The thrombin epitope recognizing thrombomodulin is a highly cooperative hot spot in exosite I

The functional epitope of thrombin recognizing thrombomodulin was mapped using Ala-scanning mutagenesis of 54 residues located around the active site, the Na(+) binding loop, the 186-loop, the autolysis loop, exosite I, and exosite II. The epitope for thrombomodulin binding is shaped as a hot spot in exosite I, centered around the buried ion quartet formed by Arg(67), Lys(70), Glu(77), and Glu(80), and capped by the hydrophobic residues Tyr(76) and Ile(82). The hot spot is a much smaller subset of the structural epitope for thrombomodulin binding recently documented by x-ray crystallography. Interestingly, the contribution of each residue of the epitope to the binding free energy shows no correlation with the change in its accessible surface area upon formation of the thrombin-thrombomodulin complex. Furthermore, residues of the epitope are strongly coupled in the recognition of thrombomodulin, as seen for the interaction of human growth hormone and insulin with their receptors. Finally, the Ala substitution of two negatively charged residues in exosite II, Asp(100) and Asp(178), is found unexpectedly to significantly increase thrombomodulin binding.     

In vitro chloroquine resistance modulation study on fresh isolates of Brazilian Plasmodium falciparum: intrinsic antimalarial activity of phenothiazine drugs

Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination.     

Niemann-Pick type C disease: mutations of NPC1 gene and evidence of abnormal expression of some mutant alleles in fibroblasts

We analyzed Niemann-Pick type C disease 1 (NPC1) gene in 12 patients with Niemann-Pick type C disease by sequencing both cDNA obtained from fibroblasts and genomic DNA. All the patients were compound heterozygotes. We found 15 mutations, eight of which previously unreported. The comparison of cDNA and genomic DNA revealed discrepancies in some subjects. In two unrelated patients carrying the same mutations (P474L and nt 2972del2) only one mutant allele (P474L), was expressed in fibroblasts. The mRNA corresponding to the other allele was not detected even in cells incubated with cycloheximide. The promoter variants (-1026T/G and -1186T/C or -238 C/G), found to be in linkage with 2972del2 allele do not explain the lack of expression of this allele, as they were also found in control subjects. In another patient, (N1156S/Q922X) the N1156S allele was expressed in fibroblasts while the expression of the other allele was hardly detectable. In a fourth patient cDNA analysis revealed a point mutation in exon 20 (P1007A) and a 56 nt deletion in exon 22 leading to a frameshift and a premature stop codon. The first mutation was confirmed in genomic DNA; the second turned out to be a T-->G transversion in exon 22, predicted to cause a missense mutation (V1141G). In fact, this transversion generates a donor splice site in exon 22, which causes an abnormal pre-mRNA splicing leading to a partial deletion of this exon. In some NPC patients, therefore, the comparison between cDNA and genomic DNA may reveal an unexpected expression of some mutant alleles of NPC1 gene.     

Two distinctly HLA-associated contiguous linear epitopes uniquely expressed within the islet antigen 2 molecule are major autoantibody epitopes of the diabetes-specific tyrosine phosphatase-like protein autoantigens

The related tyrosine phosphatase-like proteins islet Ag (IA)-2 and IA-2beta are autoantigens of type 1 diabetes in humans. Autoantibodies are predominantly against IA-2, and IA-2-specific epitopes are major autoantibody targets. We used the close homology of IA-2 and IA-2beta to design chimeras and mutants to identify humoral IA-2-specific epitopes. Two major IA-2 epitopes that are absent from the related autoantigens IA-2beta and IA-2Delta 13 splice variant ICA512.bdc were found contiguous to each other within IA-2 juxtamembrane amino acids 611-620 (epitope JM1) and 621-630 (epitope JM2). JM1 and JM2 are recognized by sera from 67% of patients with IA-2 Abs, and relatives of patients with type 1 diabetes having Abs to either JM epitope had a >50% risk for developing type 1 diabetes within 6 years, even in the absence of diabetes-associated HLA genotypes. Remarkably, the presence of Abs to one of these two epitopes was mutually exclusive of the other; JM2 Abs and not JM1 Abs were found in relatives with HLA DR3/4, DR4/13, or DR1/4 genotypes; and the binding of autoantibodies to the JM2 epitope, but not the JM1 epitope, markedly affected proteolysis of IA-2. This is a unique demonstration of HLA-associated B cell responses to epitopes within a single autoantigen in humans and is consistent with modification of Ag processing by specific Ab-influencing peptide presentation by HLA molecules.     

Extracellular adenine nucleotides regulate Na+/H+ exchanger NHE3 activity in A6-NHE3 transfectants by a cAMP/PKA-dependent mechanism

As potential autocrine or paracrine factors, extracellular nucleotides are known to be important regulators of renal ion transporters by activating cell surface receptors and intracellular signaling pathways. We investigated the influence of extracellular adenine nucleotides on Na+/H+ exchanger isoform 3 (NHE3) activity in A6-NHE3 cells. This is a polarized cell line obtained by stable transfection of A6 cells with the cDNA encoding the rat isoform of NHE3, which is expressed on the apical membrane. Basolateral addition of the P2Y(1) agonist, 2-MeSADP, induced an inhibition of NHE3 activity, which was prevented by preincubation with selective P2Y(1) antagonists, MRS 2179 (N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and MRS 2286 (2-[2-(2-chloro-6-methylamino-purin-9-yl)-ethyl]-propane-1,3-bisoxy(diammoniumphosphate)). NHE3 activity was also significantly inhibited by ATP and ATP-gamma-S but not by UTP. 2-MeSADP induced a P2Y(1) antagonist-sensitive increase in both [Ca2+]i and cAMP production. Pre-incubation with a PKC inhibitor, Calphostin C, or the calcium chelator BAPTA-AM, had no effect on the 2-MeSADP-dependent inhibition of NHE3 activity, whereas this inhibition was reversed by either incubation with the PKA inhibitor H89 or by mutation of two PKA target serines (S552 and S605) on NHE3. Pre-incubation of the A6-NHE3 cells with the synthetic peptide, Ht31, which prevents the binding between AKAPs and the regulatory PKA subunits RII, also prevented the 2-MeSADP-induced inhibition of NHE3. We conclude that only the cAMP/PKA pathway is involved in the inhibition of NHE3 activity.     

Crystal structure and anion binding in the prokaryotic hydrogenase maturation factor HypF acylphosphatase-like domain

[NiFe]-hydrogenases require a set of complementary and regulatory proteins for correct folding and maturation processes. One of the essential regulatory proteins, HypF (82kDa) contains a N-terminal acylphosphatase (ACT)-like domain, a sequence motif shared with enzymes catalyzing O-carbamoylation, and two zinc finger motifs similar to those found in the DnaJ chaperone. The HypF acylphosphatase domain is thought to support the conversion of carbamoylphosphate into CO and CN(-), promoting coordination of these ligands to the hydrogenase metal cluster. It has been shown recently that the HypF N-terminal domain can aggregate in vitro to yield fibrils matching those formed by proteins linked to amyloid diseases. The 1.27A resolution HypF acylphosphatase domain crystal structure (residues 1-91; R-factor 13.1%) shows a domain fold of betaalphabetabetaalphabeta topology, as observed in mammalian acylphosphatases specifically catalyzing the hydrolysis of the carboxyl-phosphate bonds in acylphosphates. The HypF N-terminal domain can be assigned to the ferredoxin structural superfamily, to which RNA-binding domains of small nuclear ribonucleoproteins and some metallochaperone proteins belong. Additionally, the HypF N-terminal domain displays an intriguing structural relationship to the recently discovered ACT domains. The structures of different HypF acylphosphatase domain complexes show a phosphate binding cradle comparable to the P-loop observed in unrelated phosphatase families. On the basis of the catalytic mechanism proposed for acylphosphatases, whereby residues Arg23 and Asn41 would support substrate orientation and the nucleophilic attack of a water molecule on the phosphate group, fine structural features of the HypF N-terminal domain putative active site region may account for the lack of acylphosphatase activity observed for the expressed domain. The crystallographic analyses here reported were undertaken to shed light on the molecular bases of inactivity, folding, misfolding and aggregation of the HypF N-terminal acylphosphatase domain.     

Diverging effects of human recombinant anti-hepatitis C virus (HCV) antibody fragments derived from a single patient on the infectivity of a vesicular stomatitis virus/HCV pseudotype

Hepatitis C virus (HCV) is the major causative agent of blood-borne non-A, non-B hepatitis. Although a strong humoral response is detectable within a few weeks of primary infection and during viral persistence, the role played by antibodies against HCV envelope glycoproteins in controlling viral replication is still unclear. We describe how human monoclonal anti-HCV E2 antibody fragments isolated from a chronically HCV-infected patient differ sharply in their abilities to neutralize infection of HepG2 cells by a vesicular stomatitis virus pseudotype bearing HCV envelope glycoproteins. Two clones were able to neutralize the pseudotype virus at a concentration of 10 micro g/ml, while three other clones completely lacked this activity. These data can explain the lack of protection and the possibility of reinfection that occur even in the presence of a strong antiviral antibody response.     

Identification of 8-iso-prostaglandin F(2alpha) in rat brain neuronal endings: a possible marker of membrane phospholipid peroxidation

Isoprostanes are a family of prostaglandin (PG) F and E isomers generated by free-radical attack from membrane bound arachidonic acid. We measured detectable levels of 8-iso-PGF(2alpha) in the perfusates of synaptosomes obtained from different areas of the rat brain cortex. A small but significant release of this isoprostane was found under basal conditions from all the areas explored; being lower in the dorsal cortex in respect to the frontal, parietal and occipital areas. Exposure of synaptosomes to a phospholipase A(2) activator, i.e. calcium-ionophore A23187, an oxidant agent, such as hydrogen peroxide or amyloid beta-peptide did not modify 8-iso-PGF(2alpha) release when these stimuli were applied separately. However, either hydrogen peroxide or amyloid beta-peptide increased 8-iso-PGF(2alpha) release in a dose-dependent manner, when given in the presence of the calcium-ionophore A23187. Synaptosome treatment with a non-selective cyclooxygenase inhibitor (fenoprofen) did not modify 8-iso-PGF(2alpha) release in any way, but treatment with a water soluble antioxidant (Trolox C) completely suppressed isoprostane release under basal conditions, as well as after the oxidant injury induced either by hydrogen peroxide or amyloid beta-peptide. We conclude that, in neuronal endings, 8-iso-PGF(2alpha) is generated under basal conditions and its formation may be increased in a dose-dependent fashion by oxidant stimuli through a cyclooxygenase-independent mechanism involving free radical-catalyzed oxidation of arachidonic acid on membrane phospholipids.     

Causal association between human papilloma virus infection and head and neck and esophageal squamous cell carcinoma

HPVs commonly cause proliferative lesions of squamous epithelium, and infection with certain HPV types carries a high risk of malignant transformation. We used molecular techniques to detect and type HPV in papillomas and carcinomas in the oral cavity and esophagus. DNA was extracted from 150 fresh or paraffin embedded biopsy specimens, and analyzed for HPV by PCR with 15 sets of consensus primers directed to conserved regions of L1 gene, three sets of HPV16E6 primers (specific for the HPV 16 prototype and L83V variant), and sets of primers specific for the E6 gene of other mucosa type HPVs including HPV 6, 11, 16, 18, 52, 58, 66 and 73. Overall, HPV sequences were detected in 61 of 150 specimens. HPV DNA sequences were detected in 16/32 specimens in the oropharyngeal region, in 13/36 specimens in larynx and 32/82 specimens in esophagus. Papillomas contained only the episomal form of HPV 16. In the esophagus, the most common type was HPV 73. In all specimens examined, HPV 6/11 (4/150), HPV 16 (23/150), HPV 35 (1/150), HPV 45 (1/150), HPV 54 (1/150), HPV 58 (1/150), HPV 61 (1/150), HPV 66 (1/150), HPV 68 (2/150), HPV 70 (3/150), HPV 72 (1/150), HPV 73 (16/150), double HPV infection (2/150), and unidentified HPV type (4/150) was detected. Interestingly, HPV was found in all verrucous carcinomas and in 18/22 basaloid squamous cell carcinomas. HPV16E6 T350G mutant were observed only in two of eight carcinomas. Using correspondence analysis, a segregation of specific virus types in specific clinico-pathologic lesions (verrucous carcinoma and basaloid squamous cell carcinoma) was proved. It was shown that the relative rates of the HPV positive tumors were significantly higher in women than in men. The synergic action of mucosal irritation and HPV infection may be necessary for the development of the papillomas and the specific types of carcinomas in the oral cavity and in the esophagus.