Tetrahydrobiopterin in striatum: localization in dopamine nerve terminals and role in catecholamine synthesis.

The hydroxylase cofactor, tetrahydrobiopterin, and its biosynthetic system are localized in dopaminergic nerve terminals in the striatum. This conclusion is based on the nearly equivalent loss of tyrosine hydroxylase and tetrahydrobiopterin and its initial biosynthetic enzyme, guanosine triphosphate cyclohydrolase, after injection of 6-hydroxydopamine into the substantia nigra. The role of the hydroxylase cofactor in the regulation of dopamine synthesis is reassessed.

Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis.

We developed a new approach to DNA sequence analysis that uses fluorogenic reporter molecules--molecular beacons--and demonstrated their ability to discriminate alleles in real-time PCR assays of genomic DNA. A set of overlapping molecular beacons was used to analyze an 81-bp region of the Mycobacterium tuberculosis rpoB gene for mutations that confer resistance to the antibiotic rifampin. In a blinded study of 52 rifampin-resistant and 23 rifampin-susceptible clinical isolates, this method correctly detected mutations in all of the resistant strains and in none of the susceptible strains. The assay was carried out entirely in sealed PCR tubes and was simple to perform and interpret. This approach can be used to analyze any DNA sequence of moderate length with single base pair accuracy.

Morphologic, immunologic, and cytogenetic classification of acute myeloid leukemia and myelodysplastic syndrome in childhood: a report from the Childrens Cancer Group.

The purposes of this report are to reaffirm concordance difficulties with the acute myeloid leukemia (AML) French-American-British (FAB) classification, to present the frequency of previously delineated AML syndromes in pediatric patients and to describe additional characteristic AML profiles utilizing composite morphologic, cytogenetic and immunophenotypic data. Profiles of 124 children with acute myeloid leukemia (AML) and 13 children with myelodysplastic syndrome entered on the Childrens Cancer Group (CCG) pilot study CCG-2861 were examined. Concordance between institutions and reviewers for FAB designation was 65%. Discordance was found principally between M1 and M2, M2 and M4, and M4 and M5. In 49% of marrow specimens, leukemic blasts expressed at least one T lineage-related antigen; 24% expressed the B lineage-related antigen CD19. CDw14 correlated with FAB M4 or M5 morphology and was the only surface antigen associated with a specific FAB subtype. Normal karyotypes were found for 15% of the 75 children with satisfactory karyotype preparations. Recurring aberrations, found in 76% of children, included t(15;17)(q22;q11), t(8;21)(q22;q22), inv(16)(p13q22), rearrangements of band 11q23, t(6;9) (p23;q34), trisomy 8 and monosomy 7. Results from this pilot study and from the current CCG randomized trial correlating morphology, immunophenotyping and cytogenetics, will help to classify AML into unique subgroups with differing clinical consequences or therapy requirements.

Carotid artery injection technique: bounds for bolus mixing by plasma and by brain.

Estimation of Michaelis-Menten kinetic parameters (Km, Vmax) of blood-brain barrier (BBB) transport processes with the carotid artery single injection technique assumes that mixing of the bolus with unlabeled substrate either from (a) circulating plasma or (b) amino acid efflux from brain, is minimal. The maximum extent to which the bolus could mix by these two sources is quantified in the present studies by measuring 14C-phenylalanine extraction in pentobarbital-anesthetized and conscious rats after the addition of 0-80% rat serum to the arterial injection solution. An upper bound (+/- SE) of bolus mixing due to mixing from both sources, expressed in terms of percentage of rat plasma, is 8.8 +/- 1.9 and 7.0 +/- 2.1% for the anesthetized and conscious rat, respectively. The estimated contribution to bolus mixing due to amino acid efflux from brain is 3.3 and 2.1% for the anesthetized and conscious rat, respectively. Based on these estimates, the upper bound for bolus mixing with circulating rat plasma is only 5.5 and 4.9%, respectively, for the anesthetized and conscious catheterized rat. Thus, any bolus mixing after rapid carotid injection is relatively small and is comparable to the mixing effects observed with the carotid artery infusion technique. Mixing effects on the order of 5% are shown to have no significant effect on the estimation of kinetic parameters of BBB nutrient transport, except for neutral and basic amino acid transport, which are characterized by very low Km values relative to the usual amino acid plasma concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute lymphoblastic leukemia in children: current status, controversies, and future perspective.

Disease-free survival (DFS) in childhood ALL is 60%, and survival in good, average, and poor prognostic groups defined by initial WBC and age is 90, 60, and 45%, respectively. Additional immunological, morphological, biochemical, cytokinetic, and cytogenetic factors have been identified, illustrating the heterogeneity of ALL and its derivation from malignant clones at various stages of differentiation and with varying rates of proliferation. Of biologic importance, these factors may refine further the characteristic features of clinically-determined prognostic groups. Multivariate analysis of large prospective trials with homogeneous therapy will be required to determine the independent prognostic importance of these factors. Current treatment strategies in ALL include (1) tailoring therapy and its intensity to prognostic groups; (2) multiple-drug combinations in induction; (3) early use of intrathecal (IT) methotrexate (MTX); (4) CNS prophylaxis with IT MTX alone in good prognosis patients and combined cranial radiation (CXRT), 1800 rads plus IT MTX, in average and poor prognosis patients. Current studies show a CNS relapse rate of 5% in all prognostic groups. Late neuropsychological defects caused by cranial XRT and IT MTX have prompted programs designed to reduce the potential late toxicity of CNS prophylaxis. More pronounced in younger children, these abnormalities include decreased IQ, visual-motor incoordination, poor performance in mathematics, and memory dysfunction. Until 1980, more intensive induction, consolidation, and maintenance therapy had failed to prolong DFS in children with a poor prognosis. In West Germany (Berlin-Frankfurt-Muenster protocol) a 70 to 75% DFS is seen in all patients regardless of initial WBC, suggesting that effective therapy will override prognostic factors. Ultra-high-dose MTX, without cranial radiation, is also showing promise in poor prognosis patients. Other issues include the optimal duration of therapy, the role of testicular biopsies, and prophylactic testicular radiation. Recent studies suggest that prognostic factors lose their significance after 2 years of continuous complete remission and that 2 years of maintenance therapy is adequate. Bilateral open-wedge testicular biopsies have identified occult testicular disease in 8 to 10% of males. A unified approach to children with leukemia/lymphoma, a group with a particularly poor prognosis, utilizing NHL-type therapy may be more effective than conventional ALL therapy.(ABSTRACT TRUNCATED AT 400 WORDS)

Tetrahydrobiopterin in dystonia: identification of abnormal metabolism and therapeutic trials.

The pteridine cofactor of tyrosine and tryptophan hydroxylases, tetrahydrobiopterin (BH4), is concentrated in the striatum and other sites of brain monoamine synthesis and is a regulatory factor in the rate-limiting step of catecholamine synthesis. CSF content was decreased in eight patients with dystonic disorders (mean, 13.0 +/- 0.8 pmol/ml CSF compared with 20.6 +/- 1.4 in age-matched normals). We gave several trials of synthetic BH4 intravenously to 10 dystonic patients with benefit for 2 subjects with diurnally fluctuating dystonia, 1 with hemidystonia and parkinsonism, and 1 with generalized torsion dystonia. The findings of biopterin abnormality and the observed clinical improvements may point to a role for the cofactor in the pathogenesis and, possibly, the treatment of some forms of primary dystonia.

The ontogeny of excitatory amino acid receptors in rat forebrain--I. N-methyl-D-aspartate and quisqualate receptors.

The ontogeny of radioligand binding to N-methyl-D-aspartate and quisqualate receptors in rat forebrain was studied quantitatively using in vitro receptor autoradiography. Specific binding to both receptors could be detected by postnatal day 1 in hippocampus and striatum. The adult pattern of binding to N-methyl-D-aspartate receptors emerged by postnatal day 14 with high densities of binding in CA1 (stratum oriens and stratum radiatum), dentate gyrus (molecular layer) and striatum (caudate-putamen). Binding to the outer laminae of frontal cortex was as much as 45% above adult levels during development. Binding of [3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid to quisqualate receptors showed a similar overshoot during development, but also manifested a unique distribution with CA3 and medial aspects of the amygdala exhibiting transient, intense labeling. Homogenate binding studies with [3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid demonstrated a 73% increase in quisqualate receptors in whole brain at postnatal day 21 compared with adult levels. The selectivity of excitatory amino acid binding to the quisqualate site in development was similar to the selectivity in adult brain. These data taken with other recent reports, suggest that quisqualate receptors may have a role in development distinct from their function in the adult brain.

The ontogeny of excitatory amino acid receptors in the rat forebrain--II. Kainic acid receptors.

The ontogeny of [3H]kainic acid binding in rat forebrain was studied quantitatively using in vitro receptor autoradiography. Specific binding was detectable in ventral thalamus, hippocampus, striatum and olfactory bulb by postnatal day 1. In regions with high densities of receptors in adulthood, such as CA3, dentate gyrus and striatum, binding increased progressively across development peaking at postnatal day 21. In ventral thalamus and the inner lamina of the neocortex, [3H]kainic acid binding was high in the first three postnatal weeks and relatively low thereafter. Saturation studies performed on adults and 14-day-old animals suggest differences in both the affinity and the maximal binding capacity contributed to the observed developmental changes in binding of [3H]kainic acid.

Therapeutic potential for adenosine receptor activation in ischemic brain injury.

The present report reviews the biochemical and physiological responses to adenosine receptor activation and how these responses underlie the ability of adenosine to couple energy demand with energy supply. In addition, activation of adenosine receptors pharmacologically is shown to initiate various reactions which could be responsible for the observed adenosine-mediated attenuation of the neuropathological consequences of brain ischemia. Also reported is the extent to which side effects such as hypothermia can contribute to the observed efficacy of adenosine agonist administration in the small animal model of ischemia. Data from various in vitro and in vivo ischemia studies is presented showing that neuroprotection can be achieved following pharmacological activation of adenosine receptors either through agonists with high affinity for A1 adenosine receptors or drugs which potentiate endogenous adenosine levels. In general the data support utilization of adenosine receptor activation as a means of attenuating ischemic brain damage.

Combined pretrauma scopolamine and phencyclidine attenuate posttraumatic increased sensitivity to delayed secondary ischemia.

Fasted Wistar rats were given a mild level of traumatic brain injury (TBI) and then subjected to 6 min of transient forebrain ischemia 24 h posttrauma. One group was given simultaneous 1 mg/kg scopolamine and 4 mg/kg phencyclidine intraperitoneally (IP) 15 min before trauma and another group an equal volume of plasmalyte A solution. After 7 days of postinjury survival, placebo-treated rats demonstrated increased posttraumatic vulnerability to secondary ischemic CA1 neuronal death even 24 h after trauma. This finding confirmed that increased posttraumatic ischemic vulnerability persists for at least 24 h even following mild trauma. Combined muscarinic receptor and N-methyl-D-aspartate (NMDA) receptor coupled ion channel blockade given and present during the mild TBI statistically attenuated this enhanced secondary ischemic CA1 neuronal death and thus posttraumatic increased ischemic vulnerability. Placebo-treated rats had 335.3 +/- 93.6 CA1 neurons/10(6) microns 2 and drug-treated rats had 844.8 +/- 184.9 CA1 neurons/10(6) microns 2. This result suggests that muscarinic and/or NMDA receptor-mediated events confined to TBI and the early posttraumatic period are in part responsible for the phenomenon of increased posttraumatic ischemic vulnerability.

The use of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin in the analysis of nonphosphorylated and phosphorylated rat striatal tyrosine hydroxylase at pH 7.0.

The kinetics of tyrosine hydroxylase from the desalted high-speed supernatants of rat striatal homogenates were examined at pH 7.0 using different concentrations of the natural cofactor, (6R)-l-erythrotetrahydrobiopterin, ranging from 4 ?M to 1.5 mM. All analyses were performed using two different buffering solutions and their appropriate reducing systems for maintaining cofactor in the reduced state. In the presence of phosphate buffer the results show that tyrosine hydroxylase exists in two kinetically different forms with apparent K(m) values for the cofactor of 16 ?M (low K(m)) and 2.3 mM (high K(m)). Similar results were obtained using MOPS buffer. A comparative analysis of the appropriate V(max) values indicates that tyrosine hydroxylase as obtained by our standard preparation procedures is predominately (95%) in the high K(m) form. When the striatal supernatant was exposed to phosphorylating conditions and subsequently analyzed it appeared that the enzyme now existed totally in the low K(m) form with very little change in the overall V(max). A comparison of the results using the two different buffering systems, phosphate and MOPS, revealed that following phosphorylation a large percentage of enzyme was maintained in the phosphorylated state only when using phosphate buffer. In light of the present results, we can for the first time suggest a functional significance not only for the two apparently different kinetic forms of the enzyme but also for a supporting role for phosphorylation. In vivo dopamine synthesis may be accomplished to a significant extent by the phosphorylated form of the enzyme while the non-phosphorylated form may constitute a relatively inactive reservoir which can be recruited for increased dopamine synthesis by phosphorylation.

Correlation of changes in pineal gland cAMP metabolism with light-mediated decrease in N-acetyltransferase activity.

The present study investigated the correlation between the light-mediated decrease in rat pineal N-acetyltransferase (NAT) observed in vivo and changes in cAMP metabolism. While exposing dark-adapted rats to light for short time periods (0-10 min) resulted in a rapid decrease in pineal NAT activity, cAMP exhibited a biphasic response. Following light exposure for 30 s there was a 50% decrease in cAMP levels. However, after 6 min of light exposure the cyclic nucleotide levels had increased 2-3 times above control values. These responses were prevented by phenoxybenzamine pretreatment and the initial decrease was mimicked by i.v. propranolol administration. Examination of cAMP metabolic enzymes, adenylate cyclase and phosphodiesterase revealed an increase in adenylate cyclase activity following 6 min of exposure to light. We discussed how the results observed in vivo compare with those observed using cultured pinealocytes.

Analysis of fluctuations in the activity of rat pineal tyrosine hydroxylase.

Tyrosine hydroxylase was measured in pineals removed from light-adapted and dark-adapted rats. The animals were adapted to the appropriate light situation for approximately 5 h before removal of the pineals. Bilateral ganglionectomy confirmed that all detectable enzyme activity in the pineal appears present in sympathetic fibers originating in the superior cervical ganglia. An analysis of tyrosine hydroxylase at subsaturating cofactor concentrations in dark-adapted rats revealed activity which was 60% higher than in appropriate light control animals. Further kinetic analysis showed that the K(m) for the synthetic cofactor, 6-methyltetrahydropterin, for dark-adapted animals was 0.8 + or - 0.1 mM which was significantly lower than a K(m) value of 1.4 + or - 0.2 mM for light adapted animals. Thus the rate of norepinephrine synthesis in vivo could increase due to an alteration in the enzyme molecule, which affects its interaction with substrate. These findings support previously documented results which show both an increased turnover and an increase in the concentration of norepinephrine in the pineals of dark-adapted rats.

Opiate receptor subtype binding in gerbil hippocampus is altered by forebrain ischemia.

A role for endogenous opioids in trauma-induced brain injury has been supported by pharmacological studies. The present series of experiments were initiated to extend these observations by measuring opiate receptor subtype binding in gerbil hippocampus following 7 days recovery from a 10 min ischemic insult. Quantitative in vitro autoradiography was utilized to measure mu [( 3H]DAGO), kappa [( 3H]bremazocine + 10 microM morphiceptin + 100 nM DSLET), delta [( 3H]DSLET + 10 microM morphiceptin) and lambda [( 3H]naloxone + 300 nM diprenorphine) binding. While ischemic tissue samples at the level of the dorsal hippocampus showed complete loss of CA1 pyramidal cells, we observed no significant alterations in mu or delta binding suggesting a non-pyramidal cell localization of these receptors. Kappa binding decreased significantly to 88% of control in the CA1 and CA3 regions while lambda binding in the stratum lucidum (CA3) increased to 165% of control. Our results show that opiate receptor subtypes are differentially affected by an ischemic insult.

Chronic L-DOPA-pretreatment of rats: an electrophysiological and biochemical study in the basal ganglia.

Treatment of rats with L-DOPA (251.25 mg as the methyl ester HCl/kg) plus benserazide (B, 50 mg/kg) (L-DOPA+B), twice daily (i.p.) for 5 days or 12 days resulted in the dopamine (DA) neurons of the substantia nigra pars compacta becoming subsensitive to the rate-depressing effects of D-amphetamine (i.v.) 16 to 24 h after the last chronic drug dose. In contrast, pretreated rats were significantly less sensitive than control rats to the rate depressant effects of apomorphine (i.v.) after 12, but not 5 days of L-DOPA+B-pretreatment. After 5, but not 12 days of L-DOPA+B-pretreatment, a significant increase in the number of spontaneously active DA neurons was noted in the substantia nigra pars compacta. Caudate tyrosine hydroxylase was examined and a significant increase in apparent Vmax was noted after 5 days of L-DOPA+B, with no apparent change being noted in Km for cofactor. At this time, no change was noted in caudate DA or HVA concentrations. Several distinct processes may be occurring in response to the L-DOPA+B-pretreatment: (1) the DA autoreceptors located on cell bodies in the substantia nigra have become subsensitive after 12 days of L-DOPA+B-pretreatment; (2) the subsensitivity to D-amphetamine seen after both chronic schedules is probably unrelated to the subsensitive DA autoreceptors and may depend upon homeostatic alterations in neurotransmitter systems other than those utilising DA; (3) the activation of tyrosine hydroxylase may be a reflection of the increase in the number of spontaneously active units.

Forebrain ischemia in the gerbil increases lambda opiate binding in hippocampal mossy fibers.

Transient forebrain ischemia was produced in gerbils by short-term occlusion of the common carotid arteries under halothane anesthesia. Histological analysis of brains 7 days post-ischemia demonstrated characteristic destruction of CA1 pyramidal cells. lambda Opiate binding (measured with [3H]naloxone in the presence of 300 nM diprenorphine) at 7 days post-ischemia was significantly increased in the stratum lucidum of the hippocampus (the mossy fiber layer), but not in any other region measured, including other hippocampal regions, cortex, amygdala, caudate putamen, thalamus, and hypothalamus. The increase in mossy fiber lambda binding was slow to develop (no increase detected up to 48 h post-ischemia), and long-lasting (binding remained elevated at 32 days post-ischemia). While MK-801 significantly inhibited CA1 pyramidal cell destruction when administered 20 min prior to ischemia, the increase in mossy fiber lambda binding was still evident. None of seven different opioid agonists and antagonists examined had an effect on either the pyramidal cell damage or increased mossy fiber lambda binding seen 7 days after ischemia.

Excitatory amino acid receptor subtype binding following traumatic brain injury.

Sprague-Dawley rats were subjected to a moderate level (2.2 atm) of traumatic brain injury (TBI) using fluid percussion. Injured animals were allowed post-trauma survival periods of 5 min, 3 and 24 h. Regional glutamate receptor subtype binding was assessed with quantitative autoradiography in each group for N-methyl-D-aspartate (NMDA), quisqualate and kainate receptor subpopulations at approximately the -3.8 bregma level and compared to a sham control group. [3H]glutamate binding to the NMDA receptor was significantly (P less than 0.05) decreased at 3 h post-TBI in the hippocampal CA1 stratum radiatum, the molecular layers of the dentate gyri and the outer (layers 1-3) and inner (layers 5 and 6) overlying neocortex. NMDA receptor binding was significantly reduced in layers 5 and 6 of the neocortex at all post-trauma survival times but no further differences were seen in the hippocampi. No significant changes were observed with [3H]AMPA binding to quisqualate receptors and [3H]KA binding was significantly reduced only in layers 5 and 6 of the neocortex at 24 h after TBI. These data further confirm the pathological involvement of the NMDA receptor complex in brain regions selectively vulnerable to moderate levels of TBI in this model.

Pre- and peristroke treatment with the adenosine kinase inhibitor, 5'-deoxyiodotubercidin, significantly reduces infarct volume after temporary occlusion of the middle cerebral artery in rats.

The adenosine kinase inhibitor, 5'-deoxyiodotubercidin (5dITU), was examined in a rat focal stoke model with temporary (105 min) middle cerebral artery occlusion (MCAO) followed by a 24 h recovery period. Inhibition of this adenosine metabolizing enzyme indirectly enhances the actions of endogenous adenosine without inducing cardiovascular side effects. Such effects could limit the potential clinical application of any approach targeting adenosine receptor activation. MCAO was accomplished with a transluminal 4-0 nylon suture inserted through the common carotid artery to block blood flow at the origin of the MCA. Treatment with 5dITU 30 min prior to and 5 h after MCAO resulted in a dose dependent (0.1-0.5 mg/kg, i.p.) reduction in infarct volume. A significant (P = 0.02) 44% reduction (control, 265 +/- 35 mm3; treated, 149 +/- 30 mm3) was observed at 0.5 mg/kg. However, at the highest dose examined (1.0 mg/kg) infarct volume was unaffected. To assess the potential for acute (i.e. post-occlusion) treatment, 5dITU was administered (0.33 mg/kg, i.v.) successively at each of 0.5, 1.75 and 3.5 h after MCAO. Post-occlusion treatment resulted in a significant (P = 0.037) 32% reduction in infarct volume (control, 314 +/- 34 mm3; treated, 212 +/- 28 mm3). At this dose there were no apparent changes in a number of physiological parameters monitored over the period of MCAO. The present study shows that intervention with an adenosine kinase inhibitor in an ischemic brain injury model is neuroprotective whether treatment is begun prior to or just after MCAO.

Interaction of cholinergic and glutamatergic transmission in the hippocampus: an in vitro autoradiographic receptor analysis.

Quantitative in vitro receptor autoradiography was used to examine the effect of acute scopolamine administration on specific binding to components of the N-methyl-D-aspartate (NMDA) receptor complex in four regions of mouse hippocampus. The binding of [3H]glycine to the strychnine-insensitive site was increased 1 h after administration of scopolamine hydrobromide (10 mg/kg) in the ventral dentate gyrus. The study suggests that rapid alterations in strychnine-insensitive glycine binding can occur in response to cholinergic perturbations. Moreover, these data suggest a delicate interaction between cholinergic and glutamatergic projections in the hippocampus.

Sequential combination of methotrexate and vindesine in previously treated children with acute leukemia. A phase I-II study.

A therapeutic synergistic effect is seen in animal models when vinca alkaloids are administered after methotrexate. To examine further this interaction in clinical studies, a phase I-II trial was conducted in children with hematologic malignancies in the Department of Pediatrics at Memorial Sloan-Kettering Cancer Center. A schedule of sequential of methotrexate and vindesine was developed which showed effect in acute lymphoblastic leukemia in children in relapse and which was relatively nontoxic. The regimen has also been useful for reinduction for patients who are candidates for bone marrow transplant.