Identification and cloning of the second type transglutaminase from Litopenaeus vannamei, and its transcription following pathogen infection and in relation to the haemolymph coagulation.

Complementary (c)DNA encoding transglutaminaseII (TGII) messenger (m)RNA of white shrimp, Litopenaeus vannamei, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus (accession no.: BAA02134), tiger shrimp, Penaeus monodon (AAV49005; AAO33455), kuruma shrimp, Marsupenaeus japonicus (BAD36808) and Pacifastacus leniusculus (AAK69205) TG. The 2405-bp cDNA contained an open reading frame (ORF) of 2292 bp, a 31-bp 5'-untranslated region (UTR), and an 82-bp 3'-UTR containing a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (764 aa) was 85.9 kDa with an estimated pI of 5.32. The L. vannamei TGII (abbreviated LvTGII) contains a typical TG-like homologue, two putative integrin binding motif (RGD and KGD), and five calcium-binding sites; three catalytic triad is present as in arthropod TG. Sequence comparison and phylogenetic analysis revealed that shrimp TG can be separated into two groups, STGI and STGII, and LvTGII is more closely related to STGII than to STGI. LvTGII mRNA was detected in all tested tissues of L. vannamei, and was highly expressed in haemocytes. The haemocytes of L. vannamei injected with Vibrio alginolyticus showed a significant increase of LvTGI and LvTGII mRNA expression at 6 h followed by a notable decrease at 24 h in LvTGI and a continually increase in LvTGII indicating a complementary effect, which implied that both LvTGs involved in the immune response of shrimp, and LvTGII was more important in the later defense response. The gene silencing of LvTGII in shrimp significantly decreased LvTGII expression and TG activity of haemocytes, and significantly increased clotting time of haemolymph, suggests that the cloned LvTGII is a clotting enzyme involved in haemolymph coagulation of L. vannamei. In conclusion, the cloned LvTGII is a clotting enzyme involved in coagulation of haemolymp and immune response of white shrimp, L. vannamei.

Identification and cloning of a selenophosphate synthetase (SPS) from tiger shrimp, Penaeus monodon, and its transcription in relation to molt stages and following pathogen infection.

Complementary (c)DNA encoding selenophosphate synthetase (SPS) messenger (m)RNA of the tiger shrimp Penaeus monodon, designated PmSPS, was obtained from the hepatopancreas by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The 1582-bp cDNA contained an open reading frame (ORF) of 1248 bp, a 103-bp 5'-untranslated region (UTR), and a 231-bp 3'-UTR, which contained a conserved selenocysteine insertion sequence (SECIS) element, a conventional polyadenylation signal, and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (416 aa) was 45.5 kDa with an estimated pI of 4.85. It contained a putative selenocysteine residue which was encoded by the unusual stop codon, (275)TGA(277), which formed at the active site with residues Sec(58) and Lys(61). A comparison of amino acid sequences showed that PmSPS was more closely related to invertebrate SPS1, such as those of Heliothis virescens and Drosophila melanogaster a and b. PmSPS cDNA was synthesized in all tested tissues, especially in the hepatopancreas. PmSPS in the hepatopancreas of shrimp significantly increased after an injection with either Photobacterium damsela or white spot syndrome virus (WSSV) in order to protect cells against damage from oxidation, and enhance the recycling of selenocysteine or selenium metabolism, indicating that PmSPS is involved in the disease-resistance response. The PmSPS expression by hemocytes significantly increased in stage C, and then gradually decreased until stage A, suggesting that the cloned PmSPS in hemocytes might play a role in viability by renewing hemocytes and antioxidative stress response for new exoskeleton synthesis during the molt cycle of shrimp.

Identification and cloning of a transglutaminase from giant freshwater prawn, Macrobrachium rosenbergii, and its transcription during pathogen infection and moulting.

Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of the giant freshwater prawn, Macrobrachium rosenbergii, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus; tiger shrimp, Penaeus monodon; kuruma shrimp, Marsupenaeus japonicus; and crayfish, Pacifastacus leniusculus. The 2722-bp cDNA contained an open reading frame (ORF) of 2334 bp, a 72-bp 5'-untranslated region (UTR), and a 316-bp 3'-UTR containing a stop codon and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (778 aa) was 86.67 kDa with an estimated pI of 5.4. The M. rosenbergii TG (abbreviated MrTG, accession no.: JF309296) contains a typical transglutaminase-like homologue, two putative integrin-binding motifs (RGD), ten glycosylation sites, and four calcium-binding sites; a catalytic triad is present as in arthropod TGs. Sequence comparison and a phylogenetic analysis revealed that shrimp TG can be separated into three subgroups, penaeid TG1, freshwater crustacean TG2 and marine crustacean TG2, and MrTG was more closely related to TG2 than to TG1. MrTG mRNA and TG activities were detected in all tested tissues of M. rosenbergii, with MrTG mainly being synthesised by haemocytes. There was a negative correlation between clotting time of haemolymph, and MrTG expression and TG activity of haemocytes in prawn injected with Lactococcus garvieae. The pattern of MrTG mRNA expression and TG activity in haemocytes exhibited a contrary tendency with clotting time of haemolymph during the moult stages. Those results indicate that cloned MrTG is involved in the defence response, and is probably the major functional TG for haemolymph coagulation in M. rosenbergii.

Cu/Zn ratios are associated with nutritional status, oxidative stress, inflammation, and immune abnormalities in patients on peritoneal dialysis.

OBJECTIVES: We evaluated the relationship of the plasma copper/zinc (Cu/Zn) ratio with nutritional status, inflammation, oxidative stress, and immune function in peritoneal dialysis patients. DESIGN AND METHODS: Clinical and laboratory parameters were measured in patients (n=45) and age- and sex-matched healthy individuals (n=30). RESULTS: There were significant negative correlations of the Cu/Zn ratio with nutrition-related parameters (body mass index [BMI], creatinine, hemoglobin, and albumin) and antioxidant (vitamin C and E) levels and positive correlations of the Cu/Zn ratio with the levels of high sensitivity C-reactive protein (hs-CRP) and oxidation products (malondialdehyde [MDA] and protein carbonyl). The Cu/Zn ratio was negatively correlated with the percentages of B- and T-lymphocyte subsets and the ratio of CD4/CD8 antigens. CONCLUSIONS: In peritoneal dialysis patients, elevated Cu/Zn ratios are associated with malnutrition, increased oxidative stress, inflammation, and disrupted immune status.

Identification and cloning of a selenium-dependent glutathione peroxidase from tiger shrimp, Penaeus monodon, and its transcription following pathogen infection and related to the molt stages.

Complementary (c)DNA encoding glutathione peroxidase (GPx) messenger (m)RNA of the tiger shrimp Penaeus monodon was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method. The 1321-bp cDNA contained an open reading frame (ORF) of 564bp, a 69-bp 5'-untranslated region (UTR), and a 688-bp 3'-UTR containing a poly A tail and a conserved selenocysteine insertion sequence (SECIS) element. The molecular mass of the deduced amino acid (aa) sequence (188 aa) was 21.05kDa long with an estimated pI of 7.68. It contains a putative selenocysteine residue which is encoded by the unusual stop codon, (190)TGA(192), and forms the active site with residues Glu(75) and Trp(143). Comparison of amino acid sequences showed that tiger shrimp GPx is more closely related to vertebrate GPx1, in accordance with those in Litopenaeus vannamei and Macrobrachium rosenbergii. GPx cDNA was synthesised in lymphoid organ, gills, heart, haemocytes, the hepatopancreas, muscles, and intestines. After injected with either Photobacterium damsela or white spot syndrome virus (WSSV), the respiratory bursts of shrimp significantly increased in order to kill the pathogen, and induced increases in the activities of superoxide dismutase and GPx, and regulation in the expression of cloned GPx mRNA to protect cells against damage from oxidation. The GPx expression significantly increased at stage D(0/1), and then gradually decreased until stage C suggesting that the cloned GPx might play a role in the molt regulation of shrimp.

Expression and characterization of the JAK kinase and STAT protein from brine shrimp, Artemia franciscana.

In this study, we isolated and characterized both JAK and STAT genes from Artemia, Artemia franciscana. Although AfJAK showed only 19% identity (33% similarity) to the Drosophila Hop protein, AfJAK contained the characteristic JAK homology domain (JH domain) from JH1 to JH7. On the other hand, AfSTAT showed higher identity (30%) to Drosophila STAT (STAT92E). The low identities of AfJAK and AfSTAT to Drosophila Hop and STAT92E suggest that JAK and STAT proteins are unique in each different species of invertebrate. RT-PCR analysis showed that both AfJAK and AfSTAT transcripts were ubiquitously expressed in the embryo, which is similar to the expression patterns of Drosophila Hop and STAT92E mRNAs during development. In addition, we generated a constitutively active form of AfSTAT by fusing the JH1 domain of AfJAK to the C-terminal end of AfSTAT. This fusion protein, AfSTAT-HA-JH1, autophosphorylated on its tyrosine residue and was able to bind to specific DNA motifs including the STAT-binding motifs in the Drosophila Raf promoter. Both AfJAK and AfSTAT proteins elicited the transactivation potential toward the fly Raf promoter in Sf9 cells. However, tyrosine phosphorylation of AfSTAT was not detected, which is consistent with the cellular localization analysis that most AfSTAT proteins were in the cytoplasm. Our results demonstrate that both JAK and STAT are present in the genome of Artemia, which can serve as the basis for further investigations to explore the role of the JAK/STAT signal pathway in the development and immune response of brine shrimp.

cDNA cloning, identification, tissue localisation, and transcription profile of a transglutaminase from white shrimp, Litopenaeus vannamei, after infection by Vibrio alginolyticus.

Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of white shrimp, Litopenaeus vannamei, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus (accession no.: BAA02134); tiger shrimp, Penaeus monodon (AAL78166); and Pacifastacus leniusculus (AF336805). The 2638-bp cDNA contained an open reading frame (ORF) of 2172 bp, a 55-bp 5'-untranslated region (UTR), and a 411-bp 3'-UTR containing a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (757 aa) was 84.9 kDa with an estimated pI of 5.2. The L. vannamei TG (abbreviated LvTG) contains a typical transglutaminase-like homologue, a putative integrin-binding motif (RGD), and four calcium-binding sites; a catalytic triad is present as in arthropod TG. Sequence comparison and phylogenetic analysis revealed that shrimp TG can be separated into two subgroups, STGS1 and STGS2, and LvTG is more closely related to STGS1 than to STGS2. LvTG mRNA and TG activities were detected in all tested tissues of L. vannamei, with LvTG mainly being synthesised in haemocytes. However, the pattern of LvTG mRNA expression was not directly correlated with TG activity. The haemocytes of L. vannamei injected with Vibrio alginolyticus showed a significant decrease of TG activity at 3 h and a significant increase of LvTG mRNA expression at 6 h followed by a notable decrease from 12 to 24 h, which indicated that cloned LvTG was involved in the immune response of shrimp. The results also imply that more than one type of TG may be involved in the defense response in L. vannamei.

Cold shock-induced norepinephrine triggers apoptosis of haemocytes via caspase-3 in the white shrimp, Litopenaeus vannamei.

The total haemocyte count (THC), haemolymph norepinephrine (NE) level, caspase-3 mRNA expression and activity levels, and apoptotic haemocyte rate were measured when shrimp Litopenaeus vannamei (20-25 g) were transferred from 28 to 22 degrees C after 0, 2, and 7 days, and the caspase-3 mRNA expression and activity levels and the apoptotic cell rate of haemocytes, in vitro, were determined after incubation with 2 x 10(-8) M NE for 0, 30, 60, and 120 min at 27 +/- 1.0 degrees C. For shrimp transferred from 28 +/- 1.0 to 22 +/- 0.5 degrees C after 2 and 7 days, the THC decreased by 17.9% and 18.0%, but the NE concentration, caspase-3 transcription and activity levels, and apoptotic cell rate increased by 62.5% and 37.3%, 5100.0% and 446.6%, 148.6% and 152.0%, and 88.7% and 200.1%, respectively, compared to those of shrimp held at 28 +/- 0.5 degrees C which served as the control. Similar tendencies were observed for the apoptotic cell rate, and caspase-3 transcription and activity levels of haemocytes exposed to 2 x 10(-8) M NE in vitro. These results suggest that NE plays an important role in the apoptosis of haemocytes in L. vannamei under hypothermal stress, which causes depressive effects on immunological responses.

Molecular cloning and characterization of lipopolysaccharide- and beta-1,3-glucan-binding protein from the giant freshwater prawn Macrobrachium rosenbergii and its transcription in relation to foreign material injection and the molt stage.

Lipopolysaccharide (LPS)- and beta-1,3-glucan-binding protein (LGBP) complementary (c)DNA was cloned from the hepatopancreas of the giant freshwater prawn Macrobrachium rosenbergii using oligonucleotide primers and a reverse-transcription polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1389 bp encoding a protein of 378 amino acids (aa) including a 15-aa signal peptide. The calculated molecular mass of the mature protein (363 aa) was 41.2 kDa with an estimated pI of 4.73. The M. rosenbergii LGBP sequence contains (1) three putative integrin-binding motifs, (2) a glucanase motif, (3) a putative N-glycosylation site, (4) four protein kinase C phosphorylation sites, (5) four casein kinase II phosphorylation sites, and (6) a putative recognition motif. Sequence comparison showed that the deduced amino acids of LGBP of M. rosenbergii had overall similarities of 60-71% to those of known crustacean LGBPs and beta-1,3-glucan-binding proteins (BGBPs). The LGBP of M. rosenbergii was mainly expressed in the hepatopancreas. The LGBP transcript of M. rosenbergii was downregulated in haemocytes, but was upregulated in the hepatopancreas when injected with LPS and poly:IC after 12 h. The LGBP messenger (m)RNA expression of prawns in the postmolt stage was significantly upregulated in haemocytes, but downregulated in the hepatopancreas, which revealed a complementary relationship between haemocytes and the hepatopancreas in the molt cycle.

A second proPO present in white shrimp Litopenaeus vannamei and expression of the proPOs during a Vibrio alginolyticus injection, molt stage, and oral sodium alginate ingestion.

Prophenoloxidase (proPO) is a melanin-synthesising enzyme that plays important roles in immune responses by crustaceans. Previously, we cloned and characterized proPO-I from white shrimp, Litopenaeus vannamei. In the present study, a novel prophenoloxidase-II (proPO-II) cDNA was also cloned from haemocytes of L. vannamei using oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of complementary (c)DNA end (RACE) method. The 2504-bp cDNA contained an open reading frame (ORF) of 2073 bp, an 84-bp 5'-untranslated region, and a 347-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (691 amino acids) was 78.8 kDa with an estimated pI of 6.07. It contains two putative tyrosinase copper-binding motifs and a conserved C-terminal region common to all known proPOs. Comparisons of the amino acid sequences showed that white shrimp proPO-II is more closely related to the proPO of other penaeids than to that of crayfish, lobsters, crab, or a freshwater prawn, and is the ancestor type of known penaeid proPOs. proPO-I and proPO-II messenger (m)RNAs of shrimp were located on different loci, and were constitutively expressed mainly in haemocytes. The transcriptional regulation of these two proPOs in shrimp at different molt stages, those administered dietary sodium alginate, and those challenged with Vibrio alginolyticus were surveyed. The results showed that the proPOs may be directly involved in the acute-phase immune defence, and proPO-II may contribute earlier to immune defence in shrimp injected with V. alginolyticus, and it may be regulated by ecdysone. However, a similar effect was found by stimulating proPO-I and proPO-II mRNA expression in shrimp fed a sodium alginate-containing diet. Results of this study provide a basis for developing a comprehensive understanding of expression/function relationships of individual proPOs in shrimp.

The effect of Vibrio alginolyticus infection on caspase-3 expression and activity in white shrimp Litopenaeus vannamei.

Complementary (c)DNA encoding cysteine aspartate protease-3 (caspase-3) messenger (m)RNA of the white shrimp Litopenaeus vannamei was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the caspase sequences of Penaeus merguiensis, Spodoptera frugiperda, Spodoptera littoralis, and Penaeus monodon. The 1212-bp cDNA contained an open reading frame (ORF) of 951bp, a 56-bp 5'-untranslated region (UTR), and a 205-bp 3'-UTR containing the poly(A) tail. The molecular mass of the deduced amino acid (aa) sequence (316aa) was 28.7kDa with an estimated pI of 6.46. It contains two hits in the caspase family, including a p20 domain profile and p10 domain profile. From aa residues 183 to 194, there was a single pattern hit for QACRG, a caspase family cysteine active site. Caspase-3 mRNA was widely distributed in all tissues; especially in haemocytes and lymphoid organ, but was weakly expressed in muscles, ganglia, spermaries, and ovaries. Shrimp injected with Vibrio alginolyticus induced transcription of caspase-3 mRNA and increased caspase-3 activity which contributed to the occurrence of apoptosis and DNA fragmentation in haemocytes.

Expression of clottable protein of tiger shrimp (Penaeus monodon) in gonads and its possible role as nutrient source for the embryo.

We have investigated the expression of clottable protein (CP) in gonad of tiger shrimp (Penaeus monodon) and extent of its phosphorylation. Polyclonal antibodies against purified CP were prepared from rabbit serum. Using this anti-CP antiserum, the temporal expression of CP in gonads of tiger shrimp was analyzed. It was found that the CP occurs only in mature ovaries but not in immature ovaries and testes. Results of RT-PCR confirmed that these tissues expressed low levels of CP mRNA transcripts. Upon eyestalk-ablation, the ovaries in female shrimps were induced to develop, and the CP expression levels in ovaries were traced chronically by RT-PCR analyses. The expression level peaked on day 3 with an increase of about 40 folds relative to the basal level and returned to normal level (as the control shrimp) at day 12. The shrimp embryos at different intervals from spawning to 16h post-spawning were also collected, and it was found that CP contents were gradually decreased in the embryos until the nauplii were hatched. In addition, purified CP was shown to react with specific anti-phosphoserine, anti-phosphothreonine, and anti-phosphotyrosine antibodies suggesting that CP is a phosphoprotein with all types of phosphorylations. Taken together the results suggest that expression of CP in shrimp ovaries is coupled to ovarian development and CP possibly supply nutrition for shrimp embryo.

Expression and characterization of two STAT isoforms from Sf9 cells.

In invertebrates, the JAK-STAT signaling pathway is involved in the anti-bacterial response and is part of an anti-viral response in Drosophila. In this study, we show that two STAT transcripts are generated by alternative splicing and encode two isoforms of Sf-STAT with different C-terminal ends. These two isoforms were produced and purified using the recombinant baculovirus technology. Both purified isoforms showed similar DNA-binding activity and displayed weak but significant transactivation potential toward a Drosophila promoter that contained a STAT-binding motif. No significant activation of the Sf-STAT protein in Sf9 cells was found by infection with baculovirus AcMNPV.

Cloning and characterization of hemolymph clottable proteins of kuruma prawn (Marsupenaeus japonicus) and white shrimp (Litopenaeus vannamei).

The hemolymph clottable protein (CP) of Marsupenaeus japonica (designated as Mj-CP) was purified by a DEAE anion-exchanger and a Sepharose CL-6B gel filtration column. In the presence of Ca(2+), it formed stable clots in vitro upon the addition of the hemocytes lysate containing transglutaminase. Results of gel filtration chromatography and SDS-PAGE indicated that Mj-CP mainly existed as disulfide-linked homodimers of 390 kDa. Specific primers were designed; PCR as well as RACE help to clone and sequence Mj-CP cDNA of 5660 bp. The predicted CP-precursor contains a signal peptide followed by a subunit of 1671 amino acids (isoelectric point 5.6), including two RGD motifs and three potential N-glycosylation sites. Mj-CP is structurally 80% and 38% identical to the CPs of tiger shrimp and crayfish, respectively. Likewise, CP cDNA of white shrimp (Litopenaeus vannamei) was also cloned and sequenced; the predicted CP has 1666 amino acid residues and an isoelectric point of 5.2. Both CPs bear potential transglutaminase cross-linking sites, i.e. seven Ser-Lys-Thr repeats near the N-terminus, a Ser- and Gln-rich region in the middle, and polyGln (n=8-11) near the C-terminus. Phylogenetic analyses of crustacean CPs and vitellogenins revealed divergent evolution of the two protein families. By RT-PCR, the sub-cuticular epidermis was identified as one of the major tissues that express CP in M. japonica.

Tissue-specific expression and regulation of the haemolymph clottable protein of tiger shrimp (Penaeus monodon).

The clottable protein (CP) involved in Penaeus monodon haemolymph coagulation has previously been characterized and cloned. Polyclonal antibodies against purified CP were also prepared from rabbit serum. By Western blot analyses, we showed occurrence of CP in the shrimp central nervous system, gill, and lymphoid organ. Results of RT-PCR further indicated that the central nervous system, gill, and lymphoid organ transcribed more CP, heart and hepatopancreas transcribed less, while the haemocytes and the muscle did not. We further analyzed the CP distribution within shrimp lymphoid organ by immunohistochemical method, CP was found to localise in stromal cells of lymphoid organ rather than in the developing haemocytes. In addition, concentrations and regulation of the plasma CP under normal and artificially traumatic conditions were studied with rocket immunoelectrophoresis. The average plasma CP concentration in normal intermolt shrimps was elevated from 3 mg ml(-1) to above 12 mg ml(-1) after successive blood-withdrawing for a week. The production and secretion of CP apparently were increased more than 4 folds to compensate its loss. Our result also suggested that the shrimp sinus gland endocrine system is not directly required for the expression and up-regulation of CP.

Biochemical characterization and cloning of transglutaminases responsible for hemolymph clotting in Penaeus monodon and Marsupenaeus japonicus.

To investigate the shrimp blood clotting enzyme, a transglutaminase in the hemocytes of Penaeus monodon (abbreviated as TGH) was purified. TGH is an abundant homodimeric cytosolic protein with 84.2 kDa subunits. It clotted shrimp plasma and incorporated fluorescent dansylcadaverine into succinyl casein upon activation by CaCl(2) in vitro. IC(50) for the activation was 3 mM, which is below the shrimp plasma Ca(2+) level. Showing similar properties as other type II transglutaminase, TGH was particularly unstable after activation. MALDI-TOF/TOF mass-analyses of tryptic peptides of P. monodon TGH confirmed its identity to STG I (AY074924) previously cloned. A possible allele of the other isozyme STG II (AY771615) has also been cloned from the P. monodon cDNA and designated as PmTG. The predicted PmTG protein sequence is 58% similar to that of STG I and 99.2% to that of STG II. Likewise, a novel enzyme Mj-TGH was purified and cloned from Marsupenaeus japonicus hemocytes. Results of sequence alignment and phylogenetic analyses of these transglutaminases suggest that STG I and Mj-TGH are 83% identical and orthologous to each other, while PmTG/STG II and a previously cloned M. japonicus transglutaminase (AB162767) are their paralogs. Protein of the latter two could not be isolated, their regulated expression was discussed.

Blood micronutrient, oxidative stress, and viral load in patients with chronic hepatitis C.

AIM: To assess the extent of micronutrient and oxidative stress in blood and to examine their linkages with viral loads in chronic hepatitis C patients. METHODS: Hepatitis C virus (HCV)-RNA levels were quantified in the serum from 37 previously untreated patients with chronic hepatitis C. The plasma and erythrocyte micronutrients (zinc, selenium, copper, and iron) were estimated, and malondialdehyde (MDA) contents were determined as a marker to detect oxidative stress. Antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione reductase (GR) activities in blood were also measured. The control group contained 31 healthy volunteers. RESULTS: The contents of zinc (Zn), and selenium (Se) in plasma and erythrocytes were significantly lower in hepatitis C patients than in the controls. On the contrary, copper (Cu) levels were significantly higher. Furthermore, plasma and erythrocyte MDA levels, and the SOD and GR activities in erythrocytes significantly increased in hepatitis C patients compared to the controls. However, the plasma GPX activity in patients was markedly lower. Plasma Se (r = -0.730, P<0.05), Cu (r = 0.635), and GPX (r = -0.675) demonstrated correlations with HCV-RNA loads. Significant correlation coefficients were also observed between HCV-RNA levels and erythrocyte Zn (r = -0.403), Se (r = -0.544), Cu (r = 0.701) and MDA (r = 0.629) and GR (r = 0.441). CONCLUSION: The levels of Zn, Se, Cu, and oxidative stress (MDA), as well as related anti-oxidative enzymes (GR and GPX) in blood have important impact on the viral factors in chronic hepatitis C. The distribution of these parameters might be significant biomarkers for HCV.

The effect of zinc supplementation on the treatment of chronic hepatitis C patients with interferon and ribavirin.

OBJECTIVES: To evaluate the effects of zinc supplementation on serum zinc and copper levels, and the severity of adverse reactions and virologic responses in chronic hepatitis C patients undergoing interferon (IFN)/ribavirin therapy. DESIGN AND METHODS: Forty subjects were randomly assigned to receive IFN-alpha-2a/ribavirin with or without zinc gluconate for 24 weeks, then a period of 6 months for follow-up. Twenty healthy controls were also enrolled in the study. Blood samples were collected at different time points during therapy and at 6 months after the completion of therapy and were analyzed for zinc and copper levels. The adverse reactions and the virologic responses were also examined accordingly. RESULTS: Serum zinc levels were significantly lower in chronic hepatitis C patients than in healthy controls and further depressed by IFN/ribavirin treatment. However, serum zinc levels in patients were remediable by zinc supplements. No apparent difference was seen in virologic responses between subjects with or without zinc supplements, but certain adverse side effects associated with the zinc therapy were significantly decreased. CONCLUSIONS: Zinc supplementation may be a complementary therapy in chronic hepatitis C patients to increase the tolerance to IFN-alpha-2a and ribavirin.

Aluminum-induced suppression of testosterone through nitric oxide production in male mice.

Excessive nitric oxide (NO) production in mice serum and testis due to aluminum (Al) exposure has been shown in previous studies. The aim of this study was to further investigate the role of NO on aluminum-suppressed testosterone level in male CD-1 mice. Each animal in six groups, was given intraperitoneal injections of either saline, aluminum chloride (AlCl(3)), l-N(6)-(1-iminoethyl) lysine (NO synthase inhibitor, l-NIL), or Al chloride along with l-NIL for a period of 12 days. These groups were denoted as C (control, saline), AL (35mg Al/kg/day, saline), NIL240 (total 240mg l-NIL/kg, saline), ALNIL240 (35mg Al/kg/day, total 240mg l-NIL/kg), ALNIL60 (35mg Al/kg/day, total 60mg l-NIL/kg), and NIL60 (total 60mg l-NIL/kg, saline). Results indicated that serum/testicular aluminum levels increased significantly in aluminum-treated animals compared to the controls, whereas the values observed from groups ALNIL240 than AL/ALNIL60 were markedly lower. Aluminum administration significantly increased NO production and decreased both testicular adenosine 3',5'-cyclic monophosphate (cAMP) and testosterone levels. A lower level of NO and higher concentrations of cAMP and testosterone observed in the ALNIL240 group indicated that the protective effect of NO synthase blockage was significant, although incomplete. In addition, aluminum induction significantly elevated the testicular cholesterol, but the values were lower in the ALNIL240 group than the AL or the ALNIL60 group. Finally, it was suggested that aluminum compounds exerted a significant adverse effects on the steroidogenesis and cAMP, which aided in the transport of cholesterol to the inner mitochondrial membrane. Furthermore, nitric oxide synthase blockage prevented aluminum-induced reproductive toxicity.

Distribution patterns of trace metals and of lipid peroxidation in plasma and erythrocytes of rat exposed to aluminum.

Significant decreases of the hematocrit, hemoglobin, and plasma iron levels were observed in rats receiving daily intraperitoneal injections of aluminum at a dose of 27 mg Al/kg body wt for 3 wk, as compared to untreated controls. The activity of alkaline phosphatase was also significantly lower in the treated animals as a result of the accumulation of aluminum in the liver (p<0.05). Following aluminum administration, the plasma concentrations of aluminum and copper were also significantly increased, whereas the plasma zinc levels and oxidative stress measured through thiobarbituric acid reaction products showed nonsignificant differences between the two groups (p>0.05). The erythrocyte concentrations of aluminum, copper, zinc, and iron and of superoxide dismutase activity were found to be significantly higher in the study group as compared to controls. The treated animals also showed evidence of higher oxidative stress in comparison to controls. These results suggest that erythrocyte aluminum accumulation could result in abnormal trace element homeostasis and increasing oxidative stress, which might be a mechanism of aluminum-induced anemia.