Biomedical subjects

R Tremblay

Publications and source records attributed to R Tremblay.

At least 19 recordsLinked to original sources

PubMed2005

Rapid fabrication and chemical patterning of polymer microstructures and their applications as a platform for cell cultures.

Much of the current knowledge regarding biological processes has been obtained through in-vitro studies in bulk aqueous solutions or in conventional Petri-dishes, with neither methodology accurately duplicating the actual in-vivo biological processes. Recently, a number of innovative approaches have attempted to address these shortcomings by providing substrates with controlled features. In particular, tunable surface chemistries and topographical micro and nanostructures have been used as model systems to study the complex biological processes. We herein report a versatile and rapid fabrication method to produce a variety of microstructured polymer substrates with precise control and tailoring of their surface chemistries. A poly(dimethylsiloxane) (PDMS) substrate, produced by replication over a master mold with specific microstructures, is modified by a fluoro siloxane derivative to enhance its anti-adhesion characteristics and used as a secondary replication mold. A curable material, deposited by spin coating on various substrates, is stamped with the secondary mold and crosslinked. The removal of the secondary mold produces a microstructured surface with the same topographical features as the initial master mold. The facile chemical patterning of the microstructured substrates is demonstrated through the use of microcontact printing methods and these materials are tested as a platform to guide cell attachment, growth and proliferation. The master mold and flexible fluorinated PDMS stamps can be used in a repeated manner without any degradation of the anti-adhesion characteristics opening the way to the development of high-throughput fabrication methods that can yield reliable and inexpensive microstructured and chemically patterned substrates.

Animals↗

PubMed2001

Insulin-like growth factor binding proteins (IGFBPs) as potential physiological substrates for human kallikreins hK2 and hK3.

Insulin-like growth factors (IGFs) are important growth regulators of both normal and malignant prostate cells. Their action is regulated by six insulin-like growth factor binding proteins (IGFBPs). The proteolytic cleavage of IGFBPs by various proteases decreases dramatically their affinity for their ligands and therefore enhances the bioavailability of IGFs. To elucidate the putative biological role of prostatic kallikreins hK2 and hK3 (prostate-specific antigen) in tumour progression, we analyzed the degradation of IGFBP-2, -3, -4 and -5 by these two tissue kallikreins. We found that hK3, already characterized as an IGFBP-3 degrading protease, cleaved IGFBP-4 but not IGFBP-2 and -5, whereas hK2 cleaved all of the IGFBPs much more effectively, and at concentrations far lower than those reported for other IGFBP-degrading proteases. The proteolytic patterns after cleavage of IGFBPs by hK2 and hK3 were similar and were not modified in the presence of IGF-I. Heparin, but not other glycosaminoglycans, enhanced dramatically the ability of hK3 but not hK2 to degrade IGFBP-3 and IGFBP-4. More importantly, the IGFBP fragments generated by hK2 and hK3 had no IGF-binding capacity, as assessed by Western ligand blotting. Our results suggest that the prostatic kallikreins hK2 and hK3 may influence specifically the tumoral growth of prostate cells through the degradation of IGFBPs, to increase IGF bioavailability.

Binding Sites↗

PubMed2001

Seroprevalence of infection with Mycobacterium avium subspecies paratuberculosis, bovine leukemia virus, and bovine viral diarrhea virus in maritime Canada dairy cattle.

The purpose of this study was to survey the seroprevalence of infection with the agents of production-limiting diseases in dairy cattle in New Brunswick, Nova Scotia, and Prince Edward Island. In 30 randomly selected herds per province, 30 cattle per herd were randomly selected and tested for antibodies to bovine leukemia virus (BLV) and Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), while 5 unvaccinated cattle over 6 months of age were tested for antibodies to bovine viral diarrhea virus (BVDV). For BLV, 20.8% (15.8% to 27.0%) of cows were positive, and 70.0% (60.3% to 79.7%) of herds had at least one positive cow. In BLV-positive herds, the average BLV prevalence was 30.9% (24.8% to 37.2%). For M. paratuberculosis, 2.6% (1.8% to 3.9%) of cows were positive, and 16.7% (8.8% to 24.5%) of herds had at least 2 M. paratuberculosis-positive cows. In M. paratuberculosis-positive herds, the average M. paratuberculosis prevalence was 8.5% (6.9% to 10.1%). For BVDV, 46.1% (35.5% to 56.7%) of herds had at least 1 BVDV-positive animal with a titer greater than or equal to 1:64.

Acute Disease↗

PubMed2000

Transient NMDA receptor inactivation provides long-term protection to cultured cortical neurons from a variety of death signals.

NMDA receptor antagonists, such as (+)-5-methyl-10, 11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (MK-801), potently block glutamate-induced neuronal death in myriad in vitro cell models and effectively attenuate ischemic damage in vivo. In this report, a novel role for MK-801 and other NMDA receptor antagonists in preconditioning neurons to withstand a wide range of subsequent lethal insults is described. A brief 30 min exposure to 0.1 microM MK-801, applied up to 96 hr before a "lethal" insult, protected primary cortical neurons from a diverse group of neurotoxic agents, including NMDA, beta-amyloid, staurosporine, etoposide, and oxygen-glucose deprivation. This neuroprotective preconditioning by MK-801 arose from transient NMDA receptor inactivation, because the noncompetitive NMDA receptor antagonists memantine and nylindin and the competitive antagonist AP-5 gave similar effects. MK-801 protection was dependent on new protein synthesis during the first 2 hr, but not from 2 to 5 hr, after MK-801 exposure. The MK-801 transient did not alter the ability of NMDA to trigger normally lethal [Ca(2+)](i) influx 48 hr later, but it did block early downstream signaling events coupled to NMDA neurotoxicity, including PKC inactivation and the activation of calpain. Moreover, MK-801 protected neurons from staurosporine-induced apoptosis, although caspase activation in these cells was unimpeded. It is likely that the stress associated with transient inactivation of NMDA receptors triggered a rapid compensatory survival response that provided long-term protection from a spectrum of insults, inducing apoptotic and nonapoptotic death. The possibility that MK-801 preconditioning blocks an event common to seemingly diverse death mechanisms suggests it will be an important tool for obtaining a clearer understanding of the salient molecular events at work in neuronal death and survival pathways.

Adrenergic beta-Agonists↗

PubMed2000

Fetal biometric parameters, twin type and birth weight difference. A longitudinal study.

OBJECTIVE: To examine the relationship between twin type, divided by zygosity, chorionicity, and birth weight difference [more or less than 15% intertwin weight difference at delivery] on fetal biometric measurements, including biparietal diameter, transverse abdominal diameter, head and abdominal circumferences (AC) and head and abdominal areas, femur length (FL), transverse cerebellar diameter, cerebellar circumference and cerebellar area at 18, 23, 28 and 32 weeks of gestational age. STUDY DESIGN: A prospective and longitudinal study was done on 75 twin pairs that did not present the twin-twin transfusion syndrome (150 fetuses) divided into birth weight difference group I (<15%) and group II (>/=15%). The twin pairs were divided into three groups: dizygotic, monochorionic, and dichorionic-monozygotic. In each group the differences in various ultrasound indices were evaluated in reference to discordant growth or concordant growth. RESULTS: In our sample, there were no monozygotic twin pairs with dichorionic placentation with a birth weight difference of more than 15%. Significant associations of group and gestational age were found in dizygotic and monochorionic twins. Intrapair differences were significantly higher in group II (>/=15%) than in group I (<15%) for all fetal parameters studied except for AC, abdominal area and all cerebellar parameters. The value of these discrepancies increased according to gestational age except for FL. Significant interactions between group and zygosity type indicated that intrapair FL differences were more associated with group in monochorionic twins, whereas intrapair transverse cerebellar diameter differences were more associated with group in dizygotic twins. CONCLUSION: Our data show that most fetal biometric parameters are associated with birth weight discordancy. Abdominal area could be a relevant marker for twins with obstetric complications. Note that this is the first research that has studied a twin sample divided by both twin type and birth weight group.

Biometry↗

PubMed2000

Veno-venous continuous renal replacement therapy for burned patients with acute renal failure.

From 1995 to 1998, 12 burned patients with acute renal failure (ARF) were treated by veno-venous continuous renal replacement therapy (CRRT) at the Burn Unit of Hôtel-Dieu de Montréal. Their mean (+/-SD) age was 51+/-12 years, and the mean burned surface covered 48.6+/-15.8% of total body surface area. All patients were mechanically ventilated and presented evidence of sepsis. The mean delay before occurrence of ARF was 15+/-6 days and ARF was mainly related to sepsis and hypotension. Main reasons for CRRT initiation were azotemia and fluid overload. A total of 15 CRRT modalities were applied (12 continuous veno-venous hemodiafiltration, CVVHDF; two continuous veno-venous hemofiltration, CVVH; and one continuous veno-venous hemodialysis, CVVHD) over 14+/-13 days. For CRRT, nine patients received heparin and three were not anticoagulated. Mean values for dialysate and reinjection flow rates were 1134+/-250 ml/h and 635+/-327 ml/h, respectively. Admission weight was 78.8+/-12.7 kg with a mean weight gain before CRRT initiation of 10.0+/-5.8 kg and a mean weight loss during CRRT of 8.9+/-5.5 kg. Nine patients received enteral plus parenteral nutrition, and three, parenteral nutrition only; the total caloric intake was 31.5+/-7.0 kcal/kg/day and protein intake, 1.8+/-0.4 g/kg/day. The normalized protein catabolic rate (nPCR) was evaluated at 2.28+/-0.78 g/kg/day during CRRT. The mortality rate was 50%. The six survivors all recovered normal renal function with four of them requiring intermittent hemodialysis for short periods. In conclusion, veno-venous CRRT is particularly well suited for this selected population allowing smooth fluid removal and aggressive nutritional support.

Acute Kidney Injury↗

PubMed2000

Hyperhomocysteinemia in hemodialysis patients: effects of 12-month supplementation with hydrosoluble vitamins.

BACKGROUND: High-efficiency hemodialysis may induce a deficiency in hydrosoluble vitamins. Supplementation with B-complex vitamins has been shown to lower serum homocysteine concentrations in several groups, but relatively few studies have concerned hemodialysis patients. Our objectives were to determine the status in B-complex vitamins in a large cohort of unsupplemented hemodialysis patients and to assess the effects of supplementation with hydrosoluble vitamins on serum homocysteine over one year. METHODS: Serum total homocysteine (tHcy), vitamin B12, folate, pyridoxal-5'-phosphate (P-5'-P; the active moiety of vitamin B6), as well as red blood cell folate concentrations, were measured in 168 chronic dialysis patients on three times weekly high-efficiency hemodialysis and not supplemented with hydrosoluble vitamins. Their methylenetetrahydrofolate reductase C677T (MTHFR) genotypes were also determined (homozygotes TT, heterozygotes CT, without mutation CC). All involved patients were then supplemented with hydrosoluble vitamins (once daily by mouth, DiaVite; R&D Laboratories, Minneapolis, MN, USA), and half of them were randomized to receive in addition 10 mg intravenously of folic acid posthemodialysis (30 mg intravenously per week). Serum tHcy was monitored after 6 and 12 months of supplementation in the 140 and 128 patients available for follow-up. RESULTS: At baseline, serum and red blood cell folate concentrations were within normal limits in all patients except for two with borderline serum folate (mean values of 21 +/- 8 and 1195 +/- 454 nmol/L), whereas serum vitamin B12 and P-5'-P were below normal in 11 and 65 patients, respectively (mean values of 327 +/- 215 pmol/L and 19 +/- 16 nmol/L for the 168 patients). Initial tHcy levels were increased in all patients (mean 33.3 +/- 16.6 for a normal below 11.8 +/- 1.5 micromol/L); tHcy significantly decreased to 23.5 +/- 7.6 micromol/L after six months (P < 0.0001 vs. baseline) and to 21.7 +/- 6.1 micromol/L after 12 months (P < 0.0001 vs. baseline) for the entire group, but was normalized in only four patients at 12 months. After six months, the mean reduction in tHcy was slightly but significantly greater for patients receiving intravenous folic acid (12.2 +/- 18.5 micromol/L) compared with patients not receiving it (8.3 +/- 9.8 micromol/L, P < 0.05). However, at 12 months, no difference between both subgroups persisted. When considering the different genotypes, tHcy at baseline tended to be higher for TT than CT and CC (39.8 +/- 30.9 vs. 31.4 +/- 10.5 vs. 31.6 +/- 11.8 micromol/L) and decreased to respective values of 21.1 +/- 6.9 versus 21.4 +/- 6.1 versus 22.2 +/- 5.9 micromol/L at 12 months. The impact of the addition of folic acid to DiaVite appeared particularly significant in TT patients at six months. CONCLUSIONS: (1) Hyperhomocysteinemia was present in 100% of our hemodialysis patients. (2) Nearly 40% of our unsupplemented hemodialysis patients were deficient in vitamin B6. (3) Supplementation with DiaVite(R) has resulted in significant tHcy reductions for all three genotypes. (4) The impact of the proposed supplementation protocol was found after six months and was maintained, but did not increase further after 12 months of the same regimen. (5) The addition of intravenous folic acid has been associated with a more pronounced decrease in tHcy in TT patients.

Aged↗

PubMed2000

Pharmacological and molecular characterization of glutamate receptors in the MIN6 pancreatic beta-cell line.

The MIN6 pancreatic beta-cell line responds to glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, but not N-methyl-D-aspartate (NMDA) or 1S,3R-trans-ACPD, with increases in [Ca2+]i. This correlates with MIN6 expression of AMPA receptor subunits (GluR1-4) but only weak expression of NMDA NR2 receptor subunits, as determined by reverse transcriptase polymerase chain reaction (RT-PCR). Pharmacological characterization of the MIN6 AMPA receptors showed that AMPA-triggered [Ca2+]i responses were blocked by GYKI 52466, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and pentobarbital. AMPA-triggered [Ca2+]i responses were also blocked in Na(+)-free medium and by the voltage-sensitive Ca2+ channel antagonist La3+. Unlike cortical neuronal cultures, which show a loss of membrane-associated protein kinase C (PKC) activity and die in response to excitatory amino acid exposure, glutamate was not toxic to MIN6 cells and it did not decrease PKC activity. These studies indicate that MIN6 cells possess Ca(2+)-impermeable AMPA receptors that secondarily allow Ca2+ influx following AMPA-induced depolarization and that, despite elevating [Ca2+]i, AMPA is not toxic to these cells. The effects of glutamate and glutamate receptor antagonists on pancreatic cells needs to be better understood if these compounds are to be used as therapeutic agents to treat stroke.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1999

Factors associated with being a bovine-virus diarrhoea (BVD) seropositive dairy herd in the Møre and Romsdal County of Norway.

Factors associated with being a bovine-virus diarrhoea (BVD) seropositive dairy herd were studied in a case-control study of 314 dairy herds in the Møre and Romsdal County of Norway. Information was collected through a mailed questionnaire, and associations were modeled using conditional logistic regression after selecting variables by a best-subset procedure. Purchasing of animals, use of common pasture, herd-to-herd contact over pasture fences, purchasing cattle with insufficient health (about BVD) documentation, and not using dairy advisors were associated with higher risk. In addition, younger farmers were more likely to have a bovine-virus diarrhoea seropositive herd than older farmers. 'Other animal traffic', including use of common animal housing in the summer months and exchange of calves between farmers were also risk factors. Collectively, these factors could explain 51% of the seropositive herds.

Animals↗

PubMed1999

Evidence that brain-derived neurotrophic factor neuroprotection is linked to its ability to reverse the NMDA-induced inactivation of protein kinase C in cortical neurons.

Several lines of evidence indicate that a rapid loss of neuronal protein kinase C (PKC) activity is a characteristic feature of cerebral ischemia and is a necessary step in the NMDA-induced death of cultured neurons. Exposing embryonic day 18 primary rat cortical neurons to 50 microM NMDA or 50 microM glutamate for 10 min caused approximately 80% cell death over the next 24 h, but excitotoxic death was largely averted, i.e., by 70-80%, in cells pretreated with brain-derived neurotrophic factor (BDNF). An 8-h preexposure to BDNF (50-100 ng/ml) maximally protected cortical cells from the effects of NMDA and glutamate, although the transient application of BDNF between 8 and 4 h before NMDA was equally protective. These effects of BDNF were abolished at supralethal, i.e., >100 microM, NMDA concentrations. It is significant that BDNF pretreatment prevented the inactivation of PKC in cortical cells normally seen 30 min to 2 h following lethal NMDA or glutamate exposure. This BDNF effect did not arise from changes in NMDA channel activity because neither whole-cell NMDA current amplitudes nor increases in intracellular free Ca2+ concentration were altered by the 8-h BDNF pretreatment. Furthermore, BDNF offered no neuroprotection to cells treated with the PKC inhibitors staurosporine (10-20 nM), calphostin C (1-2.5 microM), or GF-109203X (100 nM) at the time of NMDA addition. These results underscore the importance of PKC inactivation in glutamate-induced neuronal death. They also suggest that BDNF neuroprotection arises, at least in part, via its ability to block the mechanism by which pathophysiological Ca2+ influx through the NMDA receptor causes membrane PKC inactivation.

Animals↗

PubMed1999

AR-R15896AR blocks the early NMDA-induced loss of MAP2 in primary cortical cultures.

The low-affinity use-dependent N-methyl-D-aspartate (NMDA) receptor antagonist AR-R15896AR is neuroprotective in primary rat cortical cultures exposed to toxic concentrations of NMDA and reduces the magnitude of NMDA-triggered increases in [Ca2+]i. Here we show using fluorescence staining and measurements of microtubule-associated protein-2 (MAP2) levels, that AR-R15896AR inhibits the NMDA-induced loss of MAP2 that occurs within 2 min following NMDA exposure. Understanding the multiple, Ca(2+)-triggered intracellular events that occur following NMDA receptor stimulation is important to the development of safe and effective neuroprotective agents.

Animals↗

PubMed1998

Comparison of the changes in protein kinase C induced by glutamate in primary cortical neurons and by in vivo cerebral ischaemia.

Changes in protein kinase C (PKC) were compared in primary cortical neurons exposed to glutamate and in the CA-1 hippocampal region of rats subjected to transient cerebral ischaemia. After a 15-min exposure of cortical neurons to excitotoxic levels of glutamate, a 50-60% loss of membrane PKC activity but only about a 20% loss in the amount of enzyme was observed, suggesting that in addition to enzyme loss other mechanisms also contributed to the overall loss of membrane PKC activity. Glutamate induced a 25-40% decrease in immunodetectable levels of PKC alpha, beta, gamma, and lambda but no detectable changes in PCK epsilon and zeta. The loss of PKC activity coincided with a shift in electrophoretic mobility of PKC gamma, epsilon, and lambda, but not of PKC alpha, beta, or zeta, suggesting post-translational modification of some PKC isoforms. By comparison, in rats subjected to transient (15-min) global ischaemia, a similar 50-60% decrease in membrane PKC activity, a 20-25% loss in the amount of PKC, and a shift in PKC mobility were observed in CA-1 neurons 6 h post-reperfusion. In both the in vivo and the in vitro "ischaemic" models, administration of the AMPA receptor antagonist NBQX prevented the loss of PKC activity. These results indicate that the loss of PKC observed in in vivo ischaemia is likely to be due to excitotoxic damage and that this event can be closely mirrored in primary neuronal cultures damaged by glutamate.

Animals↗

PubMed1998

Severe acute bovine viral diarrhea in Ontario, 1993-1995.

In 1993, noncytopathic bovine viral diarrhea virus (BVDV) strains with enhanced virulence caused unprecedented outbreaks of severe acute bovine viral diarrhea (BVD) in dairy, beef, and veal herds in Ontario (Canada). Fever, pneumonia, diarrhea, and sudden death occurred in all age groups of cattle. Abortions often occurred in pregnant animals. Gross lesions in the alimentary tract were similar to those associated with mucosal disease, especially in animals >6 months of age. Cattle of all age groups had microscopic lesions in the alimentary tract similar to those seen with mucosal disease. The epidemic peaked in the summer of 1993, with 15% of all bovine accessions from diseased cattle presented to the diagnostic laboratory being associated with BVDV. The virus strains involved in the outbreak were analyzed using monoclonal and polyclonal antibodies and the polymerase chain reaction. The virus isolates from these outbreaks of severe disease were determined to be type 2 BVDV. Type 2 BVDV has been present in Ontario at least since 1981 without causing widespread outbreaks of severe acute BVD, which suggests that type 2 designation in itself does not imply enhanced virulence. Cattle properly vaccinated with type 1 BVDV vaccines appear to be protected from clinical disease.

Abortion, Veterinary↗

PubMed1997

Evidence that the early loss of membrane protein kinase C is a necessary step in the excitatory amino acid-induced death of primary cortical neurons.

A rapid loss of protein kinase C (PKC) activity is a prognostic feature of the lethal damage inflicted on neurons by cerebral ischemia in vivo and by hypoxic and excitotoxic insults in vitro. However, it is not known if this inactivation of PKC is incidental or is an essential part of the neurodegenerative process driven by such insults. To address this issue, the effects of glutamate on PKC activity and neurotoxicity were studied in immature [8 days in vitro (DIV)] and mature (15-20 DIV) embryonic day 18 rat cortical neuronal cultures. Exposing 16 DIV neurons to as little as 20-50 microM glutamate for 15 min was neurotoxic and induced a rapid (approximately 1-2 h) Ca(2+)-dependent inactivation of membrane PKC. By contrast, neurons 8 DIV were resistant to > 800 microM glutamate, and no evidence of PKC inactivation was observed. Reverse transcription-polymerase chain reaction analysis of NMDA and AMPA receptor subtypes and fluorometric intracellular Ca2- concentration measurements of the effects of NMDA, AMPA, kainate, and metabotropic glutamate receptor activation demonstrated that this striking difference in vulnerability was not due to an absence of functional glutamate receptors on neurons 8 DIV. However, 8 DIV neurons became highly vulnerable to low (< 20 microM) concentrations of glutamate when PKC activity was inhibited by 50 nM staurosporine, 1 microM calphostin C, 5 microM chelerythrine, or chronic exposure to 100 nM PMA. A 15-min coapplication of 50 nM staurosporine with glutamate, NMDA, AMPA, or kainate killed between 50 and 80% of 8 DIV cells within the ensuing 24 h. Moreover, cell death was observed in these cells even when PKC inactivation was delayed up to 4 h after glutamate removal. The evidence indicates that a loss of PKC activity is an essential element of the excitotoxic death of neurons 8 DIV and that cellular event(s) responsible for linking glutamate-mediated Ca2+ influx to PKC inactivation in vulnerable neurons 16 DIV are undeveloped in resistant cells 8 DIV. These results also suggest that the loss of neuronal PKC activity observed in cerebral ischemia may indeed be an important part of the neurodegenerative process. The 8 DIV/16 DIV cortical cell model may prove to be valuable in discerning those intracellular signaling events critical to glutamate-mediated neuronal death.

Alkaloids↗

PubMed1997

[Prevalence of positive tuberculin test in a population of patients requiring long-term care in a hospital setting].

OBJECTIVE: To determine the prevalence of positive tuberculin tests in a population of patients requiring long-term care in a hospital setting. DESIGN: Prevalence study: to evaluate reaction to the test among patients who agreed to be included in the study. SETTING: The study took place in two units of the Enfant-Jésus hospital where patients are admitted for chronic care. PARTICIPANTS: A total of 108 patients were eligible for the study; 56 agreed to take part. One patient died before the study was completed. INTERVENTIONS: The tuberculin test consisted of an injection of PPD-T and reading the reaction 48 to 78 hours later. A reaction > 10 mm was considered significant. Patients with insignificant reactions were injected again 2 weeks later in order to evaluate positive response secondary to reactivation of the immune system (booster effect). MAIN OUTCOME MEASURES: Indications of previous tuberculosis, risk factors for tuberculosis, immunosuppressive medication, length of stay in hospital, size of reaction. RESULTS: Seventeen tuberculin tests were positive (30.9%); of these, six were positive after the second injection. CONCLUSION: The prevalence of positive tuberculin tests was high in our elderly population; this finding is comparable to the findings of American studies.

Aged↗

PubMed1996

An early loss in membrane protein kinase C activity precedes the excitatory amino acid-induced death of primary cortical neurons.

Several lines of evidence indicate that a rapid loss of protein kinase C (PKC) activity may be important in the delayed death of neurons following cerebral ischemia. However, in primary neuronal cultures, cytotoxic levels of glutamate have been reported not to cause a loss in PKC as measured by immunoblot and conventional activity methods. This apparent contradiction has not been adequately addressed. In this study, the effects of cytotoxic levels of glutamate, NMDA, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on membrane PKC activity was determined in cortical neurons using an assay that measures only PKC that is active in isolated membranes, which can be used to differentiate active enzyme from that associated with membranes in an inactive state. A 15-min exposure of day 14-18 cortical neurons to 100 microM glutamate, AMPA, or NMDA caused a rapid and persistent loss in membrane PKC activity, which by 4 h fell to 30-50% of that in control cultures. However, the amount of enzyme present in these membranes remained unchanged during this period despite the loss in enzyme activity. The inactivation of PKC activity was confirmed by the fact that phosphorylation of the MARCKS protein, a PKC-selective substrate, was reduced in intact neurons following transient glutamate treatment. By contrast, activation of metabotropic glutamate receptors by trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid was not neurotoxic and induced a robust and prolonged activation of PKC activity in neurons. PKC inactivation by NMDA and AMPA was dependent on extracellular Ca2+, but less so on Na+, although cell death induced by these agents was dependent on both ions. The loss of PKC activity was likely effected by Ca2+ entry through specific routes because the bulk increase in intracellular free [Ca2+] effected by the Ca2+ ionophore ionomycin did not cause the inactivation of PKC. The results indicate that the pattern of PKC activity in neurons killed by glutamate, NMDA, and AMPA in vitro is consistent with that observed in neurons injured by cerebral Ischemia in vivo.

Animals↗

PubMed1996

The desglycinyl metabolite of remacemide hydrochloride is neuroprotective in cultured rat cortical neurons.

The neuroprotective actions of remacemide and its anticonvulsant metabolite 1,2-diphenyl-2-propylamine monohydrochloride (desglycinylremacemide; DGR) a low-affinity NMDA receptor antagonist, were investigated using primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (100 microM) for 15 min killed 85% of the neurons during the next 24 h. This neurotoxicity was blocked in a concentration-dependent manner by adding DGR (5-20 microM), but not its remacemide precursor (10-100 microM), to the cultures during the time of NMDA exposure. This suggests that the neuroprotective, as well as the anticonvulsant, activity of remacemide is mediated by DGR. Neuroprotective concentrations of DGR also inhibited two of the principal acute effects of NMDA. DGR (5-20 microM) prevented the loss of membrane-associated protein kinase C (PKC) activity that developed by 4 h after transient exposure to 100 microM NMDA and reduced the NMDA-triggered increases in intracellular free Ca2+ concentration ([Ca2+]i) by up to 70%. By contrast, remacemide (50 and 100 microM) did not prevent the NMDA-induced loss of PKC activity or reduce the [Ca2+]i responses. These data suggest that DGR protection against NMDA-mediated toxicity in cultured cortical neurons is associated with a reduction of NMDA-triggered [Ca2+]i surges and a prevention of the loss of membrane-associated PKC activity. In addition, the inhibition of NMDA-triggered [Ca2+]i responses by DGR was qualitatively different from the inhibition of these responses by the high-affinity NMDA-receptor antagonists MK-801 and phencyclidine. This may be a consequence of DGR's lower affinity for the NMDA receptor.

Acetamides↗

PubMed1996

DSM-III-R and DSM-III criteria for conduct disorder in preadolescent girls: specific but insensitive.

OBJECTIVE: To determine whether DSM-III and DSM-III-R criteria for conduct disorder identify girls in the general population with early-onset, persistent, and pervasive antisocial behavior. METHOD: 2,251 girls, representative of all girls entering kindergarten in Qúebec, were assessed using parent and teacher ratings of antisocial behavior; a subsample was then rated for the next 6 years (ages 7 to 12) by parent and teacher. At age 10 years, the girls who been rated as antisocial in kindergarten, along with a random sample of those not rated as antisocial, were assessed for DSM-III and DSM-III-R diagnoses of conduct and oppositional defiant disorder using a structured psychiatric interview (Diagnostic Interview Schedule for Children) administered to the parent, teacher, and/or child (n = 381). RESULTS: Of the girls with early-onset, persistent, and pervasive antisocial behavior, 3% met DSM-III-R criteria and 22% met DSM-III criteria for conduct disorder. Conduct disorder was not diagnosed at all in girls who had not been initially rated as antisocial in kindergarten. Lowering the threshold for a DSM-III-R conduct disorder diagnosis to two symptoms and adding the criterion of violation of rules increased the rate of diagnosis to 35% in the pervasively antisocial girls but only to 1% in girls who did not have persistent antisocial behavior. CONCLUSIONS: DSM-III-R criteria for conduct disorder do not identify most preadolescent girls with early-onset, pervasive, and persistent antisocial behavior. Modifications to the DSM-III-R criteria resulted in increased sensitivity without a loss of specificity.

Age of Onset↗