Progress in predicting protein function from structure: unique features of O-glycosidases.

The Structural Genomics Initiative promises to deliver between 10,000 and 20,000 new protein structures within the next ten years. One challenge will be to predict the functions of these proteins from their structures. Since the newly solved structures will be enriched in proteins with little sequence identity to those whose structures are known, new methods for predicting function will be required. Here we describe the unique structural characteristics of O-glycosidases, enzymes that hydrolyze O-glycosidic bonds between carbohydrates. O-glycosidase function has evolved independently many times and enzymes that carry out this function are represented by a large number of different folds. We show that O-glycosidases none-the-less have characteristic structural features that cross sequence and fold families. The electrostatic surfaces of this class of enzymes are particularly distinctive. We also demonstrate that accurate prediction of O-glycosidase function from structure is possible.

Thiopurine methyltransferase activity in the Jewish population of Israel.

OBJECTIVE: 6-Mercaptopurine is used therapeutically for its immunosuppressant and cytotoxic properties. It is deactivated by thiopurine methyltransferase (TPMT), which shows genetic polymorphism in many populations. In North American populations, TMPT activity exhibits a trimodal activity pattern. In Oriental populations, TPMT shows almost a unimodal pattern of activity. The purpose of the present study was to assess the activity of TPMT in a Jewish male population sample in Israel. METHODS: The study was approved by the Israeli Ministry of Health. Blood samples of 2.5 ml were collected in heparinized tubes from 134 males. The red blood cell (RBC) fraction of each individual was washed and hemolyzed. TPMT activity in the RBC hemolysate was determined using a radioactive assay with tritiated S-adenosyl methionine as a methyl donor. RESULTS: The activity of TPMT ranged from 3.2 nmol/h/ml to 42.9 nmol/h/ml packed RBCs with mean and median activities of 18.6 nmol/h/ml and 17.9 nmol/h/ml packed RBCs, respectively. The distribution frequency of TPMT was very close to the unimodal by analysis of normal distribution. CONCLUSION: The pattern of distribution of TPMT in the Jewish population of Israel is closer to that of East Asian populations than European and North American populations. This observation may have relevance for the usage of 6-mercaptopurine and azathioprine as therapeutic agents in the Jewish population.

Multicenter double-blind, randomized, placebo-controlled trial of levetiracetam as add-on therapy in patients with refractory partial seizures. European Levetiracetam Study Group.

PURPOSE: To evaluate the efficacy and tolerability of levetiracetam (LEV, Keppra) as add-on therapy in patients with refractory partial seizures. METHODS: In this European multicenter, double-blind, randomized, placebo-controlled trial, LEV (500 or 1,000 mg twice daily) was compared with placebo as add-on therapy in 324 patients with uncontrolled simple or complex partial seizures, or both, with or without secondary generalization. After enrollment, three parallel groups were assessed during a baseline period of 8 or 12 weeks, followed by a 4-week titration interval and a 12-week evaluation period. RESULTS: LEV significantly decreased partial seizure frequency compared with placebo. A reduction in seizure frequency of > or =50% occurred in 22.8% of patients in the 1,000-mg group and 31.6% of patients in the 2,000-mg group, compared with 10.4% of patients in the placebo group. Administration of LEV did not affect plasma concentrations of concomitant antiepileptic drugs or alter vital signs or laboratory parameters. No significant difference in the incidence of adverse events was observed between treatment groups (70.8% for the 1,000-mg group and 75.5% for the 2,000-mg group), or between the LEV and placebo groups (73.2% for placebo group). The most commonly reported adverse effects in the LEV group were asthenia, headache, and somnolence. CONCLUSIONS: The antiepileptic efficacy and tolerability of LEV (1,000 mg/d and 2,000 mg/d, administered in two divided doses) as add-on therapy was established in patients with refractory partial seizures in this clinical study.

Cytosolic phospholipase A2 is required for the activation of the NADPH oxidase associated H+ channel in phagocyte-like cells.

The NADPH oxidase producing-superoxide is the major mechanism by which phagocytes kill invading pathogens. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A2 (cPLA2) antisense mRNA and stable clones were selected which lack detectable cPLA2. cPLA2-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid, DMSO or 1,25 dihydroxyvitamin D3 indicating that cPLA2 is not involved in the differentiation process. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA2-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but addition of exogenous arachidonic acid (AA) fully restores oxidase activity. This establishes an essential requirement of cPLA2 generated AA for activation of phagocyte NADPH oxidase. In order to elucidate the mechanism by which cPLA2 regulates the oxidase, the role of cPLA2 in NADPH oxidase associated H+ channel was studied. Activation of differentiated PLB cells resulted in a Zn+2 sensitive alkalization, indicating H+ channel activity. In contrast, differentiated PLB-D cells failed to activate the H+ channel, but addition of exogenous AA fully restored this activity, indicating an essential and specific physiological requirement of cPLA2-generated AA for activation of the H+ channel. The presence of the H+ channel inhibitor, Zn+2, caused significant inhibition of NADPH oxidase activity, suggesting a role of the NADPH oxidase associated H+ channel in regulating oxidase activity.

Antiplatelet treatment does not reduce the severity of subsequent stroke. European Stroke Prevention Study 2 Working Group.

OBJECTIVE: To assess the the effect of antiplatelet therapy on the severity of subsequent stroke in patients with stroke and TIA. BACKGROUND: The Second European Stroke Prevention Study (ESPS2) recruited 6,602 patients in four treatment groups: placebo, 2 x 25 mg acetylsalicylic acid (ASA), 2 x 200 mg dipyridamole (DP), and the combination of 50 mg ASA and 400 mg DP per day. Seventy-six percent of the patients had had a stroke as the qualifying event, whereas 24% had a TIA. All patients were followed at 3-month intervals for 2 years. ESPS2 showed a benefit from antiplatelet treatment compared with placebo and an additional benefit using ASA and DP together compared with either of these antiplatelet agents alone. METHODS: In the ESPS2, the study protocol included assessment of severity of end point stroke with the modified Rankin scale once the stroke had clinically stabilized, and no further impairment was observed. There were 824 new stroke events during follow-up. In 701 of them, the initial Rankin scale was known, and this was also evaluated after each nonfatal recurrent stroke. The difference in Rankin scale between treatment groups was analyzed after recurrent stroke, and the progress in Rankin scale between entry and recurrent stroke was quantified by calculating the number of patients with a change of one or more degrees in the scale. RESULTS: There were no significant differences in these changes in Rankin scale between the treatment groups. The mean time to reach an end point of stroke was longest in patients who used ASA + DP (p = 0.057). However, there was no difference among the treatment groups in the time to death during follow-up. CONCLUSION: This study suggests that antiplatelet therapy does not influence the severity of recurrent stroke as evaluated with the Rankin scale. However, antiplatelet therapy seems to lengthen the time the patient remains free from a recurrent stroke.

Essential requirement of cytosolic phospholipase A(2) for activation of the H(+) channel in phagocyte-like cells.

The NADPH oxidase-producing superoxide is the major mechanism by which phagocytes kill invading pathogens. We previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation (Dana, R., Leto, T., Malech, H., and Levy, R. (1998) J. Biol. Chem. 273, 441-445). In the present study, we used this model to determine the physiological role of cPLA(2) in the regulation of both the H(+) channel and the Na(+)/H(+) antiporter and to study whether NADPH oxidase activation is regulated by either of these transporters. PLB-D cells and two controls: parent PLB-985 cells and PLB-985 cells transfected with the vector only (PLB cells) were differentiated using 1.25% Me(2)SO or 5 x 10(-8) M 1, 25-dihydroxyvitamin D(3). Activation of differentiated PLB cells resulted in a Zn(2+)-sensitive alkalization, indicating H(+) channel activity. In contrast, differentiated PLB-D cells failed to activate the H(+) channel, but the addition of exogenous AA fully restored this activity, indicating the role of cPLA(2) in H(+) channel activation. The presence of the H(+) channel inhibitor Zn(2+) caused significant inhibition of NADPH oxidase activity, suggesting a role of the H(+) channel in regulating oxidase activity. Na(+)/H(+) antiporter activity was stimulated in differentiated PLB-D cells, indicating that cPLA(2) does not participate in the regulation of this antiporter. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA for activation of the H(+) channel and suggest the participation of this channel in the regulation of NADPH oxidase activity.

Second European Stroke Prevention Study: antiplatelet therapy is effective regardless of age. ESPS2 Working Group.

BACKGROUND: The Second European Stroke Prevention Study (ESPS2) was a randomized, placebo-controlled trial that investigated the efficacy of low-dose acetylsalicylic acid (ASA) and modified-release dipyridamole (DP), alone or in combination, in the secondary prevention of ischemic stroke. The trial demonstrated that the combination was significantly more effective than either agent used alone. The aim of the present study was to evaluate the influence of age on the efficacy of ASA and DP, alone or in combination, in the secondary prevention of stroke in the ESPS2 population. METHODS AND RESULTS: A total of 6602 patients were recruited to the ESPS2 and there were 4 treatment groups: ASA (25 mg twice daily), DP (200 mg twice daily), ASA and DP in a combined formulation, or placebo. Primary endpoints were stroke, death, and stroke or death together. The endpoints evaluated in the present study were stroke, stroke and/or death, and vascular events. Stroke was the qualifying event in 76% of the patients, while 24% had a transient ischaemic attack. Patients were reviewed at 3-month intervals for 2 years. The study population consisted of 2565 (39%) patients aged less than 65 years, 2240 (34%) patients aged between 65 and 74 years, and 1797 (27%) patients aged 75 years and over. Advancing age was associated with an increased incidence of endpoints in all 4 treatment groups. The combination of ASA and DP significantly reduced the incidence of all endpoints, compared with placebo, in each age group. There was no influence of age on the efficacy of antiplatelet therapy for any of the evaluated endpoints. Relative risk reductions of treatment compared with placebo were 11.1-27.6% in the ASA group, 8.0-18.7% in the DP group, and 20.3-45.2% in patients receiving combination therapy. CONCLUSION: This study clearly demonstrates that combination therapy with DP and ASA is superior to either agent used alone in the secondary prevention of ischemic stroke, irrespective of the age of the patient.

GABAergic neuroaxonal dystrophy and other cytopathological alterations in feline Niemann-Pick disease type C.

Feline Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal storage disease which shares many of the clinical, biochemical and pathological features of the corresponding human disorder. Cytopathological alterations in distinct neuronal cell populations were investigated in this animal model to gain a better understanding of the pathogenesis of brain dysfunction. Golgi and immunocytochemical methods were employed to characterize the cell architectural changes occurring in neuronal somata, dendrites and axons at different stages of disease progression. Cortical pyramidal neurons in laminae II, III, and V exhibited various degrees of meganeurite and/or swollen axon hillock formation with or without ectopic dendritogenesis. Enlarged axon hillock regions with neuritic processes and spines were recognized early in the progression of feline NPC but were less prevalent in mid to late stages of the disease. Glutamic acid decarboxylase (GAD) immunocytochemistry demonstrated immunoreactive spheroids in numerous GABAergic axons in neocortex, subcortical areas, and cerebellum. Parvalbumin-immunoreactive axonal spheroid distribution in brain closely mirrored results from the GAD studies, whereas calbindin D-28k-immunoreactive spheroids were conspicuously absent in most cortical and subcortical areas examined. Purkinje cell axonal spheroid formation progressed in a distal to proximal direction, with eventual involvement of recurrent axon collaterals. Purkinje cell death and a concomitant decrease in the numbers of spheroids in the cerebellum were observed late in the disease course. Clinical neurological signs in feline NPC occur in parallel with neuronal structural alterations and suggest that GABAergic neuroaxonal dystrophy is a contributor to brain dysfunction in this disease.

European Stroke Prevention Study. 2. Dipyridamole and acetylsalicylic acid in the secondary prevention of stroke.

In 1988, we undertook a randomized, placebo-controlled, double-blind trial to investigate the safety and efficacy of low-dose acetylsalicylic acid (ASA), modified-release dipyridamole, and the two agents in combination for secondary prevention of ischemic stroke. Patients with prior stroke or transient ischemic attack (TIA) were randomized to treatment with ASA alone (50 mg daily), modified-release dipyridamole alone (400 mg daily), the two agents in a combined formulation, or placebo. Primary endpoints were stroke, death, and stroke or death together. TIA and other vascular events were secondary endpoints. Patients were followed on treatment for two years. Data from 6,602 patients were analysed. Factorial analysis demonstrated a highly significant effect for ASA and for dipyridamole in reducing the risk of stroke (p < or = 0.001) and stroke or death combined (p < 0.01). In pairwise comparisons, stroke risk in comparison to placebo was reduced by 18% with ASA alone (p = 0.013); 16% with dipyridamole alone (p = 0.039); and 37% with combination therapy (p < 0.001). Risk of stroke or death was reduced by 13% with ASA alone (p = 0.016); 15% with dipyridamole alone (p = 0.015); and 24% with the combination (p < 0.001). The treatment had no statistically significant effect on the death rate alone. Factorial analysis also demonstrated a highly significant effect of ASA (p < 0.001) and dipyridamole (p < 0.01) for preventing TIA. The risk reduction for the combination was 36% (p < 0.001) in comparison with placebo. Headache was the most common adverse event, occurring more frequently in dipyridamole-treated patients. All-site bleeding and gastrointestinal bleeding were significantly more common in patients who received ASA in comparison to placebo or dipyridamole. We conclude that (1) ASA 25 mg twice daily and dipyridamole, in a modified-release form, at a dose of 200 mg twice daily have each been shown to be equally effective for the secondary prevention of ischemic stroke and TIA; (2) when co-prescribed the protective effects are additive, the combination being significantly more effective than either agent prescribed singly; (3) low-dose ASA does not eliminate the propensity for induced bleeding.

Defective measles virus in human subacute sclerosing panencephalitis brain.

Evidence is presented showing that the brain cells of patients with subacute sclerosing panencephalitis (SSPE) contain mutant measles (MV) genomes having the characteristics of 5' copy-back defective interfering (DI) RNAs. Using a polymerase chain reaction-based amplification specific for copy-back DIs, abundant, discrete cDNAs representing different-sized MV defective RNA species were generated from each SSPE brain. The defective genomes were cloned in two portions. The most common of these defective species were sequenced, confirming their MV genome origin and 5' copy-back nature. We deduced that the minimum DI stem length of these species was 95 nucleotides, further delimiting the prerequisite 5' regulatory region sequences specifying MV genomic replication/encapsidation functions. This calculation assumes a precise copy-back mechanism and complete complementarity of the panhandle structure. Since the SSPE-derived viral genome encodes dysfunctional viral envelope proteins, we hypothesize that SSPE brains may lack the high degree of selective pressure encountered in tissue culture MV infections. This allows for the coexistence of numerous replication-competent defective particles in each SSPE brain. A role for viral defective particles as modulators of this persistent measles virus infection of humans is proposed.

Cerebrospinal fluid in multiple sclerosis.

The diagnosis of multiple sclerosis (MS) is a clinical one which should be made by a neurologist. Examination of the cerebrospinal fluid (CSF) [Lowenthal (1991): Neurol Neurosurg 4:914-918], the results of evoked potential studies, and magnetic resonance imaging serve as confirmatory tests. CSF is a window which permits a glimpse into the metabolism of the nervous system. The detailed investigation of the CSF proteins has provided important clues about our understanding of the pathogenesis of MS.

Secondary prevention of stroke: does dipyridamole add to aspirin?

BACKGROUND AND PURPOSE: The purpose of this paper is to evaluate, in the light of all available evidence, the place of aspirin alone and of aspirin combined with dipyridamole in the secondary prevention of cerebrovascular accidents. METHODS: We performed a meta-analysis of all identified double blind, controlled, studies in secondary prevention of cerebrovascular accidents for the following categories: studies comparing aspirin with placebo; studies comparing aspirin plus dipyridamole with placebo; studies comparing aspirin plus dipyridamole with aspirin alone. An indirect comparison was carried out to compare the results obtained with aspirin alone and those obtained with aspirin combined with dipyridamole. RESULTS: The meta-analysis of trials involving aspirin alone against placebo showed a risk reduction on strokes (17% reduction, p = 0.02), "important vascular events", i.e. a combination of vascular deaths, non-fatal strokes and non-fatal myocardial infarction (18% reduction, p = 0.003). Fatal vascular events (vascular deaths and fatal strokes) did not seem to be reduced at all. The overall mortality was reduced by 10%, but this reduction failed to reach statistical significance (p = 0.23). The meta-analysis of trials involving aspirin combined with dipyridamole showed more important risk reductions on every outcome whether fatal or not. Strokes were reduced by 42% (p < 0.001), fatal strokes by 43% (p = 0.02) and vascular deaths by 24% (p = 0.07, not significant). The overall mortality was reduced by 30% (p = 0.004). Direct comparisons of aspirin with aspirin plus dipyridamole did not indicate differences between the two treatment regimens. However the sample sizes involved in these comparisons were far too small to be informative. Indirect comparisons yielded statistically significant results in favour of the combination in terms of "important vascular events" (p = 0.007), all strokes (p = 0.007) and fatal strokes (p = 0.03). The results were also in favour of the combination but not statistically significant in terms of all deaths (p = 0.10) and vascular deaths (p = 0.08). CONCLUSIONS: Aspirin used alone reduces secondary occurrence of vascular events in cerebrovascular patients. There is no evidence, however, of a reduction of fatal events (vascular deaths and fatal strokes). In contrast, aspirin in combination with dipyridamole reduces non-fatal as well as fatal events. These results as well as the indirect comparisons of the risk reductions suggest that the combination of aspirin with dipyridamole may be superior to aspirin alone; this hypothesis is presently tested in a large randomized trial.