 )     

Recombinant HoxB4 fusion proteins enhance hematopoietic differentiation of human embryonic stem cells

Enforced expression of the HoxB4 gene promotes expansion of hematopoietic stem cells (HSCs) and enhances hematopoietic development of both murine and human embryonic stem (ES) cells. HoxB4- expanded HSCs have also been shown to retain their normal potential for differentiation and longterm self-renewal in vivo without the development of leukemia, suggesting that manipulation of HoxB4 expression might represent an effective way to expand functional HSCs for use in transplantation medicine. However, the genetic modification of cells poses clinical concerns, including a potentially increased risk of tumor genicity. Constitutive high-level ectopic viral expression of HoxB4 can also produce perturbations in the lineage differentiation of HSCs, an indication that uncontrolled HoxB4 manipulation may not be a satisfactory therapeutic strategy. Here we demonstrate that recombinant HoxB4 protein fused with a triple protein transduction domain (tPTD) promotes hematopoietic development of hES cells. The tPTD-HoxB4 protein enhanced the development of erythroid, myeloid, and multipotential progenitors in both early- and late-stage embryoid bodies (EBs). This effect varied considerably between different hES cell lines. Addition of the tPTD-HoxB4 protein did not alter the globin gene expression pattern; progeny derived from hES cells expressed high levels of embryonic (epsilon) and fetal (gamma) globin genes with or without tPTD-HoxB4 treatment. CD34+ cells derived from hES cells engrafted in bone marrow when transplanted into fetal CD1 mice, although supplementation of the differentiation medium with tPTD-HoxB4 protein did not result in increased repopulating capacity. This suggests that other gene(s), together with HoxB4, are required for generating more competitive HSCs. In summary, our study demonstrates that the tPTD-HoxB4 protein can be used with other recombinant proteins to efficiently generate transplantable HSCs from human ES cells.     

Combined effects of glutathione S-transferase polymorphisms and thyroid cancer risk

Since exposure to ionizing radiation, a risk factor for thyroid cancer, may produce genotoxins potentially eliminated by glutathione-S-transferases, we conducted a case control study to evaluate the role of the GSTM1- and GSTT1-null genotypes and GSTP1 polymorphisms in thyroid cancer. The frequency of GSTP1 Ile/Ile, GSTM1-, and GSTT1-null genotypes was increased in cancer patients when compared with control population. Considering the genotypes over-represented in thyroid cancer patients as potential risk genotypes, we carried out an odds ratio (OR) analysis considering the presence of none, one, two, or three risk genotypes. The results obtained showed that the presence of three potentially risk alleles (GSTM1 null, GSTT1 null, and GSTP1 Ile/Ile) lead to a significant OR increase for all the cases, irrespective of the type of tumor (OR=2.91), for papillary (OR=3.64) but not for follicular tumors. The presence of GSTP1 Ile/Ile leads to a significant later age of tumor onset when compared with GSTP1 Ile/Val and Val/Val (P<0.05), suggesting a possible association between GSTP1 Ile/Ile and the age of disease manifestation. These results suggest that combined GST polymorphisms lead to a moderate increased risk for thyroid cancer, especially for the papillary type, and GSTP1 polymorphisms might modulate the age of onset of the disease.     

Polymorphisms of glutathione S-transferase M1 and male infertility in Taiwanese patients with varicocele

BACKGROUND: To examine glutathione S-transferase M1 (GST M1) gene polymorphism and male infertility in Taiwanese patients with varicocele, 80 young male patients with varicocele (group 1), 62 young male patients with subclinical varicocele (group 2) and 60 normal young males (group 3) were recruited in this study. METHODS: GST M1 null homozygous genotype [GST M1-] and the occurrence of a 4977 bp deletion of sperm mitochondrial DNA (mtDNA) were determined by polymerase chain reaction. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) content of sperm DNA was measured by high-performance liquid chromatography. RESULTS: The frequencies of GST M1- genotype were 43.8, 41.9 and 45% for patients in groups 1, 2 and 3 respectively. In group 1 patients with GST M1- genotype, the frequency of the presence of the 4977 bp deletion in sperm mtDNA (54.3%) was significantly higher than that of the patients without the 4977 bp deletion in sperm mtDNA (45.7%, OR: 2.63, P = 0.04). Patients of groups 1 and 2 with GST M1- genotype had significantly higher 8-OHdG content in sperm DNA and lower protein thiols and ascorbic acid in seminal plasma than those with GST M1+ genotype. CONCLUSION: GST M1- genotype predisposes to increased oxidative damage to sperm of patients with varicocele.     

Expression of glutathione S-transferase pi and glutathione synthase correlates with survival in early stage non-small cell carcinomas of the lung

The glutathione S-transferase (GST) family of genes encode for detoxification enzymes that protect against reactive oxygen species and influence host susceptibility to carcinogens, including tobacco smoke. It has not been determined whether isoenzyme GST-pi or glutathione synthase (GSH2) expression by tumor cells bears a relationship to survival. A total of 201 non-small cell lung cancers (NSCLC) with long-term follow-up were immunostained with antibodies to GST-pi and GSH2 using standard immunostaining techniques. Results were graded semiquantitatively using a scale of 0 to 3 (0 < or = 10%; 1 = 10%-50%; 2 = 51%-80%; 3 > or = 80%) for both nuclear and cytoplasmic staining. Results were correlated with patient survival using Kaplan-Meier analysis. Nuclear staining with GST-pi in greater than 10% of the cells was closely associated with decreased survival (P = .02) in stage I and II squamous cell carcinomas (n = 40). Cytoplasmic staining showed a similar trend that did not reach statistical significance. No significant correlation between GST-pi staining and survival was determined for other histologic types of NSCLC. Cytoplasmic GSH2 staining in greater than 80% of tumor cells was associated with a trend toward improved survival for stage I adenocarcinoma (P = .08) but did not show a relationship to survival for other histologic types of NSCLC. GST-pi expression predicts prognosis in stage I and II squamous cell lung carcinoma, and GSH2 expression may indicate better survival in early stage adenocarcinoma of the lung. Manipulation of GST-pi and GSH2 may be a potential basis for treatment of some NSCLC.     

Augmented expression of metallothionein and glutathione S-transferase pi as unfavourable prognostic factors in cisplatin-treated ovarian cancer patients

Resistance to cis- or carboplatin represents the principal cause of therapeutic failures in ovarian carcinoma. The phenomenon of resistance to platinum-based drugs is partly related to expression of metallothionein (MT) and of glutathione S-transferase pi (GST-pi), but opinion on the subject is discordant. Documentation of a negative predictive effect of MT and GST-pi expression for the therapy employing platinum-based drugs would permit to select resistant cases in which other therapeutic approaches could be employed. The present study aimed at examining the relation between intensities of MT and GST-pi expressions in ovarian carcinomas and dynamics of the clinical course in the neoplastic disease in a group of cisplatin-treated patients. The analyses were performed on samples of ovarian carcinoma originating from 43 first-look laparotomies (FLLs) and, in 30 cases, from second-look laparotomies (SLL) from the same patients. Immunohistochemical reactions were performed on paraffin sections of studied tumors, using monoclonal antibodies to MT and GST-pi. The calculations showed that in cases with augmented expression of MT, mortality was higher. On the other hand, augmented expression of GST-pi predisposed to more frequent relapses, deaths and progression of the tumor. Kaplan–Meier analysis showed that a significantly shorter survival time was linked to cases of higher expression of MT at FLL and of higher expression of GST-pi at FLL, whereas a shorter progression-free time was manifested by cases with higher expression of GST-pi at FLL. The performed investigations indicate that augmented expressions of MT at FLL and GST-pi at FLL in ovarian cancer represent an unfavourable predictive factor in cisplatin-treated patients.     

Recombinant fusion proteins for haemagglutination-based rapid detection of antibodies to HIV in whole blood

Recombinant fusion proteins, consisting of a monovalent anti-human RBC monoclonal antibody B6, and conserved immunodominant peptide of HIV-1 envelope glycoprotein gp41 or HIV-2 envelope glycoprotein gp36, have been designed and purified after over-expression in E. coli. These fusion proteins are Fab-based and were obtained by assembling the light chain with Fd (variable domain and the first constant domain of the heavy chain) or Fd fusions containing HIV-derived peptide, and following a protocol of in vitro denaturation of inclusion bodies and subsequent renaturation to assemble functional Fab. Using a multistep column chromatographic procedure, monomeric Fab and Fab fusion proteins containing HIV-derived peptide were purified to high degree, free of aggregates. The yield of various proteins on the laboratory scale (1-2 l of shake flask culture) was in the range of tens of milligram. Purified anti-human RBC Fab fusion proteins containing sequences derived from HIV-1 gp41 and HIV-2 gp36 were highly specific for detection of antibodies to HIV-1 and HIV-2, respectively. The described design, expression and purification protocols will make it possible to produce specific recombinant reagents in large quantities for agglutination-based rapid detection of antibodies to HIV in whole blood.     

Cloning, expression and characterization of human glutathione S-transferase Omega 2

The class of Omega glutathione transferases is newly identified with novel structural and functional characteristics. Human GSTO 1-1 (glutathione S-transferase Omega 1) is the first member of the GST Omega class. It was found to play a role in apoptosis and be in association with age-at-onset of AD and PD. In order to improve the understanding of the properties of other Omega class members, we screened a human fetal brain cDNA library and obtained the human GSTO2 (glutathione S-transferase Omega 2) cDNA. The full-length cDNA of human GSTO2 is 1179 bp long and encodes a protein of 243 amino acid residues. Expression pattern analysis revealed that GSTO2 was ubiquitously expressed at a low level, with a higher expression in pancreas and prostate. Enzyme assays showed that GSTO2 protein had activities similar to Omega class GSTs. It has detectable glutathione-dependent thiol transferase activity and glutathione-dependent dehydroascorbate reductase activity. But different from GSTO1-1, GSTO2 exhibits a high catalytic activity with CDNB. Subcellular localization analysis of GSTO2-EGFP fusion protein revealed that GSTO2 distributed to cytoplasm of COS-7 cells and both cytoplasm and nucleus of L-02, QGY-7703 and SMMC-7721 cells. Overexpression of GSTO2 induced apoptosis of L-02 cells detected by Annexin V-PE staining. The results suggest that GSTO2 may play an important role in cellular signaling.