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Biomedical subjects

A Villa

Publications and source records attributed to A Villa.

At least 19 recordsLinked to original sources
PubMed1996

Monocyte function in a severe combined immunodeficient patient with a donor splice site mutation in the Jak3 gene.

Janus kinase-3 (Jak3) is a nonreceptor tyrosine kinase functionally coupled to cytokine receptors which share a "common" gamma chain (gamma c). Mutations in gamma c and Jak3 genes have been identified in X-linked and autosomal severe combined immuno deficiency (SCID), respectively. Jak3 is expressed and activated in myelomonocytic cells. The present study was designed to define the structural alteration responsible for lack of Jak3 in a patient with autosomal SCID and to characterize monocyte function in the absence of this signal transduction element, as well as to establish the whole exon-intron structure. Polymerase chain reaction analysis, performed with primers designed on exon sequences, identified 20 exons spanning approximately 15 kb. These primers, or others designed on the flanking sequences provided in the present report, can be used to amplify the whole gene, allowing the definition of the molecular defects in all cases, including prenatal diagnosis, in which transcript analysis is not possible. On this basis, the deletion transcript found at the homozygous state in patient CM, with both his consanguineous parents being heterozygous for the deletion, was associated with mutation (T to C) of a splice donor site of intron 16 that was also detected in his mother's DNA. Monocytes from Jak3-SCID showed normal cytokine production in response to interleukin-4 (IL-4) (release of IL-1 receptor antagonist) and IL-2 (release of tumor necrosis factor-alpha and IL-8). Lipopolysaccharide-induced cytokine production was also normal and was blocked by IL-4 in Jak3- SCID monocytes. Interferon-gamma induced augmented expression of major histocompatibility class II in Jak3-SCID monocytes. These data indicate that Jak3, expressed and activated in myelomonocytic cells, is dispensable for monocyte differentiation and responsiveness to cytokines that interact with gamma c receptors as well as to other regulatory signals.

Base Sequence↗
PubMed1996

The ZNF75 zinc finger gene subfamily: isolation and mapping of the four members in humans and great apes.

We have previously reported (Villa et al. (1993), Genomics 18: 223) the characterization of the human ZNF75 gene located on Xq26, which has only limited homology (less than 65%) to other ZF genes in the databases. Here, we describe three human zinc finger genes with 86 to 95% homology to ZNF75 at the nucleotide level, which represent all the members of the human ZNF75 subfamily. One of these, ZNF75B, is a pseudogene mapped to chromosome 12q13. The other two, ZNF75A and ZNF75C, maintain an ORF in the sequenced region, and at least the latter is expressed in the U937 cell line. They were mapped to chromosomes 16 and 11, respectively. All these genes are conserved in chimpanzees, gorillas, and orangutans. The ZNF75B homologue is a pseudogene in all three great apes, and in chimpanzee it is located on chromosome 10 (phylogenetic XII), at p13 (corresponding to the human 12q13). The chimpanzee homologue of ZNF75 is also located on the Xq26 chromosome, in the same region, as detected by in situ hybridization. As expected, nucleotide changes were clearly more abundant between human and orangutan than between human and chimpanzee or gorilla homologues. Members of the same class were more similar to each other than to the other homologues within the same species. This suggests that the duplication and/or retrotranscription events occurred in a common ancestor long before great ape speciation. This, together with the existence of at least two genes in cows and horses, suggests a relatively high conservation of this gene family.

Amino Acid Sequence↗
PubMed1996

A mutant cytochrome b5 with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane.

Many resident membrane proteins of the endoplasmic reticulum (ER) do not have known retrieval sequences. Among these are the so-called tail-anchored proteins, which are bound to membranes by a hydrophobic tail close to the C terminus and have most of their sequence as a cytosolically exposed N-terminal domain. Because ER tail-anchored proteins generally have short (< or = 17 residues) hydrophobic domains, we tested whether this feature is important for localization, using cytochrome b5 as a model. The hydrophobic domain of cytochrome b5 was lengthened by insertion of five amino acids (ILAAV), and the localization of the mutant was analyzed by immunofluorescence in transiently transfected mammalian cells. While the wild-type cytochrome was localized to the ER, the mutant was relocated to the surface. This relocation was not due to the specific sequence introduced, as demonstrated by the ER localization of a second mutant, in which the original length of the membrane anchor was restored, while maintaining the inserted ILAAV sequence. Experiments with brefeldin A and with cycloheximide demonstrated that the extended anchor mutant reached the plasma membrane by transport along the secretory pathway. We conclude that the short membrane anchor of cytochrome b5 is important for its ER residency, and we discuss the relevance of this finding for other ER tail-anchored proteins.

Amino Acid Sequence↗
PubMed1996

Survival of women with advanced ovarian cancer and complete pathologic response at second-look laparotomy.

BACKGROUND: The purpose of the study was to analyze the determinants of long term survival in women with advanced ovarian cancer and negative second-look laparotomy. METHODS: A series of 140 advanced (Stage III-IV) ovarian cancer patients (median age, 54 years; range, 22-74 years) with negative second-look laparotomy after primary surgery and chemotherapy is included in the analysis. At first diagnosis, all patients were treated with radical or debulking surgery. After primary surgery, the patients were treated with a chemotherapy regimen based on cisplatin or carboplatin alone or in combination with other drugs. All second-look laparotomies were performed 6-8 months after first surgery. RESULTS: The overall survival rates were 76% at 3 years, 66% at 5 years, and 51% at 8 years. The corresponding rates for disease free survival were 57, 50, and 43%, respectively. Survival rates were better for women with a residual tumor 1 cm or less after primary surgery. The 5-year probability of survival was 78% in this group, compared with 55% in women with a residual tumor more than 1 cm (log rank test, P < 0.05). Survival rates for women with tumor Grade 3 tended to be worse than Grades 1-2, but the difference was only of borderline statistical significance. No relationship emerged between survival and age, histotype, and presence of ascites at diagnosis. Women with a residual tumor 1 cm or less and positive lymph nodes had a 66% 5-year probability of survival, compared with 85% for women with a residual tumor 1 cm or less and negative lymph nodes. This difference was significant (log rank test, P = 0.05). The 5-year survival probabilities were 47 and 58%, respectively, in women with a residual tumor more than 1 cm and positive or negative lymph nodes. CONCLUSIONS: This analysis shows a favorable long term survival rate for women with advanced ovarian cancer and complete pathologic response after debulking surgery and postoperative chemotherapy. It further suggests that lymph nodal status is a prognostic factor for women with minimal residual tumor after surgery.

Adult↗
PubMed1996

Uterine myomas and smoking. Results from an Italian study.

OBJECTIVE: To evaluate the relationship between smoking and uterine myomas requiring surgery. STUDY DESIGN: We conducted a case-control study in Milan between 1986 and 1992. Cases were 476 patients under 55 years of age with histologically confirmed myomas. Controls were 1,283 women admitted to the hospital for a spectrum of acute, other-than-gynecologic, hormonal or neoplastic conditions (30% trauma, 25% nontraumatic orthopedic conditions, 25% surgical, 20% other miscellaneous). RESULTS: Cases were less frequently current smokers (22%) than controls (32%). In comparison with never smokers, the multivariate relative risk (RR) for myomas was 0.5 (95% confidence interval [CI], 0.4-0.7) in smokers. Ex-smokers were 10% of cases versus 7% of controls (RR 1.2, 95% CI 0.9-1.8). No clear trend in risk was observed with the number of cigarettes smoked per day or duration of smoking and risk of fibroids. The estimated RRs were largely consistent when separate analyses were performed in strata of age and other selected covariates, including indices of body weight. CONCLUSION: Current smoking seems to reduce the risk of myomas.

Adult↗
PubMed1995

The N-terminal domain of human DNA ligase I contains the nuclear localization signal and directs the enzyme to sites of DNA replication.

DNA replication in mammalian cells occurs in discrete nuclear foci called 'replication factories'. Here we show that DNA ligase I, the main DNA ligase activity in proliferating cells, associates with the factories during S phase but displays a diffuse nucleoplasmic distribution in non-S phase nuclei. Immunolocalization analysis of both chloramphenicol acetyltransferase (CAT)-DNA ligase I fusion proteins and epitope tagged DNA ligase I mutants allowed the identification of a 13 amino acid functional nuclear localization signal (NLS) located in the N-terminal regulatory domain of the protein. Furthermore, the NLS is immediately preceded by a 115 amino acid region required for the association of the enzyme with the replication factories. We propose that in vivo the activity of DNA ligase I could be modulated through the control of its sub-nuclear compartmentalization.

3T3 Cells↗
PubMed1995

Mutations of Jak-3 gene in patients with autosomal severe combined immune deficiency (SCID).

Severe combined immune deficiency (SCID) represents a heterogenous group of hereditary diseases. Mutations in the common gamma-chain (gamma c), which is part of several cytokine receptors including those for interleukin (IL)-2, IL-4, IL-7, IL-9 and IL-15, are responsible for X-linked SCID, which is usually associated with a lack of circulating T cells and the presence of B lymphocytes (T- B+ SCID). The gene(s) responsible for autosomal recessive T- B+ SCID is still unknown. The Jak-3 protein kinase has been found to associate with the gamma c-chain-containing cytokine receptors. Therefore Jak-3 or other STAT proteins with which it interacts are candidate genes for autosomal recessive T- B+ SCID. Here we investigate two unrelated T- B+ SCID patients (both from consanguineous parents) who have homozygous mutations in the gene for Jak-3. One patient carries a mutation (Tyr100-->Cys) in a conserved tyrosine residue in the JH7 domain of Jak-3 which is absent in more than 150 investigated chromosomes. The other patient carries a homozygous 151-base-pair deletion in the kinase-like domain, leading to a frameshift and premature termination. Both mutations resulted in markedly reduced levels of Jak-3. These findings show that abnormalities in the Jak/STAT signalling pathway can account for SCID in humans.

B-Lymphocytes↗
PubMed1995

The genomic organization of the human transcription factor 3 (TFE3) gene.

We have determined the exon-intron structure of the human TFE3 gene located on Xp11.22-23. By designing PCR primers, we were able to amplify various segments of the TFE3 genomic region, thus establishing that this gene is composed of seven exons, the first six of which are small (from 56 to 159 nt). The 5' UT region is contained entirely in the first exon, while the 3' UT region is contained in the seventh exon. The comparison of the genomic and the published cDNA versions revealed that the deduced amino acid sequence of TFE3 in the C-terminus region is 125 amino acids shorter than previously reported. This eliminates most of the putative proline- and arginine-rich domain and makes the human sequence more similar to its mouse homolog. The activation domain at the N-terminus is contained in exon 2, as has been described for the mouse. The basic helix-loop-helix (BHLH) motif is spread over exons 4 to 6, while the leucine zipper (LZ) is almost all contained in the last portion of exon 6. This split BHLH is different from other BHLH-LZ genes whose genomic structures have been determined up to now.

Animals↗
PubMed1995

Presynaptic component of long-term potentiation visualized at individual hippocampal synapses.

Long-term potentiation has previously been studied with electrophysiological techniques that do not readily separate presynaptic and postsynaptic contributions. Changes in exocytotic-endocytotic cycling have now been monitored at synapses between cultured rat hippocampal neurons by measuring the differential uptake of antibodies that recognize the intraluminal domain of the synaptic vesicle protein synaptotagmin. Vesicular cycling increased markedly during glutamate-induced long-term potentiation. The degree of potentiation was heterogeneous, appearing greater at synapses at which the initial extent of vesicular turnover was low. Thus, changes in presynaptic activity were visualized directly and the spatial distribution of potentiation could be determined at the level of single synaptic boutons.

Amino Acid Sequence↗
PubMed1995

De novo interstitial deletion q16.2q21 on chromosome 6.

A de novo interstitial deletion of 6q16.2q21 was observed in a 23-month-old boy with mental and psychomotor delay, obese appearance, minor craniofacial anomalies, and brain anomalies. We compare clinical manifestations of this patient with those observed in previously reported cases with similar 6q interstitial deletions. It is interesting to note the clinical similarities between some patients with interstitial deletions of 6q16 or q21 bands and patients with Prader-Willi syndrome (PWS) and it may help to keep in mind cytogenetic studies of patients with some PWS findings.

Abnormalities, Multiple↗
PubMed1995

Antifungal agents, Part 11. Biphenyl analogues of naftifine: synthesis and antifungal activities.

A series of naftifine analogues having the biphenyl instead of the naphthyl moiety have been synthesized in a search devoted to study bioanalogues of clinically efficacious antifungal agents. The new derivatives were tested against Candida albicans by the direct contact method. They were also assayed against Gram-positive and Gram-negative bacteria and against some isolates of plant pathogenic fungi. Derivatives 8a, 8c, and 9a were found to be active against Candida albicans, derivative 5a was active against E. coli, a very resistant species to antimycotic agents, and derivatives 8a and 8b inhibited the plant pathogenic Rhizoctonia solani.

Allylamine↗
PubMed1995

Autonomic neuropathy and prolongation of QT interval in human immunodeficiency virus infection.

Autonomic neuropathy has been reported in human immunodeficiency virus positive (HIV+) patients. Since alterations in cardiac innervation may determine QT interval prolongation, this interval was studied in a group of HIV+ subjects to evaluate if it is prolonged and to compare this measurement with other diagnostic tests for autonomic neuropathy. Fifty-seven HIV+ and 23 human immunodeficiency virus negative (HIV-) subjects were studied. Autonomic function was tested by noninvasive cardiovascular reflex tests, and the QT interval on the electrocardiogram was measured at rest, at maximum tachycardia during Valsalva manoeuvre, and afterwards at maximum bradycardia. QT intervals were corrected for heart rate according to Bazzett's formula (QTc). Autonomic neuropathy was found in 37 HIV+ subjects: 25 had moderate autonomic neuropathy (HIV+/mAN) and twelve had severe autonomic neuropathy (HIV+/sAN). The 23 HIV- and 20 HIV+ (HIV+/AN-) patients did not have autonomic neuropathy. QTc intervals were significantly longer in HIV+/sAN and HIV+/mAN than in HIV- at rest; in HIV+/sAN than in HIV- at maximum tachycardia; in HIV+/sAN and HIV+/mAN than in HIV+, in HIV+/sAN and HIV+/mAN than in HIV+/AN- and in HIV+/sAN than in HIV+/mAN at maximum bradycardia. QTc was > or = 440 ms in 24 out of 37 (64.8%) patients with autonomic neuropathy and in five out of 20 (25%) HIV+/AN- patients (sensitivity 65%, specificity 75%). A significant correlation was observed between scores of autonomic involvement and QTc interval prolongation. This study confirms that the QTc measurement is a reliable parameter indicating the presence of autonomic neuropathy. Since QT prolongation may determine ventricular arrhythmias, such patients must be followed because they may be at increased risk of sudden death.

Adult↗
PubMed1995

Risk factors for benign ovarian teratomas.

Risk factors for benign ovarian teratomas have been analysed in a case-control study conducted in Milan. Cases were women aged less than 65 years with a histologically confirmed diagnosis of benign ovarian teratoma who were admitted to a network of Obstetrics and Gynecology Departments in Milan. A total of 77 women aged 16-64 years were interviewed. Controls were women admitted to hospital for acute, non-gynaecological, non-hormonal and non-neoplastic diseases; 231 controls were interviewed (age range 15-64 years). Cases tended to be more educated: in comparison with women with less than 7 years of education, the estimated relative risk (RR) of ovarian benign teratoma was 1.6 and 2.5 respectively in women with 7-11 and 12 or more years of schooling, the trend in risk being statistically significant (chi 2(1) trend 5.39, P < 0.01). Four of the 77 cases (5.2%) and two of the 231 controls (0.9%) reported a history of infertility, with a corresponding RR of 8.3 (95% confidence interval 1.3-54.0). There was no clear relation between parity and risk of ovarian benign teratomas: in comparison with nulliparae, the estimated RRs were 1.1 and 0.7 respectively in women reporting one or two or more births (chi 2(1) trend 0.53, P = not significant). No relation emerged between marital status, age at menarche, menstrual cycle pattern, menopausal status, abortions, age at first pregnancy, oral contraceptive use and risk of ovarian benign teratomas.

Adolescent↗
PubMed1995

X-linked thrombocytopenia and Wiskott-Aldrich syndrome are allelic diseases with mutations in the WASP gene.

X-linked thrombocytopenia (XLT) is a rare recessive hereditary disorder characterized by isolated thrombocytopenia with small-sized platelets. The XLT locus has been located to chromosome Xp11 by linkage analysis, which is also where the recently cloned Wiskott-Aldrich syndrome (WAS) gene, maps. The relationship between XLT and WAS has long been debated; they might be due to different mutations of the same gene or to mutations in different genes. We now show that mutations in the WAS gene, different from those found in WAS patients, are present in three unrelated male patients with isolated thrombocytopenia and small-sized platelets. Our results demonstrate that XLT and WAS are allelic forms of the same disease, but the causes of the differences need to be further investigated.

Alleles↗
PubMed1995

Differential localization and functional role of calsequestrin in growing and differentiated myoblasts.

Calsequestrin (CSQ) is the low affinity, high capacity Ca(2+)-binding protein concentrated within specialized areas of the muscle fiber sarcoplasmic reticulum (a part of the ER) where it is believed to buffer large amounts of Ca2+. Upon activation of intracellular channels this Ca2+ pool is released, giving rise to the [Ca2+]i increases that sustain contraction. In order to investigate the ER retention and the functional role of the protein, L6 rat myoblasts were infected with a viral vector with or without the cDNA of chicken CSQ, and stable clones were investigated before and after differentiation to myotubes. In the undifferentiated L6 cells, expression of considerable amounts of heterologous CSQ occurred with no major changes of other ER components. Ca2+ release from the ER, induced by the peptide hormone vasopressin, remained however unchanged, and the same occurred when other treatments were given in sequence to deplete the ER and other intracellular stores: with the Ca2+ pump blocker, thapsigargin; and with the Ca2+ ionophore, ionomycin, followed by the Na+/H+ ionophore, monensin. The lack of effect of CSQ expression on the vasopressin-induced [Ca2+]i responses was explained by immunocytochemistry showing the heterologous protein to be localized not in the ER but in large vacuoles of acidic content, positive also for the lysosomal enzyme, cathepsin D, corresponding to a lysosomal subpopulation. After differentiation, all L6 cells expressed small amounts of homologous CSQ. In the infected cells the heterologous protein progressively decreased, yet the [Ca2+]i responses to vasopressin were now larger with respect to both control and undifferentiated cells. This change correlated with the drop of the vacuoles and with the accumulation of CSQ within the ER lumen, where a clustered distribution was observed as recently shown in developing muscle fibers. These results provide direct evidence for the contribution of CSQ, when appropriately retained, to the Ca2+ capacity of the rapidly exchanging, ER-located Ca2+ stores; and for the existence of specific mechanism(s) (that in L6 cells develop in the course of differentiation) for the ER retention of the protein. In the growing L6 myoblasts the Ca(2+)-binding protein appears in contrast to travel along the exocytic pathway, down to post-Golgi, lysosome-related vacuoles which, based on the lack of [Ca2+]i response to ionomycin-monensin, appear to be incompetent for Ca2+ accumulation.

Animals↗