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Biomedical subjects

C Sala

Publications and source records attributed to C Sala.

At least 19 recordsLinked to original sources

Expression of phage P4 integrase is regulated negatively by both Int and Vis.

Phage P4 int gene encodes the integrase responsible for phage integration into and excision from the Escherichia coli chromosome. Here, the data showing that P4 int expression is regulated in a complex manner at different levels are presented. First of all, the Pint promoter is regulated negatively by both Int and Vis, the P4 excisionase. The N-terminal portion of Int appears to be sufficient for such a negative autoregulation, suggesting that the Int N terminus is implicated in DNA binding. Second, full-length transcripts covering the entire int gene could be detected only upon P4 infection, whereas in P4 lysogens only short 5'-end covering transcripts were detectable. On the other hand, transcripts covering the 5'-end of int were also very abundant upon infection. It thus appears that premature transcription termination and/or mRNA degradation play a role in Int-negative regulation both on the basal prophage transcription and upon infection. Finally, comparison between Pint-lacZ transcriptional and translational fusions suggests that Vis regulates Int expression post-transcriptionally. The findings that Vis is also an RNA-binding protein and that Int may be translated from two different start codons have implications on possible regulation models of Int expression.

Artificial Gene Fusion↗

Prevalence and correlates of advanced retinopathy in a large selected hypertensive population. The Evaluation of Target Organ Damage in Hypertension (ETODH) study.

OBJECTIVE: To describe the prevalence of advanced retinal microvascular lesions and their associations with cardiac and extracardiac signs of target organ damage (TOD) in a large selected hypertensive population. METHODS: A total of 2172 non-diabetic untreated and treated uncomplicated essential hypertensives consecutively attending for the first time our hospital outpatient hypertension clinic and included in the Evaluation of Target Organ Damage in Hypertension (ETODH), an observational ongoing registry of hypertension-related TOD, were considered for this analysis. Advanced hypertensive retinopathy was defined by the presence of any of the following lesions: flame-shaped haemorrhages, soft exudates or cotton wool spots and papilloedema. Left ventricular hypertrophy (LVH), carotid structural abnormalities, such as plaques and intima media (IM) thickening, and microalbuminuria were diagnosed according to the 2003 ESH/ESC guidelines criteria. RESULTS: Among the whole study population, 33 patients (1.5%) were found to have advanced hypertensive retinopathy. Patients with these retinal lesions were similar to those without for age, body mas index, known duration of hypertension, smoking habit, total serum cholesterol, fasting blood pressure and prevalence of antihypertensive treatment; whereas mean systolic and diastolic blood pressures were higher in the former group. The prevalence rates of LVH, carotid plaques, carotid IM thickening and microalbuminuria in patients with and without retinopathy were 57%, 67%, 69%, 19% and 25%, 47%, 44%, 12%, respectively. In a multivariate logistic regression analysis, advanced retinopathy was significantly associated with LVH (OR = 4.0), carotid IM thickening (OR = 2.9), carotid plaques (OR = 2.8), but not with microalbuminuria. CONCLUSIONS: Our study indicated that: (i) advanced retinopathy is a rare finding in non-diabetic hypertensive patients seen in a specialist setting; (ii) a strong relation exists between retinal microvascular lesions and cardiac and macrovascular markers of TOD.

Adult↗

Mutation analysis of two candidate genes for premature ovarian failure, DACH2 and POF1B.

BACKGROUND: Balanced X;autosome translocations interrupting the 'critical region' of the long arm of the human X chromosome are often associated with premature ovarian failure (POF). However, the mechanisms leading to X-linked ovarian dysfunction are largely unknown, as the majority of the X chromosome breakpoints have been mapped to gene-free genomic regions. A few genes have been found to be interrupted, but their role has never been clarified. METHODS AND RESULTS: By fine mapping of the X chromosome breakpoint of an X;autosome balanced translocation, we identified a new interrupted gene, POF1B. We performed a mutation analysis of POF1B and of another gene previously identified, DACH2, localized approximately 700 kb distal in Xq21, in a cohort of >200 Italian POF patients. Rare mutations were found in patients in both genes. CONCLUSIONS: Our findings could not demonstrate any involvement of POF1B, but suggest that rare mutations in the DACH2 gene may have a role in the POF phenotype.

Adolescent↗

Awareness of hypertension guidelines in primary care: results of a regionwide survey in Italy.

Unsatisfactory blood pressure (BP) control so often described in treated hypertensive populations is also explained by insufficient physicians' awareness of experts' guidelines. We assessed awareness of current recommendations about hypertension management in a general practice setting, using the World Health Organization/International Society of Hypertension (WHO/ISH) 1999 guidelines as reference. In a regionwide survey, a total of 5133 physicians (three-quarters of all active general practitioners in Lombardy, a region of north-western Italy) were contacted by letter and received a multiple choice 10-item questionnaire. Data on physicians' demographic characteristics, information on hypertension prevalence and their perception of BP control among their patients were also requested in an additional form. The number of answers in agreement with WHO/ISH guidelines was used as an awareness measure. This was considered adequate if correct answers to six out of 10 questions, including an appropriate definition of hypertension, were provided. Of the 1256 returned questionnaires (a 24.5% response rate), 1162 were suitable for analysis. The mean score of correct answers was 5.3 points and only 20.1% of the study population correctly answered at least six of the questions (including that on the definition of hypertension in the elderly). Guidelines awareness was negatively related to increase in physicians' age and duration of clinical practice and by the male gender. Finally, the physicians reported a high rate of achieved BP control, and those who were considered to have achieved highest rates (>75%) of control in their patients had a lower rate of adequate knowledge than those who reported less-successful therapeutic results (18 vs 25%, P<0.01), suggesting that they ignored or disregarded the goal BP recommended by the guidelines. This large regional survey shows that a sufficient degree of guidelines awareness is present in a minority of primary-care physicians in the Italian region of Lombardy. Therefore, further efforts are required to intensify information strategies for improving professional education, training and practice organization aimed at achieving therapeutic goals. Physicians with longer duration of clinical practice represent a particularly relevant target group for these interventions.

Awareness↗

Lack of effect of acute repaglinide administration on postprandial lipaemia in patients with type 2 diabetes mellitus.

The effect of acute repaglinide administration (2 mg) on postprandial glycaemia and lipaemia has been examined in 20 subjects with type 2 diabetes mellitus. Each subject received in the morning, after a 12 to 14 h fast, a standard mixed meal (total energy 783 kcal), preceded by one tablet of 2 mg repaglinide or placebo. Chylomicrons and chylomicron-deficient plasma were prepared by ultracentrifugation. Triglyceride levels in CM fraction (CM-triglycerides) in total plasma as well as in CM-deficient plasma (non-CM-triglycerides) were determined. A significant reduction in postprandial glycaemia was observed after repaglinide administration compared to placebo ( p < 0.001). Plasma concentrations of total triglycerides, CM-triglycerides, non-CM-triglycerides, free fatty acids and the other plasma lipids measured, were not significantly different between the two phases of the study. It is concluded that, in contrast to sulphonylureas, acute repaglinide administration does not improve postprandial lipaemia in patients with type 2 diabetes.

Analysis of Variance↗

Headspace solid-phase microextraction analysis of 3-alkyl-2-methoxypyrazines in wines.

A procedure to determine 3-alkyl-2-methoxypyrazines in wines is described. It is based on the headspace solid-phase microextraction (HS-SPME) technique after a clean-up of the sample by distillation (previously acidified to pH 0.5) to remove ethanol and other volatile compounds that can interfere in the SPME. Determination is performed by means of capillary gas chromatography using a nitrogen-phosphorus detector. The method allows quantification of 3-isobutyl-2-methoxypyrazine, 3-sec-butyl-2-methoxypyrazine and 3-isopropyl-2-methoxypyrazine at their natural concentration levels and below their sensory thresholds in Cabernet Sauvignon and Merlot wines. The method was successfully applied to experimental red wines and the evolution of their pyrazine contents during the winemaking process was monitored. Pyrazine content increased during the first maceration day but did not change significantly during alcoholic and malolactic fermentation. Final contents in wines were 12-27 ng/l of 3-isobutyl-2-methoxypyrazine and 5-10 ng/l of 3-sec-butyl-2-methoxypyrazine.

Chromatography, Gas↗

Left ventricular concentric remodelling and extracardiac target organ damage in essential hypertension.

Left ventricular (LV) concentric remodelling is an adaptive change in cardiac geometry frequently observed in arterial hypertension. This study was addressed to investigate the extent of extracardiac target organ damage (TOD) in patients with LV concentric remodelling. Two groups of never-treated essential hypertensives, 31 with normal LV geometry (group I, relative wall thickness: 0.39) and 31 with LV concentric remodelling (group II, relative wall thickness: 0.47) matched for age, sex, body mass index and mean 24-h systolic blood pressure (BP), were included in the study. They underwent clinical and laboratory examination, 24-h ambulatory BP monitoring (ABPM), 24-h urinary collection for microalbuminuria, non-mydriatic photography of ocular fundi, echocardiography and carotid ultrasonography. In both groups age (I: 51 +/- 11 years; II: 51 +/- 11 years), body mass index (I: 25 +/- 3 kg/m(2); II: 26 +/- 3 kg/m(2)), clinic and 24-h ABPM values (I: 149 +/- 11/95 +/- 8, 142 +/- 11/91 +/- 7 mm Hg; II: 150 +/- 11/98 +/- 9, 142 +/- 12/92 +/- 9 mm Hg) were similar by design. There were no differences between patients with normal LV geometry and with LV concentric remodelling in LVM index (97 +/- 16 vs 99 +/- 16), carotid intima-media thickness (0.7 +/- 0.02 vs 0.7 +/- 0.02) and carotid plaques prevalence (35% vs 35%). Furthermore, no significant differences among the two groups were found in the prevalence of retinal changes and microalbuminuria. These results suggest that in hypertensive patients with similar BP and LVMI levels, LV concentric remodelling is not associated with more prominent TOD.

Albuminuria↗

A comparison of blood pressure control in a hypertension hospital clinic between 1997 and 2000.

AIMS: (1) To evaluate the prevalence of clinic blood pressure (BP) control in a large sample of treated hypertensives followed in our hypertension clinic during the year 2000, and to compare it with our 1997 data. (2) To investigate the prevalence of left ventricular hypertrophy (LVH) according to different levels of BP control. METHODS AND RESULTS: One thousand consecutive hypertensive patients who attended our hypertension hospital clinic in a period of 7 months during the year 2000 and who had regularly been followed by the same medical team were included in the study. LVH was assessed using two different electrocardiographic criteria (Sokolow-Lyon and Cornell). This population had similar clinical characteristics of a cohort including 700 patients seen at our centre during 1997 in which BP control rate was 34% (Cuspidi et al., J Hypertens 1999; 17: 835-41). During follow-up, 441 of the treated patients had clinic BP < 140/90 mmHg, 283 < 150/95 mmHg and 276 > or = 150/95 mmHg, indicating that BP control was satisfactory in 44.1%, borderline in 28.3% and unsatisfactory in 27.6% of the cases. Thirty-five patients (3.6%) had LVH according to Cornell criteria and 25 (2.6%) according to Sokolow criteria. A significantly lower prevalence of LVH was detected in patients with optimal BP control (< 130/ 85 mmHg) compared to those with unsatisfactory BP (2.3% vs 9.4%, respectively, p < 0.01). CONCLUSION: The study demonstrates that: (1) hypertensive patients in a hypertension clinic have satisfactory BP control in 44.1% of cases, indicating a significant improvement of BP control in this clinical setting during the last 3 years; (2) prevalence of LVH is significantly related to BP control.

Aged↗

Modulation of nicotinic acetylcholine receptor turnover by tyrosine phosphorylation in rat myotubes.

The muscle nicotinic acetylcholine receptor (AChR) turns over at different rates depending on stage of synaptogenesis and innervation. Tyrosine phosphorylation modulates desensitization, interaction with cytoskeleton and lateral mobility in the membrane of AChR. To determine whether tyrosine phosphorylation also modulates the turnover of AChR, myotubes in vitro were exposed to the tyrosine phosphatase inhibitor pervanadate. Our data indicate that a transient increase of phosphotyrosine levels stabilized a fraction of AChRs. The effects were limited to the non-epsilon subunit-containing AChRs already present in the membrane. Tyrosine phosphorylation of the receptor occurred on the beta subunit, was transient and stable molecules were not selectively tyrosine phosphorylated. The data indicate that modulation of phosphotyrosine levels in muscle cells provides signals to control AChR metabolic stability.

Animals↗

Regulation of dendritic spine morphology and synaptic function by Shank and Homer.

The Shank family of proteins interacts with NMDA receptor and metabotropic glutamate receptor complexes in the postsynaptic density (PSD). Targeted to the PSD by a PDZ-dependent mechanism, Shank promotes the maturation of dendritic spines and the enlargement of spine heads via its ability to recruit Homer to postsynaptic sites. Shank and Homer cooperate to induce accumulation of IP3 receptors in dendritic spines and formation of putative multisynapse spines. In addition, postsynaptic expression of Shank enhances presynaptic function, as measured by increased minifrequency and FM4-64 uptake. These data suggest a central role for the Shank scaffold in the structural and functional organization of the dendritic spine and synaptic junction.

Adaptor Proteins, Signal Transducing↗

Sharpin, a novel postsynaptic density protein that directly interacts with the shank family of proteins.

The Shank family of proteins (also termed CortBP, ProSAP, or Synamon) is highly enriched in the postsynaptic density (PSD) of excitatory synapses in brain. Shank contains multiple domains for protein-protein interactions, including ankyrin repeats, SH3 domain, PDZ domain, SAM domain, and an extensive proline-rich region. We have identified a novel protein, termed Sharpin, that directly interacts with the ankyrin repeats of Shank. Sharpin is enriched in the PSD and forms a complex with Shank in heterologous cells and brain. Immunostaining reveals the presence of Sharpin at excitatory synapses and its colocalization with Shank. While the C-terminal half of Sharpin interacts with Shank, the N-terminal half of Sharpin mediates homomultimerization. Considering the fact that the ankyrin repeats and the SH3 domain of Shank can be truncated by alternative splicing, these results define Sharpin as a novel PSD protein that may regulate the complexity of the Shank-based protein network in an alternative splicing-dependent manner.

Adaptor Proteins, Signal Transducing↗

Transcriptional regulation of furA and katG upon oxidative stress in Mycobacterium smegmatis.

The DNA region upstream of katG in Mycobacterium smegmatis was cloned and sequenced. The furA gene, highly homologous to Mycobacterium tuberculosis furA, mapped in this region. The furA-katG organization appears to be conserved among several mycobacteria. The transcription pattern of furA and katG in M. smegmatis upon oxidative stress was analyzed by Northern blotting and primer extension. Although transcription of both furA and katG was induced upon oxidative stress, transcripts covering both genes could not be identified either by Northern blotting or by reverse transcriptase PCR. Specific transcripts and 5' ends were identified for furA and katG, respectively. By cloning M. smegmatis and M. tuberculosis DNA regions upstream of a reporter gene, we demonstrated the presence of two promoters, pfurA, located immediately upstream of the furA gene, and pkatG, located within the terminal part of the furA coding sequence. Transcription from pfurA was induced upon oxidative stress. A 23-bp sequence overlapping the pfurA -35 region is highly conserved among mycobacteria and streptomycetes and might be involved in controlling pfurA activity. Transcription from a cloned pkatG, lacking the upstream pfurA region, was not induced upon oxidative stress, suggesting a cis-acting regulatory role of this region.

Bacterial Proteins↗

PDZ domains and the organization of supramolecular complexes.

PDZ domains are modular protein interaction domains that bind in a sequence-specific fashion to short C-terminal peptides or internal peptides that fold in a beta-finger. The diversity of PDZ binding specificities can be explained by variable amino acids lining the peptide-binding groove of the PDZ domain. Abundantly represented in Caenorhabditis elegans, Drosophila melanogaster, and mammalian genomes, PDZ domains are frequently found in multiple copies or are associated with other protein-binding motifs in multidomain scaffold proteins. PDZ-containing proteins are typically involved in the assembly of supramolecular complexes that perform localized signaling functions at particular subcellular locations. Organization around a PDZ-based scaffold allows the stable localization of interacting proteins and enhances the rate and fidelity of signal transduction within the complex. Some PDZ-containing proteins are more dynamically regulated in distribution and may also be involved in the trafficking of interacting proteins within the cell.

Amino Acid Sequence↗

Blunted vascular and renal effects of exogenous atrial natriuretic peptide in patients with cushing's disease.

The role of atrial natriuretic peptide (ANP) in glucocorticoid hypertension is still controversial, as glucocorticoids enhance the secretion of ANP in vivo, but reduce its biological activity in vitro. In isolated cells, for example, the cGMP response to ANP is suppressed by dexamethasone. We tested the in vivo relevance of this observation by comparing the cGMP, endocrine, and renal responses to exogenous ANP in patients with Cushing's disease (CD; n = 10) and in a matched group of essential hypertensives (EH; n = 8) and normotensive controls (N; n = 4). alpha-human-ANP was infused at 0.01 microg/kg/min for 120 min with a 30-min recovery period; hormonal and arterial pressure measurements were performed at 30-min intervals, and renal parameters were measured at baseline and after infusion. There was a 4-fold increase in plasma ANP in all groups, but the increments in plasma cGMP were about 50% lower in CD than in N and EH; urinary cGMP was similarly lower in CD (247 +/- 61 vs. 529 +/- 146 and 384 +/- 54 nmol/150 min, respectively). This was associated with a reduced peak increase in hematocrit in CD (+2.6 +/- 0.9%) compared with N (+6.6 +/- 0.9%) and EH (+7.1 +/- 0.7%; P < 0.05 CD vs. both); the diuretic and natriuretic effects of ANP were also lower in CD than in EH with very similar systemic pressure levels (382 +/- 63 vs. 848 +/- 130 mL/150 min, and 61 +/- 14 vs. 113 +/- 14 mmol/150 min, respectively; P < 0.05 for both). The increments in plasma and urinary cGMP in response to physiological doses of ANP are thus blunted in CD patients compared with those in N and EH. This biochemical defect is associated with reduced capillary permeability and a lesser diuretic and natriuretic effect. These data are compatible with an impairment of the biological activity of ANP in glucocorticoid hypertension in humans.

Adult↗

Cloning and characterization of the human phosphoinositide-specific phospholipase C-beta 1 (PLC beta 1).

Phospholipase C-beta (PLC beta) catalyses the generation of inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (IP(2)), a key step in the intracellular transduction of a large number of extracellular signals, including neurotransmitters and hormones modulating diverse developmental and functional aspects of the mammalian central nervous system. Four mammalian isozymes are known (PLC beta 1-4), which differ in their function and expression patterns in vivo. We have characterized the human PLC beta 1 genomic locus (PLC beta 1), cloned two distinct PLC beta 1 cDNAs (PLC beta 1a and b) and analysed their respective expression patterns in a comprehensive panel of human tissues using quantitative TaqMan technology. The two cDNAs derive from transcripts generated through alternative splicing at their 3' end, and are predicted to encode for PLC beta 1 isoforms differing at their carboxy-terminus. The human PLC beta 1 isoforms are co-expressed in the same tissues with a distinctly CNS-specific profile of expression. Quantitative differences in PLC beta 1 isoform expression levels are observed in some tissues. Transient expression of epitope-tagged versions of the two isoforms followed by immunofluorescence revealed localization of the proteins to the cytoplasm and the inner side of the cell membrane. Finally, we characterized the structure of the PLC beta 1 locus and confirmed its mapping to human chromosome 20.

Amino Acid Sequence↗

Interaction of the postsynaptic density-95/guanylate kinase domain-associated protein complex with a light chain of myosin-V and dynein.

NMDA receptors interact directly with postsynaptic density-95 (PSD-95), a scaffold protein that organizes a cytoskeletal- signaling complex at the postsynaptic membrane. The molecular mechanism by which the PSD-95-based protein complex is trafficked to the postsynaptic site is unknown but presumably involves specific motor proteins. Here we demonstrate a direct interaction between the PSD-95-associated protein guanylate kinase domain-associated protein (GKAP) and dynein light chain (DLC), a light chain subunit shared by myosin-V (an actin-based motor) and cytoplasmic dynein (a microtubule-based motor). A yeast two-hybrid screen with GKAP isolated DLC2, a novel protein 93% identical to the previously cloned 8 kDa dynein light chain (DLC1). A complex containing PSD-95, GKAP, DLC, and myosin-V can be immunoprecipitated from rat brain extracts. DLC colocalizes with PSD-95 and F-actin in dendritic spines of cultured neurons and is enriched in biochemical purifications of PSD. Immunogold electron microscopy reveals a concentration of DLC in the postsynaptic compartment of asymmetric synapses of brain in which it is associated with the PSD and the spine apparatus. We discuss the possibility that the GKAP/DLC interaction may be involved in trafficking of the PSD-95 complex by motor proteins.

Amino Acid Sequence↗

Headspace solid-phase microextraction method for determining 3-alkyl-2-methoxypyrazines in musts by means of polydimethylsiloxane-divinylbenzene fibres.

A method for determining 2-methoxypyrazine, 3-methyl-, 3-ethyl-, 3-isopropyl-, 3-sec.-butyl- and 3-isobutyl-2-methoxypyrazine in musts is described. It involves headspace solid-phase microextraction (SPME) and determination by capillary gas chromatography using nitrogen-phosphorous detection. Pyrazines were satisfactorily separated under isothermal conditions, and quantification was carried out using 3-isopropyl-2-ethoxypyrazine as the internal standard. Ionic strength, time and temperature were studied in order to make SPME as efficient as possible. The developed method enabled detection limits at the 0.1 ng(-1) levels for some of the analytes. The method was successfully applied to identify and quantify different 3-alkyl-2-methoxypyrazines in experimental musts of Cabernet Sauvignon and Merlot. Their evolution during the ripening was also monitored.

Osmolar Concentration↗

Developmentally regulated NMDA receptor-dependent dephosphorylation of cAMP response element-binding protein (CREB) in hippocampal neurons.

Developmental changes in the signaling properties of NMDA receptors have been proposed to underlie the loss of plasticity that accompanies brain maturation. Calcium influx through postsynaptic NMDA receptors can stimulate neuronal gene expression via signaling pathways such as the Ras-MAP kinase (MAPK) pathway and the transcription factor cAMP response element-binding protein (CREB). We analyzed MAPK (Erk1/2) and CREB activation in response to NMDA receptor stimulation during the development of hippocampal neurons in culture. At all stages of development NMDA stimulation induced a rapid phosphorylation of CREB on Ser-133 (phospho-CREB). However, the time course of decline in phospho-CREB changed dramatically with neuronal maturation. At 7 d in vitro (7 DIV) phospho-CREB remained elevated 2 hr after strong NMDA stimulation, whereas at 14 DIV phospho-CREB rose only transiently and fell back to below basal levels within 30 min. Moreover, at 14 DIV, but not at 7 DIV, NMDA receptor stimulation induced a dephosphorylation of CREB that previously had been phosphorylated by KCl depolarization or forskolin, suggesting an NMDA receptor-dependent activation of a CREB phosphatase. There was no developmental change in the time course of phospho-CREB induction that followed KCl depolarization or PKA activation, nor was there a developmental change in the time course of phospho-Erk1/2 induced by NMDA receptor activation. We suggest that, during neuronal maturation, NMDA receptor activation becomes linked specifically to protein phosphatases that act on Ser-133 of CREB. Such a developmentally regulated switch in the mode of NMDA receptor coupling to intracellular signaling pathways may contribute to the changes in neural plasticity observed during brain development.

Animals↗