Attenuation of staurosporine-induced apoptosis, oxidative stress, and mitochondrial dysfunction by synthetic superoxide dismutase and catalase mimetics, in cultured cortical neurons.

Neuronal apoptosis induced by staurosporine (STS) involves multiple cellular and molecular events, such as the production of reactive oxygen species (ROS). In this study, we tested the efficacy of two synthetic superoxide dismutase/catalase mimetics (EUK-134 and EUK-189) on neuronal apoptosis, oxidative stress, and mitochondrial dysfunction produced by STS in primary cortical neuronal cultures. Exposure of cultures to STS for 24 h increased lactate dehydrogenase (LDH) release, the number of apoptotic cells, and decreased trypan blue exclusion. Pretreatment with 20 microM EUK-134 or 0.5 microM EUK-189 significantly attenuated STS-induced neurotoxicity, as did pretreatment with the caspase-1 inhibitor, Ac-YVAD-CHO, but not the caspase-3 inhibitor, Ac-DEVD-CHO. Posttreatment (1-3 h following STS exposure) with 20 microM EUK-134 or 0.5 microM EUK-189 significantly reduced STS-induced LDH release, in a time-dependent manner. Exposure of cultures to STS for 1 h produced an elevation of ROS, as determined by increased levels of 2,7-dichlorofluorescein (DCF). This rapid elevation of ROS was followed by an increase in lipid peroxidation, and both the increase in DCF fluorescence and in lipid peroxidation were significantly blocked by pretreatment with EUK-134. STS treatment for 3-6 h increased cytochrome c release from mitochondria into the cytosol, an effect also blocked by pretreatment with EUK-134. These results indicate that intracellular oxidative stress and mitochondrial dysfunction are critically involved in STS-induced neurotoxicity. However, there are additional cellular responses to STS, which are insensitive to treatment with radical scavengers that also contribute to its neurotoxicity.

Neonatal resuscitation training program in Malaysia: results of the first 2 years.

OBJECTIVES: To determine the number of providers and instructors trained by the initial 37 core instructors during the first 2 years following the launch of the Malaysian Neonatal Resuscitation Program (NRP). To identify remediable problems which interfered with the propagation of the NRP in Malaysia. METHODOLOGY: A prospective observational study carried out over a 2-year period between 2 September 1996 to 2 September 1998. For every training course conducted, the instructors completed a NRP course report form (Form A) that documented the instructors involved in the course. For every participant who attended the course and successfully completed it, the instructors submitted a record form (Form B) that contained the name, hospital address, department, profession, place of work, language used for training and the marks obtained by the individual participant. After each course, completed forms A and B were returned to the NRP secretariat for compilation. RESULTS: Of the 37 core instructors, 35 (94.6%) carried out training courses in their respective home states. A further 513 new instructors and 2256 providers were trained subsequently. A total of 2806 health personnel from all 13 states of Malaysia were NRP-certified during the first 2 years. However, 61.2% (n = 335) of the 550 instructors were inactive trainers, having trained less than four personnel per instructor a year. Most of the NRP-certified personnel were either doctors (32.0%) or nursing staff (64.4%). More than 60% of these worked either in the labour rooms, neonatal intensive care units or special care nurseries. At least one person from all three university hospitals and all general hospitals, 89.3% (92/103) of the district hospitals, 3.5% (73/2090) of the maternal and child health services, and 21% (46/219) of the private hospitals and maternity homes, were trained in the NRP. CONCLUSION: Dissemination of the NRP in Malaysia during the first 2 years was very encouraging. Further efforts should be made to spread the program to private hospitals and the maternal and child health services. In view of the large number of inactive instructors, the criteria for future selection of instructors should be more stringent.

Prevention of 1-methyl-4-phenylpyridinium- and 6-hydroxydopamine-induced nitration of tyrosine hydroxylase and neurotoxicity by EUK-134, a superoxide dismutase and catalase mimetic, in cultured dopaminergic neurons.

Oxidative stress has been implicated in the selective degeneration of dopaminergic (DAergic) neurons in Parkinson's disease (PD). In this study, we tested the efficacy of EUK-134, a superoxide dismutase (SOD) and catalase mimetic, on the nitration of tyrosine hydroxylase (TH), a marker of oxidative stress, and neurotoxicity produced by 1-methyl-4-phenylpyridinium (MPP(+)) and 6-hydroxydopamine (6-OHDA) in primary DAergic neuron cultures. Exposure of cultures to 10 microM MPP(+) reduced dopamine (DA) uptake and the number of tyrosine hydroxylase immunoreactive (THir) neurons to 56 and 52% of control, while exposure to 30 microM 6-OHDA reduced DA uptake and the number of THir neurons to 58 and 59% of control, respectively. Pretreatment of cultures with 0.5 microM EUK-134 completely protected DAergic neurons against MPP(+)- and 6-OHDA-induced neurotoxicity. Exposure of primary neuron cultures to either MPP(+) or 6-OHDA produced nitration of tyrosine residues in TH. Pretreatment of cultures with 0.5 microM EUK-134 completely prevented MPP(+)- or 6-OHDA-induced nitration of tyrosine residues in TH. Taken together, these results support the idea that reactive oxygen species (ROS) are critically involved in MPP(+)- and 6-OHDA-induced neurotoxicity and suggest a potential therapeutic role for synthetic catalytic scavengers of ROS, such as EUK-134, in the treatment of PD.

Neurotrophin-4/5 promotes dendritic outgrowth and calcium currents in cultured mesencephalic dopamine neurons.

Ca(2+) currents and their modulation by neurotrophin-4/5 were studied in cultured mesencephalic neurons. Tyrosine hydroxylase-positive neurons consistently had larger somas than tyrosine hydroxylase-negative neurons. Neurons with larger somas were therefore targeted for recording. In both control and neurotrophin-4/5-treated cultured neurons, isolation of Ca(2+) currents in cultured mesencephalic neurons revealed prominent low- and high-voltage-activated currents. These currents were separable based upon their voltage dependence of activation, the response to replacement of Ca(2+) with Ba(2+) and the response to Ca(2+) channel blockers. Replacement of Ca(2+) with Ba(2+) resulted in a slight reduction of low-voltage-activated currents and a significant enhancement of high-voltage-activated currents. Cd(2+) blocked a larger fraction of the high-voltage-activated current than Ni(2+). The synthetic conotoxins SNX-124 and SNX-230 selectively blocked high-voltage-activated currents. Morphological analysis of mesencephalic cultures pretreated with neurotrophin-4/5 revealed an increase in soma size and dendritic length in tyrosine hydroxylase-positive neurons. In agreement with the neurotrophin-4/5 induction of growth, neurotrophin-4/5 also increased cell capacitance in whole-cell recordings. Neurotrophin-4/5 significantly enhanced both low- and high-voltage-activated currents, but normalization for changes in capacitance revealed only a significant increase in high-voltage-activated current density. This study demonstrates the existence of low-voltage-activated and multiple classes of high-voltage-activated calcium currents in cultured mesencephalic neurons. Morphological and physiological data demonstrate that the increases in calcium currents due to neurotrophin-4/5 pretreatment are associated with somatodendritic growth, but an increase in high-voltage-activated Ca(2+) channel expression also occurred.

Adolescents' knowledge of human papillomavirus and cervical dysplasia.

STUDY OBJECTIVE: This study examined adolescents' knowledge of human papillomavirus (HPV) and cervical dysplasia (CD). Factors associated with knowledge and self-reported change in health-related behaviors were identified. DESIGN: Interviews were conducted at an average of 2.5 years following the diagnosis of HPV/CD. Medical charts were reviewed. SETTING: The study was conducted at a university-based adolescent dysplasia clinic. PARTICIPANTS: Fifty females, ages 15-23 participated in the study: 88% African-American, 12% Caucasian. RESULTS: On average, participants responded correctly to 86% of the questions regarding HPV/CD. However, the following key points were routinely missed: 52% did not know cigarette smoking increased the risk for cervical cancer; 42% believed that HPV/CD was always symptomatic; and 22% did not know condoms decreased the transmission of HPV. According to participants, their health care provider explained the diagnosis and treatment of HPV/CD using words they understood "some" or "most of the time." Higher academic skills significantly correlated with greater knowledge of HPV/CD. Forty-one percent of participants with a smoking history reportedly increased their smoking since the diagnosis, and only 40% used condoms "most of the time." However, 90% had maintained or increased their frequency of Pap tests. CONCLUSIONS: Adolescent girls had knowledge of most factors related to HPV/CD, but many did not understand the risks of cigarette smoking and failure to use condoms. To improve understanding and compliance, health care providers should tailor educational strategies to the functional level of adolescents.

Inhibition of phosphatidylinositol 3-kinase activity blocks cellular differentiation mediated by glial cell line-derived neurotrophic factor in dopaminergic neurons.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for midbrain dopaminergic neurons. To begin to understand the intracellular signaling pathways used by GDNF, we investigated the role of phosphatidylinositol 3-kinase activity in GDNF-stimulated cellular function and differentiation of dopaminergic neurons. We found that treatment of dopaminergic neuron cultures with 10 ng/ml GDNF induced maximal levels of Ret phosphorylation and produced a profound increase in phosphatidylinositol 3-kinase activity, as measured by western blot analysis and lipid kinase assays. Treatment with 1 microM 2-(4-morpholinyl)-8-phenylchromone (LY294002) or 100 nM wortmannin, two distinct and potent inhibitors of phosphatidylinositol 3-kinase activity, completely inhibited GDNF-induced phosphatidylinositol 3-kinase activation, but did not affect Ret phosphorylation. Furthermore, we examined specific biological functions of dopaminergic neurons: dopamine uptake activity and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. GDNF significantly increased dopamine uptake activity and promoted robust morphological differentiation. Treatment with LY294002 completely abolished the GDNF-induced increases of dopamine uptake and morphological differentiation of tyrosine hydroxylase-immunoreactive neurons. Our findings show that GDNF-induced differentiation of dopaminergic neurons requires phosphatidylinositol 3-kinase activation.

Characterization of two distinct monoclonal antibodies specific for glial cell line-derived neurotrophic factor.

Here we report the generation and characterization of two distinct monoclonal antibodies, G-90 and B-1531, specific to glial cell line-derived neurotrophic factor (GDNF). ELISA results confirmed that G-90 and B-1531 both recognize GDNF. Western blots showed that G-90 recognized only the GDNF dimer, whereas B-1531 recognized both the monomer and dimer. Peptide competition ELISA (PCE) and BIAcore data suggested that G-90 and B-1531 recognize different epitopes: PCE confirmed that B-1531 binds to NH2-terminal peptides between amino acids 18 and 37, whereas G-90 does not; BIAcore data showed that B-1531 binds to the NH2 terminus of GDNF, whereas G-90 does not. G-90, in a concentration-dependent manner, completely neutralized the GDNF-induced increases of choline acetyltransferase in cultured motoneuron and of dopamine uptake and morphological differentiation in dopaminergic neuron cultures. B-1531 had no neutralizing effects. GDNF-induced Ret autophosphorylation in NGR-38 cells was completely neutralized by G-90, whereas B-1531 had a moderate effect. These data show that G-90 and B-1531 are specific antibodies to GDNF. The data also suggest that the NH2 terminus of GDNF is not critical for activity. Partial inhibition of Ret phosphorylation is insufficient to down-regulate GDNF-induced biological activity.

Inhibition of glial cell line-derived neurotrophic factor induced intracellular activity by K-252b on dopaminergic neurons.

The c-ret protooncogene encodes Ret, the functional tyrosine kinase receptor for glial cell line-derived neurotrophic factor (GDNF). K-252b, a known protein tyrosine kinase inhibitor, has been shown earlier to inhibit the trophic activity of brain-derived neurotrophic factor on dopaminergic (DAergic) neurons and nerve growth factor on basal forebrain cholinergic neurons while potentiating neurotrophin-3 activity on central cholinergic and peripheral sensory neurons and PC12 cells. We tested whether K-252b would modulate GDNF-induced differentiation in DAergic neuron cultures. Exposure to 1 ng/ml GDNF increased dopamine (DA) uptake 80% above control, whereas treatment with 5 microM K-252b decreased the efficacy of GDNF by 60%. Concentrations of GDNF of <100 pg/ml were completely inhibited, whereas concentrations of >100 pg/ml were moderately active, between 10 and 20% above control. In addition, K-252b shifted the ED50 from 20 to 200 pg/ml. GDNF treatment increased soma size and neurite outgrowth in tyrosine hydroxylase-immunoreactive neurons. K-252b inhibited differentiation of these morphological parameters induced by GDNF. Furthermore, GDNF stimulated Ret autophosphorylation at maximal levels, whereas the inhibition of DA uptake and morphological differentiation by K-252b correlated with a significantly decreased level of Ret autophosphorylation. Therefore, K-252b is able to inhibit intracellular activities induced by GDNF on mesencephalic DAergic neurons.

Neurotrophin-4/5 treatment reduces infarct size in rats with middle cerebral artery occlusion.

The aim of this study was to determine whether neurotrophin-4/5 (NT-4/5) treatment alters infarction volume following permanent focal cerebral ischemia in the rat. Permanent focal cerebral ischemia was produced in adult male rats by intraluminal occlusion of the right middle cerebral artery. NT-4/5 was administered intraventricularly one day before and immediately following occlusion. Rats were sacrificed at 1, 4 and 7 days after occlusion. NT-4/5 treatment reduced infarction volume by 34% when compared to control rats 1 day after occlusion. Infarction volume was unaltered by treatment 4 to 7 days after occlusion. Middle cerebral artery occlusion led to a significant reduction in levels of mRNAs coding for catalytic and truncated TrkB receptors. This expression was unaffected by NT-4/5 treatment.