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Biomedical subjects

N Longo

Publications and source records attributed to N Longo.

At least 19 recordsLinked to original sources
PubMed2005

Genitourinary anomalies in Mowat-Wilson syndrome with deletion/mutation in the zinc finger homeo box 1B gene (ZFHX1B). Report of three Italian cases with hypospadias and review.

Hypospadias, when the urethra opens on the ventral side of the penis, is a common malformation seen in about 3 per 1,000 male births. It is a complex disorder associated with genetic and environmental factors and can be part of genetic syndromes. Mowat-Wilson syndrome (MWS) is a multiple congenital anomaly syndrome characterized by a distinct facial phenotype, Hirschsprung disease, microcephaly and mental retardation. It is caused by mutations in the zinc finger homeo box 1B gene, ZFHX1B (SIP1). To date, 68 deletion/mutation-positive cases have been reported. Genitourinary anomalies are common in MWS. Here we report that hypospadias is common in males with this syndrome. In 39 patients where this information was available, hypospadias was present in 46% of patients (18/39). In the 3 Italian male cases reported here, hypospadias was always present. MWS should be considered by endocrinologists in patients with hypospadias associated with developmental delays/mental retardation, in particular in the presence of a distinct facial phenotype.

Child, Preschool↗
PubMed2004

Rapid, comprehensive screening of the human medium chain acyl-CoA dehydrogenase gene.

Newborn screening by tandem mass spectrometry (MS/MS) identifies patients with medium chain acyl-CoA dehydrogenase (MCAD) deficiency the most frequently observed disorder of fatty acid oxidation. Molecular genetic analysis is becoming a common tool to confirm those identified as affected by prospective screening and for carrier detection in family studies. The A985G (K304E) mutation accounts for approximately 80% of mutant alleles in MCAD deficient patients, presenting symptomatically, while greater variability of mutant alleles is observed among cases identified through prospective screening. Aside from A985G, the mutation spectrum in MCAD deficient patients is heterogeneous such that comprehensive gene analysis is required. Traditionally the MCAD gene is assayed by sequencing the entire coding region. Although effective and definitive, this approach is expensive, turn around time is slow, and is poorly amenable to a clinical service molecular genetics laboratory. Dye-binding/high-resolution thermal denaturation is a rapid and homogeneous method by which to scan a PCR product for evidence of sequence aberration. PCR is performed in capillaries in the presence of the dsDNA-binding dye LCGreen I and subsequently the DNA/dye complexes are analyzed by high-resolution thermal denaturation. DNA sequencing was limited to fragments displaying abnormal melting profiles. Of 18 specimens analyzed, 11 have a genotype consistent with MCAD deficiency and seven have a genotype consistent with carrier status. Clinical and biochemical data corroborate that the genotype results identified the affected patients and differentiates them from carriers. The entire process is homogeneous requiring no post-PCR manipulation and is completed in under 3 h.

Acyl-CoA Dehydrogenase↗
PubMed2004

Carnitine transporter defect due to a novel mutation in the SLC22A5 gene presenting with peripheral neuropathy.

The carnitine transporter defect (McKusick 212140) is an autosomal recessive disorder caused by mutations in the SLC22A5 gene, which encodes the high-affinity carnitine transporter OCTN2 (Wang et al 2001). Diagnosis is suspected when plasma carnitine levels are extremely low and secondary causes of carnitine loss are excluded. The disease can present with recurrent Reye-like episodes of hypoketotic hypoglycaemia or with cardiomyopathy associated with myopathy (Stanley et al 1991). Here we report novel clinical findings in a 3-year-old with primary carnitine deficiency.

Carnitine↗
PubMed2003

Neurophysiologic evaluation of central-peripheral sensory and motor pudendal pathways in primary premature ejaculation.

OBJECTIVES: Pudendal nerve somatosensory evoked potentials (SEPs), the bulbocavernosus (BC) reflex, and BC perineal motor evoked potentials after transcranial magnetic cortical stimulation were performed in patients with primary premature ejaculation to investigate the somatic sensory and motor function of the genital area. METHODS: Fourteen patients with primary premature ejaculation underwent psychological counseling, urologic physical examination, transrectal ultrasound examination, laboratory testing, and the Stamey test. The spinal and cortical pudendal nerve SEPs were performed by dorsal nerve stimulation at the penile shaft (DN-SEPs) in all patients and at the glans penis (GP-SEPs) in 3 of them. The BC reflex was obtained by stimulating the base of the penis. RESULTS: The mean sensory threshold did not significantly differ between the patients and normal subjects. Cortical DN-SEPs were normal in all patients. The sensory central conduction time, calculated in 6 patients, was normal. The mean cortical DN-SEP amplitude was significantly smaller in patients than in controls. In 3 patients and in 3 controls who underwent both DN-SEP and GP-SEP testing, the glans penis sensory threshold was lower than the dorsal nerve threshold and the cortical GP-SEP latency was longer than the cortical DN-SEP latency. The BC reflex was normal in most patients. The BC motor evoked potentials were normal in all patients, but one. CONCLUSIONS: We did not confirm either a faster conduction along the pudendal sensory pathway or a greater cortical representation of the sensory stimuli from the genital area in our patients. Moreover, we did not confirm hyperexcitability of the BC reflex in them. Our results suggest that the electrophysiologic approach is probably not sufficient to clarify the causes of primary premature ejaculation. A more integrated investigation could allow better results in this field.

Adult↗
PubMed2003

Female sexual dysfunction: an update on physiopathology.

Despite the importance of sexuality for both men and women of all ages, only in the last few years extensive research has been carried out into female sexual problems. It has been discovered that sexual problems affect a considerable number of women each year, and this indicates the validity and necessity of further medical studies. We know that female genital sexual response is a combination of vasocongestive and neuromuscular events in the genital tract and pelvic floor which are controlled in part by specific neurotransmitters. Other pelvi-perineal genital structures undergo vasculogenic changes, namely the labia, periurethral glands, urethra and the Halban's fascia but much less attention has been paid to the role of these tissues in sexual response compared to the clitoris and the vagina. The most common etiologies of female sexual dysfunction are vasculogenic, neurogenic, hormonal/endocrine, muscologenic. The increasing various problems of female sexual dysfunction and the interest in the matter and the subsequent research are factors which keep the scientific community involved constantly active.

Female↗
PubMed2002

Did men with erectile dysfunction discuss their condition with partner and physicians? A survey of men attending a free call information service.

We present data collected among men attending a free call service on information on erectile dysfunction (ED) activated in Italy during the period 1997-1999. Their attitudes towards discussion with their partner and physician about the condition are considered. Each subject, was asked if he was affected by ED (defined as inability to achieve and maintain an erection sufficient for satisfactory sexual performance). In the case of a positive answer, the subject was asked if he had ever discussed his condition with partner or a physician. A total of 12 761 subjects with ED called the service: 7265 (56.9%) reported to have discussed their condition with their partner. The proportion tended to increase with duration of ED, being 47.9% in subjects reporting ED lasting <6 months and 59.9% in those reporting ED lasting >3 y (w(2)(1) trend <0.05). Likewise, the proportion of subjects reporting to have discussed ED with a physician was 50.3% (6416 subjects), being 33.6% in subjects with ED lasting <6 months and 57.9% of those with ED lasting >3 y (w(2)(1) trend, P<0.01).

Adult↗
PubMed2001

Regulatory role of tetraspanin CD9 in tumor-endothelial cell interaction during transendothelial invasion of melanoma cells.

Heterotypic interaction among tumor cells (TCs) and endothelial cells (ECs) may play a critical role during the vascular dissemination of neoplastic cells and during pathologic angiogenesis in tumors. To identify molecules involved in these processes, the distribution of vascular junctional proteins was first studied by immunofluorescence at sites of heterologous intercellular contact using TC-EC mosaic monolayers grown on 2-dimensional collagen. Several members of the tetraspanin superfamily, including CD9, CD81, and CD151, were found to localize at the TC-EC contact area. The localization of tetraspanins to the TC-EC heterologous contact area was also observed during the active transmigration of TCs across EC monolayers grown onto 3-dimensional collagen matrices. Dynamic studies by time-lapse immunofluorescence confocal microscopy showed an active redistribution of endothelial CD9 to points of melanoma insertion. Anti-CD9 monoclonal antibodies were found to specifically inhibit the transendothelial migration of melanoma cells; the inhibitory effect was likely caused by a strengthening of CD9-mediated heterotypic interactions of TCs to the EC monolayer. These data support a novel mechanism of tetraspanin-mediated regulation of TC transcellular migration independent of TC motility and growth during metastasis and a role for these molecules in the formation of TC-EC mosaic monolayers during tumor angiogenesis.

Antibodies, Monoclonal↗
PubMed2001

Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells.

Chemokines are secreted into the tumor microenvironment by tumor-infiltrating inflammatory cells as well as by tumor cells. Chemokine receptors mediate agonist-dependent cell responses, including migration and activation of several signaling pathways. In the present study we show that several human melanoma cell lines and melanoma cells on macroscopically infiltrated lymph nodes express the chemokine receptors CXCR3 and CXCR4. Using the highly invasive melanoma cell line BLM, we demonstrate that the chemokine Mig, a ligand for CXCR3, activates the small GTPases RhoA and Rac1, induces a reorganization of the actin cytoskeleton, and triggers cell chemotaxis and modulation of integrin VLA-5- and VLA-4-dependent cell adhesion to fibronectin. Furthermore, the chemokine SDF-1alpha, the ligand of CXCR4, triggered modulation of beta(1) integrin-dependent melanoma cell adhesion to fibronectin. Additionally, Mig and SDF-1alpha activated MAPKs p44/42 and p38 on melanoma cells. Expression of functional CXCR3 and CXCR4 receptors on melanoma cells indicates that they might contribute to cell motility during invasion as well as to regulation of cell proliferation and survival.

Actins↗
PubMed2001

Decreased half-life of insulin-like growth factor I in Rabson-Mendenhall syndrome.

Rabson-Mendenhall syndrome is an autosomal recessive disorder of insulin signalling caused by mutations in the insulin receptor gene. Affected patients are insensitive to exogenous insulin. Insulin-like growth factor I (IGF-I), whose receptor is similar to the one for insulin and is not impaired in this condition, is not always effective in these patients. To understand the reason for this failure, IGF-I concentrations were measured in a patient after subcutaneous injection of 0.1 and 0.2 mg/kg of rhIGF-I. IGF-I concentrations increased only transiently because of the short half-life (1.3-3 h, compared to a normal range of 17-22 h). No correlation was found between IGF-I concentrations and glucose or insulin concentrations. The short half-life of IGF-I may contribute to treatment failure in patients with inherited insulin-resistant syndromes.

Acanthosis Nigricans↗
PubMed2001

Insulin increases the turnover rate of Na+-K+-ATPase in human fibroblasts.

Insulin stimulates K+ transport by the Na+-K+-ATPase in human fibroblasts. In other cell systems, this action represents an automatic response to increased intracellular [Na+] or results from translocation of transporters from an intracellular site to the plasma membrane. Here we evaluate whether these mechanisms are operative in human fibroblasts. Human fibroblasts expressed the alpha(1) but not the alpha(2) and alpha(3) isoforms of Na+-K+-ATPase . Insulin increased the influx of Rb+, used to trace K+ entry, but did not modify the total intracellular content of K+, Rb+, and Na+ over a 3-h incubation period. Ouabain increased intracellular Na+ more rapidly in cells incubated with insulin, but this increase followed insulin stimulation of Rb+ transport. Bumetanide did not prevent the increased Na+ influx or stimulation of Na+-K+-ATPase. Stimulation of the Na+-K+-ATPase by insulin did not produce any measurable change in membrane potential. Insulin did not affect the affinity of the pump toward internal Na+ or the number of membrane-bound Na+-K+-ATPases, as assessed by ouabain binding. By contrast, insulin slightly increased the affinity of Na+-K+-ATPase toward ouabain. Phorbol esters did not mimic insulin action on Na+-K+-ATPase and inhibited, rather than stimulated, Rb+ transport. These results indicate that insulin increases the turnover rate of Na+-K+-ATPases of human fibroblasts without affecting their number on the plasma membrane or modifying their dependence on intracellular [Na+].

Androstadienes↗
PubMed2000

Maturation-dependent expression and function of the CD49d integrin on monocyte-derived human dendritic cells.

Dendritic cells (DC) are highly specialized APC that are critical for the initiation of T cell-dependent immune responses. DC exert a sentinel function while immature and, after activation by inflammatory stimuli or infectious agents, mature and migrate into lymphoid organs to prime T cells. We have analyzed integrin expression on monocyte-derived DC (MDDC) and found that expression of CD49d integrins (CD49d/CD29 and CD49d/beta7) was induced/up-regulated during TNF-alpha- or LPS-initiated MDDC maturation, reflecting the induction/up-regulation of CD49d and beta7 mRNA. CD49d mRNA steady-state level increased more than 10 times during maturation, with the highest levels observed 24 h after TNF-alpha treatment. CD49d integrin expression conferred mature MDDC with an elevated capacity to adhere to the CS-1 fragment of fibronectin, and also mediated transendothelial migration of mature MDDC. Up-regulation of CD49d integrin expression closely paralleled that of the mature DC marker CD83. CD49d integrin expression was dependent on cell maturation, as its induction was abrogated by N:-acetylcysteine, which inhibits NF-kappaB activation and the functional and phenotypic maturation of MDDC. Moreover, CD49d integrin up-regulation and MDDC maturation were prevented by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase, but were almost unaffected by the mitogen-activated protein/extracellular signal-related kinase kinase 1/2 inhibitor PD98059. Our results support the existence of a link between functional and phenotypic maturation of MDDC and CD49d integrin expression, thus establishing CD49d as a maturation marker for MDDC. The differential expression of CD49d on immature and mature MDDC might contribute to their distinct motility capabilities and mediate mature DC migration into lymphoid organs.

Acetylcysteine↗
PubMed2000

Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton.

Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter protein, binding several structural and signaling molecules. alpha-Tubulin was identified as an interacting protein in a two-hybrid screen using the paxillin C-terminal LIM domain as a bait. In vitro binding assays with glutathione S-transferase-paxillin demonstrated an interaction of alpha-tubulin with the C terminus of paxillin. Another member of the tubulin family, gamma-tubulin, bound to both the N and the C terminus of paxillin. The interaction between paxillin and both alpha- and gamma-tubulin in vivo was confirmed by co-immunoprecipitation from human T lymphoblasts. Immunofluorescence studies revealed that, in adherent T cells, paxillin localized to sites of cell-matrix interaction as well as to a large perinuclear region. Confocal microscopy revealed that this region corresponds to the lymphocyte microtubule organizing center, where paxillin colocalizes with alpha- and gamma-tubulin. The localization of paxillin to this area was observed in cells in suspension as well as during adhesion to integrin ligands. These data constitute the first characterization of the interaction of paxillin with the microtubule cytoskeleton, and suggest that paxillin, in addition to its well established role at focal adhesions, could also be associated with the lymphocyte microtubule network.

Cell Adhesion Molecules↗
PubMed2000

Abnormal sodium stimulation of carnitine transport in primary carnitine deficiency.

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the sodium-dependent carnitine cotransporter OCTN2. The majority of natural mutations identified in this and other Na(+)/solute symporters introduce premature termination codons or impair insertion of the mutant transporter on the plasma membrane. Here we report that a missense mutation (E452K) identified in one patient with primary carnitine deficiency did not affect membrane targeting, as assessed with confocal microscopy of transporters tagged with the green fluorescent protein, but reduced carnitine transport by impairing sodium stimulation of carnitine transport. The natural mutation increased the concentration of sodium required to half-maximally stimulate carnitine transport (K(Na)) from the physiological value of 11.6 to 187 mm. Substitution of Glu(452) with glutamine (E452Q), aspartate (E452D), or alanine (E452A) caused intermediate increases in the K(Na). Carnitine transport decreased exponentially with increased K(Na). The E452K mutation is the first natural mutation in a mammalian cotransporter affecting sodium-coupled solute transfer and identifies a novel domain of the OCTN2 cotransporter involved in transmembrane sodium/solute transfer.

Animals↗
PubMed2000

A missense mutation in the OCTN2 gene associated with residual carnitine transport activity.

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective carnitine transport. This disease can present early in life with hypoketotic hypoglycemia and acute metabolic decompensation, or later in life with skeletal or cardiac myopathy. Mutations abolishing the function of OCTN2, an organic cation/carnitine transporter with twelve putative transmembrane spanning domains, were recently demonstrated in patients with early- and late-onset (up to seven years of age) presentation of this syndrome. Most of the reported mutations are null alleles. Here we evaluate the OCTN2 gene in a male patient who presented at seven years of age with severe dilated cardiomyopathy. Plasma carnitine levels were undetectable and carnitine transport by his fibroblasts was reduced to about 3% of normal controls. This patient was homozygous for a single base pair change in exon 8 of the OCTN2 gene (1354G>A) converting the codon for Glu 452 to Lys (E452K) in the predicted intracellular loop between transmembrane domains 10 and 11. Stable expression of the mutant E452K-OCTN2 cDNA in Chinese hamster ovary (CHO) cells caused a partial increase in carnitine transport to 2-4% of the levels measured in the wild type transporter. This reduced transport activity was associated with normal Km toward carnitine (3.1 +/- 1.1 microM), but markedly reduced Vmax. These results indicate that primary carnitine deficiency can be caused by mutations encoding for carnitine transporters with residual activity, and that the E452K affects a domain not involved in carnitine recognition.

Adult↗
PubMed2000

Functional analysis of mutations in the OCTN2 transporter causing primary carnitine deficiency: lack of genotype-phenotype correlation.

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective carnitine transport. This disease is caused by mutations in the novel organic cation transporter OCTN2 (SLC22A5 gene). The disease can present early in life with hypoketotic hypoglycemia or later in life with skeletal myopathy or cardiomyopathy. To determine whether the variation in phenotypic severity is due to mutations retaining residual function, we extended mutational analysis of OCTN2 to four additional European families with primary carnitine deficiency. Three patients were homozygous for novel missense mutations (R169W, G242V, A301D). The fourth patient was compound heterozygous for R169W and W351R substitutions. Stable expression of all the mutations in CHO cells confirmed that all mutations abolished carnitine transport, with the exception of the A301D mutation in which residual carnitine transport was 2-3% of the value measured in cells expressing the normal OCTN2 cDNA. Analysis of the patients characterized in molecular detail by our laboratory failed to indicate a correlation between residual carnitine transport and severity of the phenotype or age at presentation.

Animals↗
PubMed2000

Insulin-induced gene 33 mRNA expression in Chinese hamster ovary cells is insulin receptor dependent.

Gene 33 (g33) is a non-tissue-specific gene regulated in rat liver and hepatoma cells by insulin and other agents. It is thought to participate in the transition from quiescence to proliferation in mitogen-treated cells. The mechanism(s) by which insulin exerts its action on g33 are not totally understood; it is unclear whether a functional insulin receptor is required for this action. In this study, we evaluate the mechanism for insulin induction of g33 mRNA in Chinese hamster ovary (CHO) cells transfected with the neomycin-resistant plasmid (CHONeoB), human insulin receptor (CHONewIRa), and a kinase-defective insulin receptor mutated at the ATP-binding site (CHOK1018A). Transfected cells had higher levels of insulin binding than that of CHONeoB cells; insulin-induced phosphorylation of the insulin receptor and its intracellular substrates were impaired in CHOK1018A cells. Maximal insulin induction of mRNA(g33) occurred 3 h after hormonal exposure in all cell lines. The degree of insulin stimulation of g33 mRNA levels was four- to sixfold higher in CHONewIRa than in CHONeoB or CHOK1018A cells, which had minimal levels of insulin-stimulated g33 mRNA levels. Half-maximal stimulation of g33 mRNA levels was observed at 0.06 +/- 0.01 nM in CHONewIRa cells, consistent with insulin interaction with its own receptor. Wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI3K), had some effects on insulin stimulation of g33 mRNA in CHO NewIRa cells. PD98059, an inhibitor of mitogen-activated kinase kinase (MAPKK), and rapamycin, a p70 S6 kinase inhibitor, had minimal effect on insulin stimulation of g33 mRNA in all cells tested. By contrast, hydroxy-2-naphthalenylmethyl)phosphonic acid triacetoxymethyl ester (HNMPA(AM)(3), a selective inhibitor of the insulin receptor tyrosine kinase, caused complete inhibition of insulin stimulation of g33 mRNA levels. These data indicate that the insulin receptor with intact kinase activity is required for insulin stimulation of g33 mRNA levels. They also suggest that AKT, a PI 3-kinase downstream effector molecule, could mediate insulin stimulation of g33 mRNA. The mechanism(s) of insulin regulation of g33 expression downstream of receptor do not seem to rely entirely on the classic insulin receptor transduction pathway, as a minor effect was observed upon inhibition of MAPKK, suggesting that multiple pathways may be involved.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Functional characterization of the carnitine transporter defective in primary carnitine deficiency.

Primary carnitine deficiency is an autosomal recessive disorder caused by defective carnitine transport which impairs fatty acid oxidation and manifests as nonketotic hypoglycemia or skeletal or heart myopathy. Here we report the functional characterization of this transporter in human fibroblasts. Carnitine enters normal cells by saturable and unsaturable routes, the latter corresponding to Na+-independent uptake. Saturable carnitine transport was absent in cells from patients with primary carnitine deficiency. In control cells, saturable carnitine transport was energized by the electrochemical gradient of Na+. Carnitine uptake was not inhibited by amino acid substrates of transport systems A, ASC, and X-AG, but was inhibited competitively (in potency order) by butyrobetaine > carnitine > palmitoylcarnitine = acetylcarnitine > betaine. Carnitine uptake was also noncompetitively inhibited by verapamil and quinidine, inhibitors of the multidrug resistance family of membrane transporters, suggesting that the carnitine transporter may share a functional motif with this class of transporters. A high-affinity carnitine transporter was present in kidney 293 cells, but not in HepG2 liver cells, whose carnitine transporter had a Km in the millimolar range. These result indicate the presence of multiple types of carnitine transporters in human cells.

Acetylcarnitine↗