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Sergio Crovella

Publications and source records attributed to Sergio Crovella.

34 records · Page 2Linked to original sources
PubMed2004

Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque.

BACKGROUND: The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis), a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. RESULTS: A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i) surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii) detection of approximately 42 and 84 kDa proteins by Western blot and (iii) the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. CONCLUSION: This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme.

ADP-ribosyl Cyclase↗
PubMed2004

A single-nucleotide polymorphism in the human beta-defensin 1 gene is associated with HIV-1 infection in Italian children.

In this study we show a significant correlation between a single-nucleotide polymorphism in the 5'-untranslated region of the DEFB1 gene, which probably regulates the gene expression of human beta defensin 1 (hBD-1) and the risk of HIV-1 infection in an Italian paediatric population (97 HIV-1 perinatally infected children), pointing to the importance of innate immunity in HIV-1 infection.

Child↗
PubMed2004

Evidence for duplication of the human defensin gene DEFB4 in chromosomal region 8p22-23 and implications for the analysis of SNP allele distribution.

Defensins constitute a primary mechanism in the innate immune system of humans and all mammals. Defensins are short, processed peptide molecules that are classified by structure into three groups: alpha-defensins, beta-defensins and theta-defensins. In humans, four beta-defensins have been described so far, corresponding to the products of the genes DEFB1 (hBD1, NM_005218), DEFB4 (hBD2, NM_004942.2), DEFB103 (hBD3, NM_018661), and DEFB104 (hBD4, NM_080389), respectively. All these genes have been mapped to chromosome 8p22-23. Much interest has been shown in genetic variation in the population at defensin loci to understand individual differences in disease susceptibility and severity. In this study, we have used an electronic search and then fluorescence in situ hybridization (FISH) on elongated chromosomes to demonstrate that the region containing the DEFB4 gene is duplicated on human chromosome 8p, making difficult the discovery of new SNPs in this gene and compromising the assessment of their allelic distribution in various ethnic populations for disease association studies.

Alleles↗
PubMed2004

Effects of positively selected sequence variations in human and Macaca fascicularis beta-defensins 2 on antimicrobial activity.

The evolution of orthologous genes coding for beta-defensin 2 (BD2) in primates has been subject to positive selection during the divergence of the platyrrhines from the catarrhines and of the Cercopithecidae from the Hylobatidae, great apes, and humans. Three peptides have been selected for a functional analysis of the effects of sequence variations on the direct antimicrobial activity: human BD2 (hBD2), Macaca fascicularis BD2 (mfaBD2), and a variant of the human peptide lacking Asp(4), (-D)hBD2, which is characteristic only of the human/great ape peptides. hBD2 and mfaBD2 showed a significant difference in specificity, the former being more active towards Escherichia coli and the later towards Staphylococcus aureus and Candida albicans. Asp(4) in the human peptide appears to be important, as (-D)hBD2 was less structured and had a markedly lower antimicrobial activity. The evolution of beta-defensin 2 in primates may thus have been driven, at least in part, by different environmental pressures so as to modulate antimicrobial activity.

Amino Acid Sequence↗
PubMed2003

A study of host defence peptide beta-defensin 3 in primates.

We have investigated the molecular evolution of the gene coding for beta-defensin 3 (DEFB103) in 17 primate species including humans. Unlike the DEFB4 genes (coding for beta-defensin 2) [Boniotto, Tossi, Del Pero, Sgubin, Antcheva, Santon and Masters (2003) Genes Immun. 4, 251-257], DEFB103 shows a marked degree of conservation in humans, Great Apes and New and Old World monkeys. Only the Hylobates concolor defensin hcBD3 showed an amino acid variation Arg17-->Trp17 that could have a functional implication, as it disrupts an intramolecular salt bridge with Glu27, which locally decreases the charge and may favour dimerization in the human congener hBD3. This is thought to involve the formation of an intermolecular salt bridge between Glu28 and Lys32 on another monomer [Schibli, Hunter, Aseyev, Starner, Wiencek, McCray, Tack and Vogel (2002) J. Biol. Chem. 277, 8279-8289]. To test the role of dimerization in mediating biological activity, we synthesized hBD3, hcBD3 and an artificial peptide in which the Lys26-Glu27-Glu28 stretch was replaced by the equivalent Phe-Thr-Lys stretch from human beta-defensin 1 and we characterized their structure and anti-microbial activity. Although the structuring and dimerization of these peptides were found to differ significantly, this did not appear to affect markedly the anti-microbial potency, the broad spectrum of activity or the insensitivity of the anti-microbial action to the salinity of the medium.

Amino Acid Sequence↗
PubMed2003

Chronic infantile neurological cutaneous articular syndrome: CD10 over-expression in neutrophils is a possible key to the pathogenesis of the disease.

UNLABELLED: Chronic, Infantile, Neurological, Cutaneous and Articular Syndrome (CINCA) or Neonatal/Infantile Onset Multisystem Inflammatory Disease (NOMID/IOMID) is a rare, multisystem inflammatory disease characterised by neonatal onset of urticarial symptoms, persistent rash, ocular inflammatory lesions, progressive articular and neurological involvement and associated with characteristic overgrowth of the ossification nucleus of the patella. The tissues involved are extensively infiltrated by inflammatory cells, mostly neutrophils. This paper describes the clinical features of three new cases as well as a study of activation markers in neutrophils and search for mutations of the CIAS1gene in these patients. Clinical records of three cases of CINCA are reported. For genetic analysis, exon 3 of the CIAS1gene was amplified and sequenced. Immunophenotype, oxidative burst and phagocytosis were analysed in neutrophils obtained from all the three CINCA patients as well as from eight juvenile idiopathic arthritis (JIA) patients and eight healthy controls. Functional assays in neutrophils were normal in all three patients with CINCA syndrome and did not differ from those of JIA patients and healthy controls. The surface density of CD10 was significantly higher on neutrophils from CINCA patients as compared to those of JIA and controls (P<0.0005). In one subject a new missense mutation in the CIAS1gene was identified. CONCLUSION: the hyper expression of the activation antigen CD10/NEP in neutrophils from these three cases of CINCA, as compared to JIA patients and healthy controls, irrespective of the presence of mutations in CIAS1, could be a marker of the inflammatory disorder typical of some patients with CINCA syndrome.

Adolescent↗
PubMed2003

Effects of maturation on RNA transcription and protein expression of four MRP genes in human placenta and in BeWo cells.

The placenta is a multifunctional organ that protects the fetus from toxic compounds and the MRPs contribute to this function. The expression of MRP1, MRP2, MRP3, and MRP5 was compared in human placental tissue and in BeWo cells by real-time RT-PCR analysis; protein expression was assessed by Western blot. MRP1 and MRP3 were the most abundantly expressed genes in placenta but only MRP1 was highly expressed in the BeWo cells. Expression of MRP1 increased 4-fold in the third as compared with first trimester placental samples, and increased 20-fold with polarization of BeWo cells. MRP2, MRP3, and MRP5 were weakly expressed both in placenta and BeWo cells. Protein expression followed mRNA quantification for MRP1 and MRP5 but not for MRP2 and MRP3. These data indicated that MRP1 and MRP5 increase with trophoblast maturation, suggesting a particular role for these proteins in the organ functional development.

Blotting, Western↗
PubMed2002

Extracellular NAD+: a novel autocrine/paracrine signal in osteoblast physiology.

Intercellular communication allows co-ordination of cell metabolism and sensitivity to extracellular stimuli. In bone cells, paracrine stimulation and cell-to-cell coupling through gap junctions induce the formation of complex intercellular networks, which favours the intercellular exchange of nutrients and second messengers, ultimately controlling the process of bone remodelling. The importance of local factors in bone remodelling is known since many years. Bone cells secrete and respond to a variety signals, among which include prostaglandins, cytokines, growth factors, and ATP. We here report evidence that extracellular NAD(+) is a novel extracellular signal stimulating osteoblast differentiation. We found that HOBIT human osteoblastic cells, which are known to express ADP-ribosyl cyclase/CD38 activity, respond to micromolar concentrations of extracellular NAD(+) with oscillatory increases of the cytosolic Ca(2+) concentration. The initial Ca(2+) response was followed by a time-dependent inhibition of cell growth, the appearance of an epithelial morphology, and by an increase of alkaline phosphatase and osteocalcin expression. Under resting condition HOBIT cells release NAD(+) in the extracellular medium and the release is significantly potentiated by mechanical stimulation. Taken together these results point to NAD(+) as a novel autocrine/paracrine factor involved in stimulation and maintenance of the osteoblast differentiated phenotype.

Adult↗
PubMed2002

Variant mannose-binding lectin alleles are associated with celiac disease.

In this study, we investigated the role of mannose-binding lectin (MBL) in celiac disease, by performing genotype analysis for the three point mutations in the first exon of the gene in 117 Italian celiac patients (characterized by flat biopsy and positive for anti-endomysium antibody and human transglutaminase antibodies) and 130 pan-ethnic healthy controls. The frequency of homozygous mutant 0/ 0 was significantly higher in the 117 Italian celiac patients (0.13) than in the 130 pan-ethnic healthy controls (0.05; P=0.0405). An increased frequency of homozygous 0/0 allele was found among patients with celiac disease compared with controls. These results suggest an involvement of MBL in the pathophysiology of celiac disease.

Alleles↗
PubMed2002

Prognostic value of the stromal cell-derived factor 1 3'A mutation in pediatric human immunodeficiency virus type 1 infection.

A mutation of the stromal cell-derived factor 1 gene (SDF-1 3'A) was shown to protect adults exposed to human immunodeficiency virus type 1 (HIV-1) from infection and to affect HIV disease progression in adults. The presence of this mutation in HIV-1-infected Kenyan children did not predict mother-to-child virus transmission. The SDF-1 3'A polymorphism was studied in 256 HIV-1-infected, 118 HIV-1-exposed but uninfected, and 170 unexposed and uninfected children of Italian origin, and the frequency of SDF-1 3'A heterozygosity and homozygosity in each of the 3 groups was similar. Of the 256 HIV-1-infected children, 194 were regularly followed up and were assigned to groups according to disease progression. The frequency of the SDF-1 3'A allele was substantially lower among children with long-term nonprogression than among children with rapid (P =.0329) or delayed (P =.0375) progression. We show that the presence of the SDF-1 3'A gene correlates with accelerated disease progression in HIV-1-infected children born to seropositive mothers but does not protect against mother-to-child HIV-1 transmission.

Adolescent↗
PubMed2002

Beta-defensin 1 gene variability among non-human primates.

Defensins are a recently described family of peptides that play an important role in innate immunity. Recent studies have shown that defensins exhibit a broad spectrum of antimicrobial activities against bacteria and fungi. Three families have been identified so far in mammals, alpha-defensins, beta-defensins and theta-defensins, presumably derived from a common ancestral defensin. A long-term study on the evolution of these multigene families among primates has been undertaken to investigate: (1) the degree of interspecific differentiation; (2) the genetic mechanisms responsible for the variability of these molecules; and (3) the possible role of different environmental factors in their evolution. Nucleotide sequences have been obtained from great and lesser apes, several African and Asian catarrhine monkeys and one New World monkey. A comparison of rates of synonymous and nonsynonymous (amino-acid changing) nucleotide substitution indicates that the primate beta-defensin 1 gene evolved under a pattern of random nucleotide substitution as predicted by the neutral theory of molecular evolution. These results are not consistent with the hypothesis that the primate beta-defensin 1 gene has diversified in response to changes in the microbial species to which a given host is exposed. Analyses of interpecific variability have yielded some insights about the pattern of molecular evolution of the gene among primates. Humans and great apes present high levels of sequence similarity, differing in only one amino acid residue in the mature peptide. Compared with these taxa, hylobatids and cercopithecids exhibit 3-4 amino acid substitutions, some of which increase the net charge of the active molecule.

Animals↗
PubMed2002

Localization of a new highly repeated DNA sequence of Lemur cafta (Lemuridae, Strepsirhini).

We have isolated and cloned an 800-bp highly repeated DNA (HRDNA) sequence from Lemur catta (LCA) and described its localization on LCA chromosomes. Lemur catta HRDNA sequences were localized by performing FISH experiments on standard and elongated metaphasic chromosomes using an LCA HRDNA probe (LCASAT). A complex hybridization pattern was detected. A strong pericentromeric hybridization signal was observed on most LCA chromosomes. Chromosomes 7 and 13 were lit in pericentromeric regions, as well as in the interspersed heterochromatin. Chromosomes 1, 3, 4, 17, 19, X, and microchromosomes (20, 25, 26, and 27) showed no signals in the pericentromeric region, but chromosomes 3 and 4 showed a positive hybridization in heterochromatic regions. The 800-bp L catta HRDNA was species specific. We performed FISH experiments with the LCASAT probe on Eulemur macaco macaco (EMA) and Eulemur fulvus fulvus (EFU) metaphases and no positive signal of hybridization was detected. These findings were also confirmed by Southern blot analysis and PCR.

Animals↗
PubMed2001

AGXT gene mutations and their influence on clinical heterogeneity of type 1 primary hyperoxaluria.

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder that is caused by a deficiency of alanine: glyoxylate aminotransferase (AGT), which is encoded by a single copy gene (AGXT). Molecular diagnosis was used in conjunction with clinical, biochemical, and enzymological data to evaluate genotype-phenotype correlation. Twenty-three unrelated, Italian PH1 patients were studied, 20 of which were grouped according to severe form of PH1 (group A), adult form (group B), and mild to moderate decrease in renal function (group C). All 23 patients were analyzed by using the single-strand conformation polymorphism technique followed by the sequencing of the 11 AGXT exons. Relevant chemistries, including plasma, urine and dialyzate oxalate and glycolate assays, liver AGT activity, and pyridoxine responsiveness, were performed. Both mutant alleles were found in 21 out of 23 patients, and 13 different mutations were recognized in exons 1, 2, 4, and 10. Normalized AGT activity was lower in the severe form than in the adult form (P < 0.05). Double heterozygous patients presented a lower age at the onset of the disease (P = 0.025), and they were more frequent in group A (75%) than in the group B (14%; P = 0.0406). The T444C mutation was more frequent in the severe form (P < 0.05), and the opposite was observed for G630A (P < 0.05). G630A mutation homozygotes had a higher AGT residual activity (P = 0.00001). This study confirms the allelic heterogeneity of the AGXT, which could to some extent be responsible for the phenotypic heterogeneity in PH1.

Adolescent↗