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N Longo

Publications and source records attributed to N Longo.

At least 55 records · Page 3Linked to original sources
PubMed1993

Altered production of PGE2, IL-1 beta and TNF-alpha by peripheral blood monocytes from HIV-positive individuals at early stages of HIV infection.

We analyzed the in vitro synthesis and release of PGE2, IL-1 beta, and TNF-alpha by peripheral blood monocytes from HIV-infected injection drug users at the early clinical stages of HIV infection. We investigated whether there is a concomitant altered production of PGE2 and proinflammatory cytokines by HIV-positive monocytes. We also evaluated T-cell subsets and lymphocyte transformation response to pokeweed mitogen (PWM) in HIV-positive patients and healthy controls. PGE2 and IL-1 beta levels in supernatants from monocyte cultures were determined by radioimmunoassay (RIA), and TNF-alpha by enzyme immunoassay (EIA). Monocytes from asymptomatic HIV-positive individuals produced spontaneous and significantly increased quantities of PGE2, IL-1 beta, and TNF-alpha. Concomitant increased production of PGE2 and IL-1 beta by monocytes from HIV-positive asymptomatic patients was significantly associated with low CD4+ T-cell numbers (< 500 cells/mm3). We also found a strong association between spontaneous and concomitantly increased production of PGE2 and cytokines by monocytes from asymptomatic HIV-positive individuals and a low lymphocyte transformation response to PWM. Further studies are necessary to establish whether this altered production of PGE2 and proinflammatory cytokines by monocytes from HIV-positive individuals might play a role in the mechanisms involved in the progressive impairment of cell-mediated immunity in HIV infection.

Cells, Cultured↗
PubMed1992

Activation of insulin receptor signaling by a single amino acid substitution in the transmembrane domain.

The insulin receptor is a ligand-activated tyrosine kinase composed of two alpha and two beta subunits. A single transmembrane domain composed of 23 hydrophobic residues is contained in each beta subunit. We examined the role of the transmembrane domain in regulating insulin receptor signaling by inserting a negatively charged amino acid (Asp) for Val938 (V938D). Chinese hamster ovary (CHO) cells were stably transfected with a plasmid containing both the neomycin-resistance gene and either the wild-type or the mutant (V938D) insulin receptor cDNA. Insulin binding increased similarly in CHO cells stably transfected with the wild-type and the V938D-mutant insulin receptor cDNA. Insulin stimulated glucose transport and cell growth in cells expressing the normal insulin receptor. By contrast, in the absence of insulin, glucose transport and cell growth in CHO-V938D cells were as high as in insulin-stimulated control cells and no longer responsive to insulin stimulation. Phosphorylation of the beta subunit of the insulin receptor was also increased in CHO-V938D cells not exposed to insulin. These results support an essential role of the transmembrane domain of the insulin receptor in the transduction of insulin signaling.

Animals↗
PubMed1992

Glucose transport by cultured human fibroblasts: regulation by phorbol esters and insulin.

The regulation of 3-O-methyl-D-glucose (OMG) uptake by insulin and phorbol esters was studied in cultured human skin fibroblasts. Insulin rapidly stimulated OMG uptake through a mechanism independent of new protein synthesis. Maximal insulin effect was reached in 30 min and remained constant up to 12 h. The protein kinase C activators 12-O-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12,13-dibutyrate (PdBU) promoted an initial rapid stimulation followed by a secondary long-term rise of OMG influx. This latter effect of phorbol esters on OMG influx began after 1 h, reached a maximum in 12-15 h, and was prevented by the simultaneous addition of protein synthesis inhibitors, suggesting that phorbol esters increased the synthesis of new glucose transporters. In accord with this interpretation, phorbol esters, but not insulin, increased mRNA levels for two distinct glucose transporters (GLUT1 and GLUT3) in human fibroblasts. Both the rapid and the long-term effects of phorbol esters on OMG influx were dose-dependent and half-maximal stimulations occurred at 15 nM for both PdBU and TPA. Kinetic analysis of OMG uptake indicated that both effects of phorbol esters were associated with an increase in the Vmax of the transport process, with no significant changes of the Km (4-6 mM). These results suggest that, in human fibroblasts, phorbol esters, unlike insulin, produce a long-term stimulation of OMG uptake, which is dependent upon protein synthesis and is associated with increased levels of GLUT1 and GLUT3 mRNA.

3-O-Methylglucose↗
PubMed1992

Glucose transporters.

The recent cloning of families of glucose transporters has made it possible to study their structure and their role in an increasing number of disease states. Two classes of glucose transporters transfer glucose across the plasma membrane of human cells. In epithelial cells lining the jejunum and proximal renal tubules, Na+/glucose cotransporters concentrate glucose inside cells using the transmembrane electrochemical potential of Na+. Molecular defects of different transporters of this class cause familial glucose-galactose malabsorption and renal glycosuria. In the basolateral membrane of epithelial cells and in nonepithelial cells, glucose flows down its concentration gradient through a family of facilitative glucose transporters. Reduced numbers of different facilitative glucose transporters may contribute to both defective insulin secretion and insulin resistance in diabetes mellitus and to neurological diseases associated with decreased cerebrospinal fluid glucose.

Blood Glucose↗
PubMed1992

Reduced mRNA and a nonsense mutation in the insulin-receptor gene produce heritable severe insulin resistance.

Leprechaunism is an autosomal recessive syndrome of severe insulin resistance and is characterized by intrauterine growth restriction, acanthosis nigricans, hirsutism, and loss of glucose homeostasis. Here we report a new female patient of Hispanic and Afro-American descent whose fibroblasts and lymphoblasts had markedly impaired insulin binding (less than 10% of that in controls). Insulin binding to lymphoblasts established from both unrelated parents was partially impaired. Insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) binding to the patient's fibroblasts were within the normal range. Insulin stimulation of receptor autophosphorylation and kinase activity was markedly reduced in the patient's fibroblasts. The patient's fibroblasts had both a reduced number of immunoreactive insulin receptor (6% of those in controls) and concomitantly reduced amounts of insulin-receptor mRNA, suggesting that both mutations inherited by the patient reduced insulin-receptor mRNA. Sequencing of the insulin-receptor gene and cDNA indicated that the patient was heterozygous for a paternally derived mutation at bp 1333, converting Arg372 to a STOP codon. This nonsense mutation was observed in the insulin-receptor gene, but not in cDNA, indicating reduced amounts of mRNA for the allele containing this mutation. The coding sequence of the maternally inherited insulin-receptor allele was normal. Both the marked reduction in insulin-receptor mRNA in the compound heterozygous fibroblasts of the proband and the partially reduced insulin binding in maternal cells suggest that the maternally derived mutation is located in an insulin-receptor gene sequence that controls cellular mRNA content.

Base Sequence↗
PubMed1991

A simple method for evaluation of Rb+ transport and Na(+)-K+ pump stoichiometry in adherent cells.

This report describes a method based on flame photometry for the evaluation of transmembrane Rb+ transport and Na(+)-K+ pump stoichiometry in adherent cells. In monolayers of cultured fibroblasts, the rates of 86Rb+, an isotope widely used as a K+ congener in transport studies, and nonradioactive Rb+ influx were equivalent when measured in the absence and presence of the transport inhibitors ouabain and bumetanide. Ouabain- and bumetanide-sensitive Rb+ fluxes were also equal with the two methods. Flame photometry allowed the simultaneous determination of intracellular [Na+] in the same sample in which Rb was measured. The incubation of human fibroblasts with ouabain for 5 min promoted a significant increase in intracellular [Na+]. Under appropriate experimental conditions, the ratio between the rate of ouabain-promoted increase in intracellular [Na+] and ouabain-sensitive Rb+ influx was 1.4, close to the theoretical value of 1.5 corresponding to a Na(+)-K+ pump stoichiometry of 3 Na+ extruded from the cell in exchange for 2 K+.

Biological Transport, Active↗
PubMed1990

Insulin-receptor autophosphorylation and kinase activity are constitutively increased in fibroblasts cultured from a patient with heritable insulin-resistance.

Mutations in the insulin receptor gene have been described in families with the inherited insulin-resistant syndrome leprechaunism. At a cellular level, these mutations result in decreased insulin binding and impaired insulin stimulation of receptor autophosphorylation and sugar transport. By contrast, we previously found that fibroblasts cultured from leprechaun patient Atl had constitutively increased sugar transport, even though insulin binding was markedly reduced. Here we report that these fibroblasts have basal insulin-receptor autophosphorylation and kinase activity constitutively increased above insulin-stimulated control cells.

Cells, Cultured↗
PubMed1990

Immunological and serological markers predictive of progression to AIDS in a cohort of HIV-infected drug users.

We have performed a prospective 33-month follow-up of the evolution of HIV infection in a cohort of 76 HIV-positive intravenous drug users (IVDUs). We report on immunological and serological variables that proved to be highly predictive of development to AIDS. In a stepwise multivariate analysis of the actuarial progression rate we found the number of CD4+ lymphocytes to be the most powerful predictor of progression to AIDS. We found no independent predictive effects associated with any other variable with predictive power: loss of antibody to p24 antigen, anergy, HIV p24 antigenaemia, loss of antibody to p53 (reverse transcriptase), decreased number of CD8+ T cells, loss of antibody to p31, loss of antibody to p17, beta 2-microglobulin level, loss of antibodies to gp41 and p64, or immunoglobulin A level. We have found that our data differ from those obtained in studies in homosexual men in the different prognostic value of those predictive markers. Our findings should help to identify high risk of progression to clinical AIDS among IVDUs, thereby assisting in the selection of patients for prophylaxis and therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Human fibroblasts express the insulin-responsive glucose transporter (GLUT4).

The transport of glucose across the plasma membrane of nonepithelial cells is mediated by a family of facilitative glucose transporters. One glucose transporter is insulin-responsive (GLUT4) and is found in muscle, heart, and fat cells, differentiated cells which are difficult to maintain and study in culture. Cultured dermal fibroblasts, on the other hand, also are insulin-responsive, can be easily maintained in culture, and retain the genetic complement of the donor. In this paper, we evaluate RNA isolated from cultured human fibroblasts for the expression of four different glucose transporters. Northern blot analysis indicated that human fibroblasts expressed the erythrocyte (GLUT1), the fetal skeletal muscle (GLUT3), and the insulin-responsive (GLUT4) glucose transporters, but not the liver glucose transporter (GLUT2). To confirm the presence of GLUT4 mRNA, cDNA was synthesized from human fibroblast RNA, amplified using primers specific for GLUT4 by the polymerase chain reaction, and sequenced. The sequence was identical to that of GLUT4 cDNA. These data indicate that cultured human fibroblasts express at least three genetically distinct facilitative glucose transporters.

Base Sequence↗
PubMed1989

Increased glucose transport by human fibroblasts with a heritable defect in insulin binding.

Insulin and IGF-I binding and their regulation of hexose transport were evaluated in skin fibroblasts cultured from a family (Atl) whose proband had leprechaunism, hypoglycemia, and severe insulin resistance. High affinity insulin binding to proband Atl cells was absent, and partially, but equally, impaired in fibroblasts from his related parents. IGF-I binding to his cultured fibroblasts was within the normal range. Cells from proband Atl had insulin receptor mRNAs similar to control fibroblasts. 3-O-Methyl-D-glucose (OMG) transport by proband Atl was threefold higher than in control fibroblasts (37.7 v 7.6-11 nmol/mL/s) and was insulin-insensitive. Proband Atl fibroblasts had a threefold increase in the Vmax for OMG entry and a concomitant increase in the number of D-glucose-inhibitable cytochalasin B binding sites on their plasma membrane. Similar levels of glucose transporter mRNA were observed in control and proband Atl fibroblasts. These results suggest that fibroblasts from patient Atl have a genetically transmitted mutation in the alpha subunit of their insulin receptor. In the homozygous affected proband, this mutation impairs insulin binding and causes elevated, insulin-insensitive glucose transport. The dysfunction resulting from this mutation is similar to that introduced in Chinese hamster ovary cells by transfection with a truncated alpha subunit.

3-O-Methylglucose↗
PubMed1989

Restriction fragment length polymorphisms of the insulin receptor gene in families with insulin resistance and leprechaunism.

Restriction fragment length polymorphisms (RFLPs) for the insulin receptor gene were compared among controls and families Atl and Ark-1, whose probands have extreme insulin resistance and leprechaunism. Previous studies indicated that fibroblasts cultured from patients Atl and Ark-1 had less than 10% of normal insulin binding. Cells cultured from the related parents of Atl had partial reduction in insulin binding, suggesting that patient Atl inherited the same mutant allele for the insulin receptor from both his parents. By contrast, the parents of Ark-1 were unrelated and their cells had different degrees of impaired insulin binding, suggesting that patient Ark-1 inherited two different, non-complementing alleles for the insulin receptor. To test these hypotheses, RFLPs were defined for the insulin receptor gene and their transmission was studied in families Atl and Ark-1. Patients affected with leprechaunism had no private polymorphisms in the insulin receptor gene, indicating that gross rearrangements were not present. A polymorphism generated with EcoRI at the 5' end of the insulin receptor gene gave fragments of 16 and 20 kb and was the only one informative in family Atl. Both parents were heterozygous, presenting a 16 and a 20 kb fragment, and transmitted the 20 kb allele to their affected offspring. Ark-1's parents were heterozygous for three different RFLPs, including this EcoRI polymorphism, and patient Ark-1 inherited a different allele for the insulin receptor from each parent. These results support the genetic hypotheses suggested by insulin-binding studies and indicate that RFLPs can be used to identify transmission of the insulin receptor gene in families with insulin resistance.

DNA Probes↗
PubMed1989

Increased synthesis and production of prostaglandin E2 by monocytes from drug addicts with AIDS.

We analysed the production of prostaglandin E2 (PGE2) by monocytes in 11 drug addicts with AIDS and opportunistic infections and in 13 controls. In three patients and six controls we isolated the PGE2 fraction from culture supernatants of purified monocytes (greater than 95%) incubated in medium containing (3H) arachidonic acid, using silicic acid columns. In two of three patients PGE2-fraction values (21.9 and 21.6 pmol/g protein) were significantly higher than controls (10 +/- 4). In eight AIDS patients and seven controls, PGE2 levels were determined by high-performance liquid chromatography and radioimmunoassay (HPLC and RIA). In three out of eight patients PGE2 levels were markedly higher (736, 419 and 208 pg/ml) than the mean + 2 s.d. values from controls (73 +/- 51). We tested the effect of PGE2 on the production of interleukin-2 by normal phytohemagglutinin-stimulated peripheral blood lymphocytes (PBL). There was a significant suppression of interleukin-2 production by PGE2 released from AIDS monocytes. PGE2 may be one of the factors involved in the severe immune dysfunction associated with AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Glycine transport by cultured human fibroblasts.

The transport of glycine was studied in cultured human fibroblasts. The amino acid entered the cell by Na+-dependent and Na+-independent mechanisms. Na+-independent glycine (0.1 mM) transport was less than 10% of total uptake and occurred by a mechanism formally indistinguishable from diffusion. Two distinct routes contributed to Na+-dependent glycine transport. The first route was identified with system A because it was inhibited by MeAIB and underwent adaptive regulation. The second route was identified with system ASC as it was inhibited by L-alanine, but not by MeAIB. Kinetic analysis revealed that the two systems operated glycine transport with the same Km of 1.6 mM, a value unusually high for system ASC.

Adult↗
PubMed1988

Influx and efflux of 3-O-methyl-D-glucose by cultured human fibroblasts.

We measured the initial rates of 3-O-methyl-D-glucose (OMG) fluxes by cultured human fibroblasts. D-Glucose (300 mM) and cytochalasin B (5 microM) inhibited approximately 80% of OMG (1 mM) influx. OMG rapidly entered human fibroblasts, and influx was linear up to 20 s. OMG influx and efflux were about equal. Cytochalasin B inhibited OMG (1 mM) influx and efflux within 20 s of exposure. Cytochalasin B half maximally inhibited OMG influx and efflux at 0.51 and 0.75 microM, respectively. In zero trans conditions, the kinetics of OMG influx and efflux were similar. However, when OMG was present on the trans side of the membrane, OMG influx but not OMG efflux was stimulated. Trans stimulation of OMG influx increased the maximal velocity of this transport process, without affecting its Km. These results suggest that 1) OMG influx and efflux occur through the same transporter, and 2) the glucose transporter of cultured human fibroblasts presents a functional asymmetry when substrate is present on the trans side of the membrane.

3-O-Methylglucose↗