Spectroscopic analysis of the stability of bothrops myotoxic phospholipases A2 to guanidine and urea denaturation

Spectrophotometric profiles representing the unfolding induced by guanidine on Bothrops moojeni myotoxins-I (MjTX-I) and II (MjTX-II), Bothrops jararacussu bothropstoxin-I (BthTX-I) and Bothrops pirajai piratoxin-I (PrTX-I) were obtained and compared with those obtained with bovine ribonuclease A (RNAse) and trypsin. The molar (epsilon(1M)) and percent (epsilon(1%)) extinction coefficients were determined for the four myotoxins as well as for RNAse and trypsin as reference parameters. These coefficients were then used throughout this work. The changes in free energy (deltaGD(H)(2)(O)) corresponding to zero guanidine concentration and the guanidine concentrations (D(1/2)) able to convert 50% of the molecules from the native to the unfolded state were determined. The values of deltaGD (H)(2)(O) ranged from 4.42 (BthTX-I) to 8.02 (MjTX-I) kcal/mole, compared with 6.47 and 6.88 kcal/mole for trypsin and RNAse, respectively. The values for deltaGD(H)(2)(O) and D1/2 showed that BthTX-I is the least stable among the four myotoxins assayed, with a D1/2 close to that of RNAse, while MjTX-II is conformationally the most stable. Monitoring of the unfolding of RNAse and PrTX-I by a 0 to 6 M urea gradient PAGE revealed transitions from the native (N) to the unfolded (U) state with deltaG(N-U)of 0.22 and 0.41 kcal/mole, respectively. Sigmoidal curves showed well-defined two-stage transitions for both proteins.     

Occult HBV infection and suppression of HCV replication in the early phase of combination therapy for chronic hepatitis C

BACKGROUND: Occult HBV infection in subjects with chronic hepatitis C is related to more severe disease outcome. It has been suggested that it might reduce sensitivity to antiviral treatment. AIMS: To assess in HBsAg negative subjects with chronic hepatitis C any effect of the presence of HBV genomes in the liver on the early kinetics of HCV-RNA under PEG-IFN plus ribavirin. PATIENTS AND METHODS: Twenty-two anti-HCV and HCV-RNA positive subjects, with biopsy-proven chronic hepatitis C (M/F 15/7; 50 +/- 8.6 years, 16 genotype 1b) were given PEG-IFN alpha 2b 1.0 microg qw plus ribavirin (800 to 1,200 mg daily according to body weight) for an intended 52 week period. Early virological response was assessed over the first 4 weeks of therapy by quantifying HCV-RNA. Occult HBV infection was assessed by testing for HBV-DNA in the liver before therapy. RESULTS: HBV genomes were found in the liver of 7 of 22 (31.4%) patients, unrelated to anti-HBc status. Kinetics of HCV-RNA during the first 4 weeks of antiviral treatment was unaffected by occult HBV infection, both in terms of absolute reduction of viral load and of number of cases with a reduction of > or = 2 log10 on treatment. CONCLUSIONS: Occult HBV infection does not affect the early phase of response to combination therapy. Further follow-up of patients into the maintenance phase of antiviral treatment and after stopping it will clarify if and when occult HBV has a role in reducing sustained virological response.     

Gene dosage of the spermidine/spermine N(1)-acetyltransferase ( SSAT) gene with putrescine accumulation in a patient with a Xp21.1p22.12 duplication and keratosis follicularis spinulosa decalvans (KFSD)

Keratosis follicularis spinulosa decalvans (KFSD) or Siemens-1 syndrome is a rare X-linked disease of unknown etiology affecting the skin and the eye. Although most affected families are compatible with X-linked inheritance, KFSD appears to be clinically and genetically heterogeneous. So far, the gene has been mapped to Xp22.13p22.2 in two extended KFSD families. Analysis of additional recombination events in the first Dutch pedigree located the gene to an interval covering approximately 1 Mb between markers DXS7163 and DXS7593/DXS7105, whereas haplotype reconstruction in the second German family positioned the gene outside the previously identified region, proximal to marker DXS274. We report here the molecular characterization of an Xp21.1p22.12 duplication present in a patient affected with dosage-sensitive sex reversal (DSS) and KFSD. The duplicated region includes both the DAX1 gene (previously demonstrated to be responsible for DSS) and the KFSD interval, in which the gene encoding spermidine/spermine N(1)-acetyltransferase ( SSAT) is located. This enzyme catalyzes the N(1)-acetylation of spermidine and spermine and, by the successive activity of polyamine oxidase, the spermine can be converted to spermidine and the spermidine to putrescine. Overexpression of the SSAT enzyme in a mouse model results in putrescine accumulation and a phenotype with skin and hair abnormalities reminiscent of human KFSD. Analysis of polyamine metabolism in the cells of the patient indicated that the levels of metabolites such as putrescine, spermidine and spermine were consistent with the overexpression of the SSAT gene as in the murine model. Thus, we propose that overexpression of SSAT and the consequent putrescine accumulation are involved in the KFSD phenotype, at least in our propositus.     

Crystal structures of BnSP-7 and BnSP-6, two Lys49-phospholipases A(2): quaternary structure and inhibition mechanism insights

Phospholipases A(2) are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. A class of PLA(2)-like proteins has been described which despite PLA(2) activity on artificial substrate, due to a D49K mutation, is still highly myonecrotic. This work reports the X-ray structure determination of two Lys49-PLA(2)s from Bothrops neuwiedi pauloensis (BnSP-7 and BnSP-6) and, for the first time, the comparison of eight dimeric Lys49-PLA(2)s. This comparison reveals that there are not just two ("open" and "closed") but at least six different conformations. The binding of fatty acid observed in three recent Lys49-PLA(2) structures seems to be independent of their quaternary conformation. Cys29 polarization by Lys122 is not significant for BnSP-7 and BnSP-6 or other structures not bound by fatty acids. These structures may be in an "active" state when nothing is bound to them and the Lys122/Cys29 interactions are weak or absent.     

CENP-G in neocentromeres and inactive centromeres

CENP-G is a novel constitutive centromere-specific protein localized to the kinetochore inner plate and subjacent region. It has been identified as associating specifically with the alpha-1 subfamily of alpha-satellite DNA. In the present work, the localization of CENP-G was compared with that of other CENPs by immunofluorescence and fluorescence in situ hybridization. Studies were carried out on four abnormal human centromeres: two neocentromeres and two inactive centromeres. CENP-G was detected in one of the two inactive centromeres but not in the other that shows a partial deletion of the alphoid DNA. Interestingly, CENP-G is also present in neocentromeres, which lack alphoid DNA sequences, and in the human Y chromosome, which lacks the alpha-1 type of satellite DNA. These data provide further evidence that CENP-G may be an essential factor in centromeric function and that in centromeres lacking the alpha-1 subfamily of alphoid DNA, other DNA sequences are able to bind CENP-G.     

Myotoxic phospholipases A(2) in bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu

Venoms from eight Bothrops spp. were fractionated by ion-exchange chromatography on CM-Sepharose at pH 8.0 for the purification of myotoxins. Chromatographic profiles showed differences regarding myotoxic components among these venoms. B. alternatus, B. atrox and B. jararaca venoms did not show the major basic myotoxic fractions identified in the other venoms. Polyacrylamide gel electrophoresis for basic proteins also showed distinct patterns for these toxins. In vivo, all the isolated myotoxins induced release of creatine kinase due to necrosis of muscle fibers, accompanied by polymorphonuclear cell infiltration, and edema in the mouse paw. In addition, the toxins showed cytotoxic and liposome-disrupting activities in vitro. B. jararacussu bothropstoxins-I (BthTX-I) and II (BthTX-II) were submitted to chemical modifications of: His, by 4-bromophenacyl bromide (BPB) or photooxidation by Rose Bengal (RB); Tyr, by 2-nitrobenzenesulphonyl fluoride (NBSF); and Trp, by o-nitrophenylsulphenyl chloride (NPSC). The myotoxic and cytotoxic activities of BthTX-I, a Lys49 PLA(2) homologue, after modification by BPB, RB, NBSF and NPSC, were reduced to 50%, 20%, 75%, 65% and 13%, 0.5%, 76%, 58%, respectively. However, the edema-inducing and liposome-disrupting activities were not significantly reduced by the above modifications. BPB-treated BthTX-II, an Asp49 PLA(2) homologue, lost most of its catalytic, indirect hemolytic, anticoagulant, myotoxic and cytotoxic activities. The edema-inducing and liposome-disrupting activities were reduced to 50% and 80%, respectively. Lethality caused by BthTX-I and -II was strongly reduced after treatment with BPB or RB, but only partially with NBSF or NPSC. BthTX-I and -II, both native or modified, migrated similarly in a charge-shift electrophoresis. Antibodies raised against BthTX-I or -II, B. asper Basp-II and the C-terminal 115-129 peptide from Basp-II did not show significant differences in their cross-reactivity with the modified toxins, except with RB photooxidized toxins.     

Splicing factors induce cystic fibrosis transmembrane regulator exon 9 skipping through a nonevolutionary conserved intronic element

In monosymptomatic forms of cystic fibrosis such as congenital bilateral absence of vas deferens, variations in the TG(m) and T(n) polymorphic repeats at the 3' end of intron 8 of the cystic fibrosis transmembrane regulator (CFTR) gene are associated with the alternative splicing of exon 9, which results in a nonfunctional CFTR protein. Using a minigene model system, we have previously shown a direct relationship between the TG(m)T(n) polymorphism and exon 9 splicing. We have now evaluated the role of splicing factors in the regulation of the alternative splicing of this exon. Serine-arginine-rich proteins and the heterogeneous nuclear ribonucleoprotein A1 induced exon skipping in the human gene but not in its mouse counterpart. The effect of these proteins on exon 9 exclusion was strictly dependent on the composition of the TG(m) and T(n) polymorphic repeats. The comparative and functional analysis of the human and mouse CFTR genes showed that a region of about 150 nucleotides, present only in the human intron 9, mediates the exon 9 splicing inhibition in association with exonic regulatory elements. This region, defined as the CFTR exon 9 intronic splicing silencer, is a target for serine-arginine-rich protein interactions. Thus, the nonevolutionary conserved CFTR exon 9 alternative splicing is modulated by the TG(m) and T(n) polymorphism at the 3' splice region, enhancer and silencer exonic elements, and the intronic splicing silencer in the proximal 5' intronic region. Tissue levels and individual variability of splicing factors would determine the penetrance of the TG(m)T(n) locus in monosymptomatic forms of cystic fibrosis.     

Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae) on actions of snake and bee venoms and on activity of phospholipases A2

The crude aqueous extract from the leaves of Casearia sylvestris, a plant found in Brazilian open pastures, was assayed for its ability to inhibit phospholipase A2 (PLA2) activity and some biological activities of bee and several snake venoms, and of a number of isolated PLA2s. The extract induced partial inhibition of the PLA2 activity of venoms containing class I, II and III PLA2s. When tested against the purified toxins, it showed the highest efficacy against class II PLA2s from viperid venoms, being relatively ineffective against the class I PLA2 pseudexin. In addition, C. sylvestris extract significantly inhibited the myotoxic activity of four Bothrops crude venoms and nine purified myotoxic PLA2s, including Lys-49 and Asp-49 variants. The extract was able to inhibit the anticoagulant activity of several isolated PLA2s, with the exception of pseudexin. Moreover, it partially reduced the edema-inducing activity of B. moojeni and B. jararacussu venoms, as well as of myotoxins MjTX-II and BthTX-I. The extract also prolonged the survival time of mice injected with lethal doses of several snake venoms and neutralized the lethal effect induced by several purified PLA2 myotoxins. It is concluded that C. sylvestris constitutes a rich source of PLA2 inhibitors.