The 14-3-3 protein as a vaccine candidate against schistosomiasis

We have previously reported on the cloning of the 14-3-3 protein of Schistosoma mansoni. Here, we evaluate the potential use of this protein as a vaccine candidate against infection by S. mansoni. Sm14-3-3 was expressed and purified either as a free protein or as a fusion protein to SjGST or MBP. Sera from mice infected with S. mansoni recognized both SjGST and 14-3-3, indicating that antibodies against these two proteins are induced in the course of the natural infection. Furthermore, mice immunized with either 14-3-3, GST or 14-3-3-GST, reacted with cercaria lysate. A cellular immune response was also detected, particularly in mice immunized with 14-3-3-GST. With respect to the effect on biological functions, antibodies to 14-3-3 and 14-3-3-GST caused 23-32% complement-mediated cytotoxcity of S. mansoni schistosomula compared to only 10-11% induced by either normal mouse serum, or GST alone. In challenge infection with S. mansoni, immunization with 14-3-3, either as a fusion protein or as a free protein, led to protection ranging from 25-46%, as determined by reduction of adult worm burden, while SjGST alone elicited only 0-8% protection and MBP alone did not elicit any protection.     

Identification of the immunogenic domains in HBsAg preS1 region using overlapping preS1 fragment fusion proteins

AIM: The incorporation of hepatitis B virus(HBV)preS1 region into epitope-based vaccines against HBV has been accepted widely, but the incorporate site and size of preS1 sequence is controversial.Therefore our purpose was to further investigate its immunogenic domains for the epitope-based hepatitis B vaccine design. METHODS: Eight GST fusion proteins containing overlapping preS1 fragments in preS1(21-119) region were expressed in E.coli. Using these purified fusion proteins, the immunogenic domains in preS1 region were identified in detail in mice and humans by Western blot analysis and ELISA. RESULTS: The results in mice showed that the immunogenic domains mainly existed in preS1(21-59) and preS1(95-109). Similarly, these fragments had strong immunogenicity in humans; whereas the other parts except for preS1(60-70) also had some immunogenicity. More importantly, a major immunogenic domain, preS1 (34-59), which has much stronger immunogenicity, was identified. Additionally, the antibodies against some preS1 fragments, especially preS1(34-59), were speculated to be virus-neutralizing. CONCLUSION: Eight GST fusion proteins containing overlapping preS1 fragments were prepared successfully. They were used for the study on the immunogenic domains in preS1(21-119) region. The preS1(34-59) fragments were the major immunogenic domains in the preS1 region, and the antibodies against these fragments were speculated to be virus-neutralizing. Therefore,the incorporation of preS1(34-59) fragments into epitope-based HBV vaccines may be efficient for enhancement of immune response. Additionally, the results also imply that there are more complex immune responses to preS1 region and more abundant immunogenic domains in humans.     

GSTM1, GSTT1 and GSTP1 polymorphisms in the Korean population

The isoenzymes of the glutathione s transferase (GST) family play a vital role in phase II of biotransformation of many substances. Using a multiplex polymerase chain reaction and a direct sequencing analysis, the frequencies of GSTM1, GSTT1, and GSTP1 polymorphisms were evaluated in 1,051 Korean male subjects. We found that 53.8% of the individuals had the GSTM1 null genotype and 54.3% had the GSTT 1 null genotype. The genotypic distribution of GSTP1 was Ile105/Ile105 in 68.4%, Ile105/Val105 in 29.1% and Val105/Va105 in 2.5%. The most frequently observed combination of GSTM1, GSTP1 and GSTT1 genotypes was Null type/Ile105/Ile105/Null type, while the combination of Non-null type/Val105/Val105/Non-Null type was not observed. We found that the genotype distributions of three GST isoenzymes in the Koreans are similar to those reported in Asians and previously reported Koreans. We believe our results, which are represented by a large population, are reliable estimates of the frequencies of the polymorphic GST alleles in the Koreans and will help future researches on GST polymorphisms.     

Polymorphism within the glutathione S-transferase P1 gene is associated with increased susceptibility to childhood malignant diseases

BACKGROUND: Glutathione S-transferases (GSTs) are involved in the metabolism of carcinogens and anticancer drugs. Functional polymorphisms exist in at least three genes that code for the GSTs, such as the GSTM1 and GSTT1 gene deletions or the A-G transition within the GSTP1 gene, which represents distinct GSTP1a and GSTP1b alleles. In the present case-control study, we aimed at estimation of the relationship between the GSTM1, GSTT1, and GSTP1 genotypes and the susceptibility to various types of childhood malignancies and the early relapses of diseases. PROCEDURE: Using the polymerase chain reaction on the DNA extracted from peripheral blood leukocytes, we identified the GSTM1, GSTT1, and GSTP1 genotypes in 234 children at the initial stage of a childhood malignancy as well as in 460 age-and sex-matched healthy subjects who served as controls. The follow-up period for the effects of the anticancer therapy ranged from 11 to 43 months. RESULTS: Compared to the controls, a significant increase in the frequency of the GSTP1b/GSTP1b genotype (odds ratio (OR) 5.7; 95% confidence limit (CL) from 2.4 to 13.8; Pearsons Chi-square P = 0.0001) was detected in the children with neoplasms. The GSTM1 and GSTT1 genotypes did not show any correlation with the risk of the de novo diagnosed neoplasms. During the observation, 62 children (26%) were found to be present with a local or disseminated recurrence of the diseases. The analysis indicated a trend in increasing risk of relapse for carriers of the GSTP1a allele (OR = 3.29; 95% CL from 0.73 to 14.67 P = 0.03). CONCLUSIONS: Our results support the hypothesis that GST genotype affects etiology and outcome of a variety of childhood malignancies.     

Selective expression of Kir6.1 protein in different vascular and non-vascular tissues

K(ATP) channels are composed of pore-forming subunits Kir6.x and auxiliary subunits SURx. These channels play important roles in modulating the contractility of vascular smooth muscle cells (SMCs) by altering membrane potentials. The molecular basis of K(ATP) channels in vascular SMCs is unclear and the expression of different K(ATP) channel subunits at protein level in various tissues still undetermined. In this study, using an anti-Kir6.1 antibody, we detected the expression of Kir6.1 proteins in rat vascular tissues including mesenteric artery, pulmonary artery, aorta, and tail artery. Kir6.1 proteins were also identified in heart and other non-vascular tissues including spleen and brain, but they were undetectable in liver and kidney. Immunocytochemical study revealed the expression of Kir6.1 proteins in cultured rat thoracic aortic SMCs. Using the whole-cell patch-clamp technique, it was found that the intracellularly applied anti-Kir6.1 antibody significantly inhibited K(ATP) channel currents in HEK-293 cells that were stably transfected with Kir6.1 cDNA. A better understanding of differential expression of Kir6.1 proteins in various vascular and non-vascular tissues may help discern different molecular basis and functions of K(ATP) channel complexes in these tissues.     

Dietary clofibrate inhibits induction of hepatic antioxidant enzymes by chronic estradiol in female ACI rats

Excess production of H₂O₂ has been implicated in oncogenesis. The object of the present study was twofold: first, to determine the influence of chronic estradiol (E₂) on the activities of selected hepatic antioxidant enzymes in female ACI rats, a strain that is highly sensitive to the induction of estrogen dependent mammary tumors; secondly, to evaluate the actions of dietary clofibrate, a peroxisome proliferator, on activities of these enzymes in control and E₂-treated ACI rats. Enzymes selected for study were: NAD(P)H quinone oxidoreductase (NQO1), glutathione S-transferase (GST) and glutathione peroxidase (GPx). Cytosolic catalase (CAT) was also measured as an index of peroxisome proferation in control and E_2- treated animals. E₂ was administered chronically over 6 and 12 week periods from cholesterol pellet implants containing either 1 or 3 mg E₂. Animals were fed AIN-76A diets with or without 0.4% clofibrate over the experimental period. NQO1 and GST but not GPx were induced to varying degrees (NQO1 about 300%, and GST about 45–97%) by chronic E₂-treatment. E₂-induced increases in these activities were completely prevented in rats exposed to dietary clofibrate. Dietary clofibrate also caused slight but significant reductions in baseline activities of NQO1, GST and GPx in control animals. Serum E₂ levels, increased approximately 540% in a dose-dependent manner, and were not altered by dietary clofibrate. It is concluded that chronic E2 treatment markedly induces several important hepatic antioxidant enzymes in female ACI rats, and induction of these activities by E2 is inhibited completely by dietary clofibrate. Both of these actions have the potential to markedly influence the profile of E₂ metabolites exported from the liver to E₂ sensitive extrahepatic tissues and influence the initiation and progression of hormone-dependent tumors.     

Enzymatic characterization of recombinant mouse retinal dehydrogenase type 1

Retinal dehydrogenases (RALDHs) convert retinal into retinoic acids (RAs), which are important signaling molecules in embryogenesis and tissue differentiation. We expressed mouse RALDH type 1 (mRALDH1) in Escherichia coli and studied the kinetic properties of the recombinant enzyme for retinal substrates. Purified recombinant mRALDH1 catalyzed the oxidation of all-trans and 9-cis retinal but not 13-cis retinal, and exhibited two pH optimums, 7.8 and 9.4, for all-trans and 9-cis retinal substrates, respectively. The K(m) for all-trans retinal (11.6 micro M) was 3-fold higher than for 9-cis retinal (3.59 micro M). However, the conversion efficiencies of either all-trans or 9-cis retinal to the respective RAs were similar. MgCl(2) inhibited the oxidation of both all-trans and 9-cis retinal. Chloral hydrate and acetaldehyde competitively suppressed all-trans retinal oxidation with inhibition constants (K(i)) of 4.99 and 49.4 micro M, respectively. Retinol, on the other hand, blocked the reaction uncompetitively. These data extend the kinetic characterization of mRALDH1, provide insight into the possible role of this enzyme in the biogenesis of RAs, and should give useful information on the determination of amino acid residues that play crucial roles in the catalysis of all-trans and 9-cis retinal.     

Slowly getting a clue on CD95 ligand biology

Since the ligand for the death factor CD95 (CD95L) was identified almost a decade ago, it has been established that this molecule (CD95L, FasL, Apo-1L, CD178, TNFSF6, APT1LG1) has multiple immunoregulatory and pathophysiologically relevant functions. CD95L does not only act as a death factor when externalized with secretory lysosomes on cytotoxic T and NK cells or when expressed on CD4(+) T cells in the course of activation-induced cell death, it is also a key molecule for the establishment of immune privilege or tumor cell survival and may serve as a costimulatory molecule during T cell activation. Moreover, alterations of expression or shedding of different forms of CD95L are associated with many diseases including various malignancies, HIV infection, autoimmune disorders (systemic lupus erythematodes, rheumatoid arthritis), acute myocardial infarction, traumatic injury and many others. In most cases, however, the physiological link between altered CD95L expression and pathophysiology is unknown. Given the potency of the molecule to regulate death and survival of many different cell types, the control of CD95L production, transport, storage, shedding and release is of tremendous biological and clinical interest. This commentary aims at briefly summarizing the current knowledge, hypotheses and controversies about CD95L as a multifunctional ligand and receptor. It touches upon the complex networks of intracellular dynamics of protein transport and trafficking and the potential bidirectional signal transduction capacity of CD95L with a focus on molecular interactions that have been worked out over the past years.