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Biomedical subjects

G Lambert

Publications and source records attributed to G Lambert.

At least 37 records · Page 2Linked to original sources
PubMed2001

Control of cystic fibrosis transmembrane conductance regulator expression by BAP31.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is stringently controlled by molecular chaperones participating in formation of the quality control system. It has been shown that about 75% of all CFTR protein and close to 100% of the [DeltaPhe(508)] CFTR variant are rapidly degraded before leaving the endoplasmic reticulum (ER). B cell antigen receptor-associated proteins (BAPs) are ubiquitously expressed integral membrane proteins that may control association with the cytoskeleton, vesicular transport, or retrograde transport from the cis Golgi to the ER. The present study delivers evidence for cytosolic co-localization of both BAP31 and CFTR and for the control of expression of recombinant CFTR in Chinese hamster ovary (CHO) cells and Xenopus oocytes by BAP31. Antisense inhibition of BAP31 in various cell types increased expression of both wild-type CFTR and [DeltaPhe(508)]CFTR and enabled cAMP-activated Cl(-) currents in [DeltaPhe(508)]CFTR-expressing CHO cells. Coexpression of CFTR together with BAP31 attenuated cAMP-activated Cl(-) currents in Xenopus oocytes. These data therefore suggest association of BAP31 with CFTR that may control maturation or trafficking of CFTR and thus expression in the plasma membrane.

Animals↗
PubMed2001

Nanoparticulate systems for the delivery of antisense oligonucleotides.

Antisense oligonucleotides are molecules that are able to inhibit gene expression being therefore potentially active for the treatment of viral infections or cancer. However, because of their poor stability in biological medium and their weak intracellular penetration, colloidal drugs carriers such as nanoparticles were developed for the delivery of oligonucleotides (ODN). ODN associated to nanoparticles were shown to be protected against degradation and to penetrate more easily into different types of cells. As a consequence, nanoparticles were shown to improve the efficiency of ODNs for the inhibition of the proliferation of cells expressing the point mutated Ha-ras gene. In vivo, polyalkylcyanoacrylate (PACA) nanoparticles were able to efficiently distribute the ODNs to the liver whereas the alginate nanosponges could concentrate the ODNs in the lungs. Finally, ODN loaded to PACA nanoparticles were able to improve in mice, the treatment of RAS cells expressing the point mutated Ha-ras gene.

Animals↗
PubMed2001

Cardiovascular and behavioural responses to psychological stress in spontaneously hypertensive rats: effect of treatment with DSP-4.

We used a model of psychological stress combining exposure to an open-field novel environment, radio-telemetric measurement of blood pressure and heart rate, and behavioural tracking analysis of behavioural parameters. All rats showed significant increases in blood pressure and heart rate for the duration of open-field exposure, with spontaneously hypertensive rats (SHR) showing markedly greater pressor responses and tachycardia when compared to either Wistar-Kyoto (WKY) or Sprague-Dawley rats (SD rats). Behavioural responses in the open-field were unrelated to the magnitude of cardiovascular responses. Open-field exposure on 4 consecutive days induced similar pressor responses and tachycardia on each day. By contrast, behavioural responses were reduced from the second day of open-field exposure. Treatment of SHR and WKY rats with DSP-4, to deplete central noradrenaline levels, did not affect cardiovascular responses in SHR, whereas WKY rats showed a trend towards inhibition. However, in WKY rats, but not SHR, DSP-4 treatment caused a marked reduction in behavioural activity in the open-field. In conclusion, these data show that: (1) SHR display marked cardiovascular hyperreactivity to psychological open-field stress when compared to two normotensive rat strains; (2) unlike behavioural responses, cardiovascular stress responses do not habituate upon repeated stress exposure; and (3) noradrenergic projections from the locus coeruleus do not appear to play a major role in cardiovascular stress responses in SHR or WKY rats, although they may be involved in behavioural responses in WKY rats.

Adrenergic Agents↗
PubMed2001

Investigating the surface expression of the renal type IIa Na+/Pi-cotransporter in Xenopus laevis oocytes.

We have combined a functional assay, surface labeling and immunocytochemical methods to compare total and surface-exposed renal type IIa Na+/Pi cotransporter protein. The wild-type type cotransporter (NaPi-IIa) and its functionally comparable cysteine mutant S460C were expressed in Xenopus oocytes. S460C contains a novel cysteine residue that, when modified by preincubation with methanethiosulfonate reagents, leads to complete suppression of cotransport function. This allowed surface labeling of the S460C using MTSEA-Biotin and confirmation by electrophysiology on the same cell. Protein was analyzed by Western blotting before and after streptavidin precipitation and by immunocytochemistry and immunogold electronmicroscopy. MTSEA-Biotin treatment resulted in a complete inhibition of S460C-mediated Na+/Pi-cotransport activity, which indicated that all transporters at the surface were biotinylated. After biotinylation, only a small fraction of total S460C protein was precipitated by streptavidin compared with the total amount of S460C protein detected in the lysate. Light- and electron-microscopy analysis of oocytes showed a large amount of WT and S460C transporter protein beneath the oocyte membrane. These data indicate that the apparent weak labeling efficiencies of surface-biotinylation-based assays of membrane proteins heterologously expressed in oocytes can be related to diminished incorporation of the protein in the oolemma.

Animals↗
PubMed2001

Polyisobutylcyanoacrylate nanocapsules containing an aqueous core for the delivery of oligonucleotides.

Antisense oligonucleotides (ODNs) with base sequences complementary to a specific RNA can, after binding to intracellular mRNA, selectively modulate the expression of a gene. However, these molecules are poorly stable in biological fluids and are characterized by a low intracellular penetration. In view of using ODNs as active molecules, the development of nanocapsules containing ODNs in their aqueous core was considered. Nanocapsules were prepared by interfacial polymerization of isobutylcyanoacrylate (IBCA) in a W/O emulsion. After ultracentrifugation and re-suspension in water, the nanocapsules displayed a size of 350+/-100 nm. Oligonucleotide loading did not significantly influence the zeta potential, suggesting that they were located within the core of the nanocapsules. Fluorescence quenching assays confirmed this localization. When encapsulated in the nanocapsules and incubated in the presence of serum, the ODNs were efficiently protected from degradation by nucleases, whereas ODNs adsorbed onto nanospheres were less efficiently protected. This paper describes, for the first time, a nanotechnology able to encapsulate ODNs, rather than adsorbing them at the surface of a solid support. Such a formulation has great potential for oligonucleotide delivery.

Cyanoacrylates↗
PubMed2001

Analysis of glomerulosclerosis and atherosclerosis in lecithin cholesterol acyltransferase-deficient mice.

To evaluate the biochemical and molecular mechanisms leading to glomerulosclerosis and the variable development of atherosclerosis in patients with familial lecithin cholesterol acyl transferase (LCAT) deficiency, we generated LCAT knockout (KO) mice and cross-bred them with apolipoprotein (apo) E KO, low density lipoprotein receptor (LDLr) KO, and cholesteryl ester transfer protein transgenic mice. LCAT-KO mice had normochromic normocytic anemia with increased reticulocyte and target cell counts as well as decreased red blood cell osmotic fragility. A subset of LCAT-KO mice accumulated lipoprotein X and developed proteinuria and glomerulosclerosis characterized by mesangial cell proliferation, sclerosis, lipid accumulation, and deposition of electron dense material throughout the glomeruli. LCAT deficiency reduced the plasma high density lipoprotein (HDL) cholesterol (-70 to -94%) and non-HDL cholesterol (-48 to -85%) levels in control, apoE-KO, LDLr-KO, and cholesteryl ester transfer protein-Tg mice. Transcriptome and Western blot analysis demonstrated up-regulation of hepatic LDLr and apoE expression in LCAT-KO mice. Despite decreased HDL, aortic atherosclerosis was significantly reduced (-35% to -99%) in all mouse models with LCAT deficiency. Our studies indicate (i) that the plasma levels of apoB containing lipoproteins rather than HDL may determine the atherogenic risk of patients with hypoalphalipoproteinemia due to LCAT deficiency and (ii) a potential etiological role for lipoproteins X in the development of glomerulosclerosis in LCAT deficiency. The availability of LCAT-KO mice characterized by lipid, hematologic, and renal abnormalities similar to familial LCAT deficiency patients will permit future evaluation of LCAT gene transfer as a possible treatment for glomerulosclerosis in LCAT-deficient states.

Animals↗
PubMed2001

The sympathetic neurobiology of essential hypertension: disparate influences of obesity, stress, and noradrenaline transporter dysfunction?

Although the importance of sympathetic nervous activation in the pathogenesis of essential hypertension is well documented, the exact pathophysiology of the sympathetic nervous dysfunction present remains to be delineated. This review details three relatively new findings of disturbed sympathetic neurobiology in hypertension. Adrenaline cotransmission is present in the cardiac sympathetic nerves of patients with essential hypertension, as it is in patients with panic disorder, providing presumptive evidence of exposure to high levels of mental stress in hypertensive patients. In lean patients with hypertension there is also evidence of faulty noradrenaline reuptake into the sympathetic nerves of the heart, an abnormality amplifying the sympathetic neural signal by impairing removal of noradrenaline from the synaptic cleft. If both abnormalities are present in the sympathetic nerves of the kidneys also (which we did not test), there would most probably be a direct contribution to hypertension development. In the kidneys the causal chain between sympathetic overactivity and the development of hypertension is stronger than for the heart. In obesity-related hypertension there is evidence that renal sympathetic tone is high, based on approximately a doubling of the measured rate of spillover of noradrenaline into the renal veins. This increase in sympathetic outflow to the kidneys appears to be a necessary but apparently not a sufficient cause for the development of clinical hypertension, commonly being present also in overweight people with blood pressure in the normotensive range. High renal sympathetic tone in the latter, of course, may well still contribute to elevation of their pressure level, although not on such a scale as to cause clinical hypertension.

Adrenergic Antagonists↗
PubMed2001

Sympathetic nervous system and insulin resistance: from obesity to diabetes.

As the world faces an obesity "epidemic," the mechanisms by which overweight is translated into insulin resistance, hypertension, and diabetes need to be better understood. Although the processes of transition remain uncertain, overactivity of the sympathetic nervous system appears pivotal. In obesity, there is stimulation of sympathetic outflow to the kidneys, evident in increased rates of spillover of noradrenaline into the renal veins, and to skeletal muscle vasculature, demonstrated with microneurography. The cause is unclear, but possibly involves the stimulatory action of leptin released from adipose tissue, or from within the brain, for which there is recent evidence in human obesity. The high renal sympathetic tone contributes to hypertension development by stimulating renin secretion and through promoting renal tubular reabsorption of sodium. Neurally mediated skeletal muscle vasoconstriction reduces glucose delivery and uptake in muscle. Impairment of glucose uptake by skeletal muscle is a hallmark of insulin resistance syndromes. Pharmacologic sympathetic nervous suppression within the central nervous system with imidazoline receptor-binding agents such as rilmenidine is a logical therapeutic approach for lowering blood pressure (BP) in patients with essential hypertension, in whom sympathetic activity is often increased. In addition, drugs of this class appear to have the capacity to favorably modify insulin sensitivity, which has particular relevance in the treatment of hypertensive diabetic patients. In the hypertension accompanying maturity onset obesity, with recent recommendations from advisory bodies setting lower goal BP, and with these lower targets often being reached only with combinations of antihypertensive agents, it is advisable that all drugs used in combination therapy have a favorable or at least a neutral effect on insulin resistance.

Antihypertensive Agents↗
PubMed2001

Subarachnoid haemorrhage-induced sympathoexcitation in rats is reversed by bosentan or sodium nitroprusside.

1. The roles played by nitric oxide (NO) and endothelin (ET) in the genesis of sympathetic nervous activation following experimental subarachnoid haemorrhage was investigated using spectral analysis of blood pressure rhythms. 2. Subarachnoid haemorrhage was induced in conscious rats by injecting 0.3 mL homologous blood via a catheter placed along the surface of the brain and directed towards the circle of Willis. Three hours after the insult and after sympathetic activation was evident, animals received either an acute injection of the ET antagonist bosentan (5 mg/kg, i.v.; n = 7), an infusion of the NO donor sodium nitroprusside (SNP; 18 microg/h; n = 7) or no treatment (n = 7). 3. Three hours following the induction of subarachnoid haemorrhage, the mid-frequency components of systolic blood pressure were markedly elevated, indicating a pronounced sympathoexcitation. However, blood pressure and heart rate levels remained unchanged at this time. In the absence of treatment, the mid-frequency components of blood pressure remained elevated for a subsequent 2 h. Treatment with a non-hypotensive dose of SNP reversed the sympathoexcitation within 1 h. Treatment with bosentan was also effective in reducing the mid-frequency oscillations in blood pressure associated with subarachnoid haemorrhage. 4. Our results indicate that subarachnoid haemorrhage is associated with an acute activation of the sympathetic nervous system. The degree of sympathoexcitation can be reversed by the use of either bosentan or SNP.

Animals↗
PubMed2001

Sympathetic nerve biology in essential hypertension.

1. Although the importance of sympathetic nervous activation in the pathogenesis of essential hypertension is well documented, the exact pathophysiology of the sympathetic nervous dysfunction present remains to be delineated. There are several possible explanations for the increased spillover of noradrenaline from the kidneys and heart to plasma, a key piece of evidence supporting the neurogenic basis of essential hypertension, in addition to the obvious one of an increased rate of sympathetic nerve firing. 2. The possibility that there may be an increase in the density of sympathetic innervation in human hypertension, well documented in the spontaneously hypertensive rat, is currently under investigation by us. 3. Adrenaline cotransmission is present in the cardiac sympathetic nerves of patients with essential hypertension, presumptive evidence of their exposure to high levels of stress and a possible basis for the observed increase in cardiac noradrenaline spillover, through presynaptic augmentation of noradrenaline release. 4. Phenotypic evidence exists also of faulty noradrenaline reuptake into the sympathetic nerves of the heart in essential hypertension, an abnormality that would amplify the sympathetic neural signal by impairing removal of noradrenaline from the synaptic cleft.

Aging↗
PubMed2001

Cysteine mutagenesis reveals novel structure-function features within the predicted third extracellular loop of the type IIa Na(+)/P(i) cotransporter.

The transport function of the rat type IIa Na(+)/P(i) cotransporter is inhibited after binding the cysteine modifying reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) to a cysteine residue substituted for a serine at position 460 (S460C) in the predicted third extracellular loop. This suggests that Ser-460 lies in a functionally important region of the protein. To establish a "structure-function" profile for the regions that flank Ser-460, the substituted cysteine accessibility method was employed. 18 mutants were constructed in which selected amino acids from Arg-437 through Leu-465 were substituted one by one for a cysteine. Mutants were expressed in Xenopus oocytes and transport function (cotransport and slippage) and kinetics were assayed by electrophysiology with or without prior treatment with cysteine modifying (methanethiosulfonate, MTS) reagents. Except for mutant I447C, mutants with cysteines at sites from Arg-437 through Thr-449, as well as Pro-461, were inactive. Cotransport function of mutants with Cys substitutions at sites Arg-462 through Leu-465 showed low sensitivity to MTS reagents. The preceding mutants (Cys substitution at Thr-451 to Ser-460) showed a periodic accessibility pattern that would be expected for an alpha-helix motif. Apart from loss of transport function, exposure of mutants A453C and A455C to MTSEA or 2-(triethylammonium)ethyl MTS bromide (MTSET) increased the uncoupled slippage current, which implicated the mutated sites in the leak pathway. Mutants from Ala-453 through Ala-459 showed less pH dependency, but generally stronger voltage dependency compared with the wild type, whereas those flanking this group were more sensitive to pH and showed weaker voltage dependence of cotransport mode kinetics. Our data indicate that parts of the third extracellular loop are involved in the translocation of the fully loaded carrier and show a membrane-associated alpha-helical structure.

Carrier Proteins↗
PubMed2001

Aging cohort of perinatally human immunodeficiency virus-infected children in New York City. New York City Pediatric Surveillance of Disease Consortium.

BACKGROUND: New York City (NYC) pediatricians are now caring for fewer HIV-infected infants and more school age children and adolescents than earlier in the epidemic. METHODS: Clinical, laboratory and demographic data were abstracted from medical records at 10 NYC centers participating in the CDC Pediatric Spectrum of HIV Disease project. Pediatric AIDS cases and HIV-related deaths reported to the NYC Department of Health were examined. RESULTS: Median age of HIV-infected children in care increased from 3 years in 1989 to 1991 to 6 years in 1995 to 1998. The number of HIV-infected women giving birth in NYC declined 50% from 1990 to 1997 (1630 to 831); increasing numbers were identified prenatally (14% in 1989; 78% after 1995); and most received prenatal zidovudine prophylaxis (73% in 1997). Estimated perinatal transmission decreased to 10% by 1997. Improved identification of seropositive status in infants was associated with an increased proportion of infected infants receiving Pneumocystis carinii pneumonia (PCP) prophylaxis, 84% in 1997. AIDS free survival was longer for children born 1995 to 1998 than for those born before 1995, P = 0.004. In 1998 among children with advanced immunosuppression (CDC category 3), 66% were prescribed 3 or more antiretroviral medicines and 88% received PCP prophylaxis. Citywide AIDS cases and HIV-related deaths fell precipitously beginning in 1996. CONCLUSIONS: Based on the observations of this study, the cohort of NYC HIV-infected children in care is aging, associated with a decline in new HIV infections, high rates of PCP prophylaxis and increased time to AIDS. Falling HIV-related deaths citywide support these observations.

Aging↗
PubMed2001

Suicide prevention in Aboriginal communities: application of community gatekeeper training.

OBJECTIVE: Concern over the high rate of suicide among Aboriginal people on the south coast of NSW led to the development of a project aimed at preventing youth suicide in the Aboriginal communities of the Shoalhaven. This paper describes the development, implementation and evaluation of the project. METHOD: Following extensive consultation with the Aboriginal community, a range of culturally appropriate interventions were developed. The main focus was a series of community gatekeeper training workshops, which aimed to increase the potential of members of the Aboriginal community to identify and support people at risk of suicide and to facilitate their access to helping services. RESULTS: Evaluation of the workshops demonstrated an increase in participants' knowledge about suicide, greater confidence in identification of people who are suicidal, and high levels of intentions to provide help. Attitudes, subjective norms and barriers predicted intentions to help. CONCLUSIONS: The project indicated community members could be successfully trained in the recognition of individuals at risk of suicidal behaviour. Gatekeepers' attitudes and perceived barriers to helping predicted intentions to help those in need. There is a need for longer-term follow-up to assess the extent to which new knowledge and skills are used in practice. IMPLICATIONS: Suicide awareness and skills training have been demonstrated to be an effective early intervention strategy. Gatekeeper training empowers Aboriginal communities and is generally accepted. There is demand for such programs outside the Shoalhaven. The project has a methodological framework that can be easily adapted by other communities.

Adult↗
PubMed2001

Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis.

The numerous functions of the liver are controlled primarily at the transcriptional level by the concerted actions of a limited number of hepatocyte-enriched transcription factors (hepatocyte nuclear factor 1alpha [HNF1alpha], -1beta, -3alpha, -3beta, -3gamma, -4alpha, and -6 and members of the c/ebp family). Of these, only HNF4alpha (nuclear receptor 2A1) and HNF1alpha appear to be correlated with the differentiated phenotype of cultured hepatoma cells. HNF1alpha-null mice are viable, indicating that this factor is not an absolute requirement for the formation of an active hepatic parenchyma. In contrast, HNF4alpha-null mice die during embryogenesis. Moreover, recent in vitro experiments using tetraploid aggregation suggest that HNF4alpha is indispensable for hepatocyte differentiation. However, the function of HNF4alpha in the maintenance of hepatocyte differentiation and function is less well understood. To address the function of HNF4alpha in the mature hepatocyte, a conditional gene knockout was produced using the Cre-loxP system. Mice lacking hepatic HNF4alpha expression accumulated lipid in the liver and exhibited greatly reduced serum cholesterol and triglyceride levels and increased serum bile acid concentrations. The observed phenotypes may be explained by (i) a selective disruption of very-low-density lipoprotein secretion due to decreased expression of genes encoding apolipoprotein B and microsomal triglyceride transfer protein, (ii) an increase in hepatic cholesterol uptake due to increased expression of the major high-density lipoprotein receptor, scavenger receptor BI, and (iii) a decrease in bile acid uptake to the liver due to down-regulation of the major basolateral bile acid transporters sodium taurocholate cotransporter protein and organic anion transporter protein 1. These data indicate that HNF4alpha is central to the maintenance of hepatocyte differentiation and is a major in vivo regulator of genes involved in the control of lipid homeostasis.

Animals↗
PubMed2001

ABCA1 overexpression leads to hyperalphalipoproteinemia and increased biliary cholesterol excretion in transgenic mice.

The discovery of the ABCA1 lipid transporter has generated interest in modulating human plasma HDL levels and atherogenic risk by enhancing ABCA1 gene expression. To determine if increased ABCA1 expression modulates HDL metabolism in vivo, we generated transgenic mice that overexpress human ABCA1 (hABCA1-Tg). Hepatic and macrophage expression of hABCA1 enhanced macrophage cholesterol efflux to apoA-I; increased plasma cholesterol, cholesteryl esters (CEs), free cholesterol, phospholipids, HDL cholesterol, and apoA-I and apoB levels; and led to the accumulation of apoE-rich HDL1. ABCA1 transgene expression delayed 125I-apoA-I catabolism in both liver and kidney, leading to increased plasma apoA-I levels, but had no effect on apoB secretion after infusion of Triton WR1339. Although the plasma clearance of HDL-CE was not significantly altered in hABCA1-Tg mice, the net hepatic delivery of exogenous 3H-CEt-HDL, which is dependent on the HDL pool size, was increased 1.5-fold. In addition, the cholesterol and phospholipid concentrations in hABCA1-Tg bile were increased 1.8-fold. These studies show that steady-state overexpression of ABCA1 in vivo (a) raises plasma apoB levels without altering apoB secretion and (b) raises plasma HDL-C and apoA-I levels, facilitating hepatic reverse cholesterol transport and biliary cholesterol excretion. Similar metabolic changes may modify atherogenic risk in humans.

ATP Binding Cassette Transporter 1↗
PubMed2000

EWS fli-1 antisense nanocapsules inhibits ewing sarcoma-related tumor in mice.

EWS Fli-1, a fusion gene resulting from a t(11;22) translocation is found in 90% of both Ewing's sarcoma and primitive neuroectodermal tumor (PNET). In the present study, we show that recently developed polyisobutylcyanoacrylate nanocapsules with an aqueous core were able to encapsulate efficiently high amounts of phosphorothioate oligonucleotides (ODN) directed against EWS Fli-1 chimeric RNA. Release of these ODN in serum medium was shown to be biphasic which was explained by the presence of two types of nanocapsules able to release ODN with different kinetics. In addition, nanocapsules were found to provide protection of these oligonucleotides from the degradation in serum. These ODN nanocapsules permitted to obtain inhibition of Ewing sarcoma-related tumor in mice after intratumoral injection of a cumulative dose as low as 14.4 nanomoles. This new type of non viral vector shows great potential for in vivo administration of oligonucleotides.

3T3 Cells↗
PubMed2000

Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.

Mice lacking the nuclear bile acid receptor FXR/BAR developed normally and were outwardly identical to wild-type littermates. FXR/BAR null mice were distinguished from wild-type mice by elevated serum bile acid, cholesterol, and triglycerides, increased hepatic cholesterol and triglycerides, and a proatherogenic serum lipoprotein profile. FXR/BAR null mice also had reduced bile acid pools and reduced fecal bile acid excretion due to decreased expression of the major hepatic canalicular bile acid transport protein. Bile acid repression and induction of cholesterol 7alpha-hydroxylase and the ileal bile acid binding protein, respectively, did not occur in FXR/BAR null mice, establishing the regulatory role of FXR/BAR for the expression of these genes in vivo. These data demonstrate that FXR/BAR is critical for bile acid and lipid homeostasis by virtue of its role as an intracellular bile acid sensor.

Animals↗