Noxa determines localization and stability of MCL-1 and consequently ABT-737 sensitivity in small cell lung cancer.

The sensitivity to ABT-737, a prototype BH3 mimetic drug, varies in a broad range in small cell lung cancer (SCLC) cells. We have previously shown that the expression of Noxa, a BH3-only pro-apoptotic BCL-2 family protein, is the critical determinant of ABT-737 sensitivity. We show here that Noxa regulates the localization and stability of MCL-1, an anti-apoptotic member, which results in modulating ABT-737 sensitivity. Mutations in Noxa within the BH3 domain, the carboxyl terminus mitochondrial targeting domain, or of ubiquitinated lysines not only change the localization and stability of Noxa itself but also affect the mitochondrial localization and phosphorylation/ubiquitination status of MCL-1 and consequently modulate sensitivity to ABT-737. Results of studies utilizing these mutant proteins indicate that Noxa recruits MCL-1 from the cytosol to the mitochondria. Translocation of MCL-1 initiates its phosphorylation and subsequent ubiquitination, which triggers proteasome-mediated degradation. The precise regulatory mechanisms of Noxa/MCL-1 expression and stability could provide alternative targets to modulate apoptosis induced by BH3 mimetic drugs or other chemotherapeutic reagents.

Mitochondrial toxin 3-nitropropionic acid induces cardiac and neurotoxicity differentially in mice.

We investigated the effects of 3-nitropropionic acid (3NPA), a previously characterized neurotoxin, in four strains of mice to better understand the molecular basis of variable host responses to this agent. Unexpectedly, we found significant cardiac toxicity that always accompanied the neurotoxicity in all strains of mice in acute and subacute/chronic toxicity testing. Caudate putamen infarction never occurred without cardiac toxicity. All mouse strains tested are sensitive to 3NPA although the C57BL/6 and BALB/c mice require more exposure than 129SVEMS and FVB/n mice. Cardiac toxicity alone was found in 50% of symptomatic mice tested and morphologically, the cardiac toxicity is characterized by diffuse swelling of cardiomyocytes and multifocal coagulative contraction band necrosis. In subacute to chronic exposure, atrial thrombosis, cardiac mineralization, cell loss, and fibrosis are combined with cardiomyocyte swelling and necrosis. Ultrastructurally, mitochondrial swelling occurs initially, followed by disruption of myofilaments. Biochemically, isolated heart mitochondria from the highly sensitive 129SVEMS mice have a significant reduction of succinate dehydrogenase activity, succinate oxygen consumption rates, and heart adenosine triphosphate after 3NPA treatment. The severity of morphological changes parallels the biochemical alterations caused by 3NPA, consistent with cardiac toxicity being a consequence of the effects of 3NPA on succinate dehydrogenase. These experiments show, for the first time, that 3NPA has important cardiotoxic effects as well as neurotoxic effects, and that cardiac toxicity possibly resulting from inhibition of the succinate dehydrogenase in heart mitochondria, contributes to the cause of death in 3NPA poisoning in acute and subacute/chronic studies in mice.

Neurobiology of hypoxic-ischemic injury in the developing brain.

Hypoxic ischemia is a common cause of damage to the fetal and neonatal brain. Although systemic and cerebrovascular physiologic factors play an important role in the initial phases of hypoxic-ischemic injuries, the intrinsic vulnerability of specific cell types and systems in the developing brain may be more important in determining the final pattern of damage and functional disability. Excitotoxicity, a term applied to the death of neurons and certain other cells caused by overstimulation of excitatory, mainly glutamate, neurotransmitter receptors, plays a critical role in these processes. Selected neuronal circuits as well as certain populations of glia such as immature periventricular oligodendroglia may die from excitotoxicity triggered by hypoxic ischemia. These patterns of neuropathologic vulnerability are associated with clinical syndromes of neurologic disability such as the extrapyramidal and spastic diplegia forms of cerebral palsy. The cascade of biochemical and histopathologic events triggered by hypoxic ischemia can extend for days to weeks after the insult is triggered, creating the potential for therapeutic interventions.

Delayed increase in neuronal nitric oxide synthase immunoreactivity in thalamus and other brain regions after hypoxic-ischemic injury in neonatal rats.

We examined the response of neuronal nitric oxide synthase (nNOS)-containing CNS neurons in rats exposed to a unilateral hypoxic-ischemic insult at 7 days of age. Animals were sacrificed at several time points after the injury, up to and including 7 days (Postnatal Day 14). Brain regions ipsilateral to the injury (including cerebral cortex, caudate-putamen, and thalamus) exhibited delayed, focal increases in nNOS immunoreactivity. The increase in nNOS immunoreactive fiber staining was prominent in areas adjacent to severe neuronal damage, especially in the cortex and the thalamus, regions that are also heavily and focally injured in term human neonates with hypoxic-ischemic encephalopathy. In cerebral cortex, these increases occurred despite modest declines in nNOS catalytic activity and protein levels. Proliferation of surviving nNOS immunoreactive fibers highlights regions of selective vulnerability to hypoxic-ischemic insult in the neonatal brain and may also contribute to plasticity of neuronal circuitry during recovery.

Apoptosis has a prolonged role in the neurodegeneration after hypoxic ischemia in the newborn rat.

Birth asphyxia can cause moderate to severe brain injury. It is unclear to what degree apoptotic or necrotic mechanisms of cell death account for damage after neonatal hypoxia-ischemia (HI). In a 7-d-old rat HI model, we determined the contributions of apoptosis and necrosis to neuronal injury in adjacent Nissl-stained, hematoxylin and eosin-stained, and terminal deoxynucleotidyl transferase-mediated UTP nick end-labeled sections. We found an apoptotic-necrotic continuum in the morphology of injured neurons in all regions examined. Eosinophilic necrotic neurons, typical in adult models, were rarely observed in neonatal HI. Electron microscopic analysis showed "classic" apoptotic and necrotic neurons and "hybrid" cells with intermediate characteristics. The time course of apoptotic injury varied regionally. In CA3, dentate gyrus, medial habenula, and laterodorsal thalamus, the density of apoptotic cells was highest at 24-72 hr after HI and then declined. In contrast, densities remained elevated from 12 hr to 7 d after HI in most cortical areas and in the basal ganglia. Temporal and regional patterns of neuronal death were compared with expression of caspase-3, a cysteine protease involved in the execution phase of apoptosis. Immunocytochemical and Western blot analyses showed increased caspase-3 expression in damaged hemispheres 24 hr to 7 d after HI. A p17 peptide fragment, which results from the proteolytic activation of the caspase-3 precursor, was detected in hippocampus, thalamus, and striatum but not in cerebral cortex. The continued expression of activated caspase-3 and the persistence of cells with an apoptotic morphology for days after HI suggests a prolonged role for apoptosis in neonatal hypoxic ischemic brain injury.

Novel treatments after experimental brain injury.

Perinatal hypoxic-ischaemic encephalopathy(HIE) is being studied in laboratory models that allow the delayed cascade of events triggered by the energetic insult to be examined in detail. The concept of the 'excitotoxic cascade' provides a conceptual framework for thinking about the pathogenesis of HIE. Major events in the cascade triggered by hypoxia-ischaemia include overstimulation of N-methyl-D-aspartate type glutamate receptors, calcium entry into cells, activation of calcium-sensitive enzymes such as nitric oxide synthase, production of oxygen free radicals, injury to mitochondria, leading in turn to necrosis or apoptosis. New experimental approaches to salvaging brain tissue from the effects of HIE include inhibition of neuronal nitric oxide synthase, administration of neuronal growth factors, and inhibition of the caspase enzymes that execute apoptosis. Recent experimental work suggests that these approaches may be effective during a longer 'therapeutic window' after the insult, because they are acting on events that are relatively delayed. Application of modest hypothermia may allow these agents to be neuroprotective at even longer intervals after hypoxia-ischaemia.

Striatal perfusion of indomethacin attenuates dopamine increase in immature rat brain exposed to anoxia: an in vivo microdialysis study.

Using in vivo microdialysis and HPLC, we examined the effects of indomethacin on extracellular dopamine (DA) in the striatum of immature rats submitted to anoxia. Rat pups in two indomethacin groups received intrastriatal perfusion of either 1 mM or 5 mM indomethacin throughout the experiment. The DA level reached 1185+/-400% of the basal level during anoxia; in contrast, the peak levels of DA were only 307+/-63%, 153+/-35% in indomethacin groups (p<0.05). We consider that this suppression would be one of the mechanisms of the protective effect of indomethacin on hypoxic ischemic encephalopathy.

Immunohistochemical study on transforming growth factor-beta1 expression in liver fibrosis of Down's syndrome with transient abnormal myelopoiesis.

A case of Down's syndrome associated with liver fibrosis is reported. The fibrosis was diffusely distributed along sinusoids, and an excess of megakaryocytes was also found in the liver. To determine the mechanism of liver fibrosis in Down's syndrome, we immunohistochemically stained the liver with markers of myofibroblast-like cells, antialpha smooth muscle actin antibodies and antidesmin antibodies. The myofibroblast-like cells were found along sinusoids, suggesting that liver fibrosis in Down's syndrome is caused by the myofibroblast-like cells derived from Ito cells/lipocytes. The expression of transforming growth factor (TGF)-betal, which is an important mediator of the activation of lipocytes, was immunohistochemically examined. The accumulation of TGF-betal was observed in cells in the sinusoidal spaces, which involve the intracellular expression of megakaryocytes. Together, these findings suggest that megakaryocyte-derived TGF-betal is one of the likely candidates in the lipocyte activation of liver fibrogenesis in Down's syndrome.

Anoxic and hypoxic immature rat model for measurement of monoamine using in vivo microdialysis.

The immature brain is considered relatively resistant to anoxia and ischemia. Although hypoxia without ischemia has not been considered to produce brain damage in immature rats as well as in adult rats (S. Levine, Anoxic-ischemic encephalopathy in rats, Am. J. Pathol., 36 (1960) 1-17 [8]; D.E. Levy, J.B. Brieley, D.G. Silverman, F. Plum, Brief hypoxia-ischemia initially damages cerebral neurons, Arch. Neurol., 32 (1975) 450-456 [9]; J.E. Rice, R.C. Vannucci, J.B., Brieriey, The influence of immaturity on hypoxic-ischemic brain damage in rat, Ann. Neurol., 9 (1981) 131-141 [14]), hypoxia in postnatal period is possible to cause a functional brain damage (T. Hender, P. Lundborg, Regional changes in monoamine synthesis in the developing rat brain during hypoxia, Acta. Physiol. Scand., 106 (1979) 139-143 [3]; W. Ihle, J. Gross, R. Moller, Effect on chronic postnatal hypoxia on dopamine uptake by synaptosomes from striatum of adult rats, Biomed. Biochem. Acta., 44 (1985) 433-437 [7]; A. Lun, J. Gross, M. Beyer, H.D. Fischer, C. Wustmann, J. Schmidt, K. Hecht, The vulnerable period of perinatal hypoxia with regard to dopamine release and behavior in adult rats, Biomed. Biochem. Acta., 45 (1986) 619-627 [10]). Using microdialysis, we studied the anoxic or hypoxic effect on catecholamine metabolism in immature rat brain by measuring extracellular concentrations of norepinephrine (NE), dopamine (DA), and its metabolites and also 5-hydroxyindole-3-acetic acid (5-HIAA), the serotonin metabolite. DA is a well established excitatory neurotransmitter (R.C. Vannucci, Experimental biology of cerebral hypoxia-ischemia: relation to perinatal brain damage, Pediatr. Res., 27 (1990) 317-326 [16]), and in the previous report using hypoxic 7-day-old rat pups increase of DA was not detected without additional stimulations (K. Gordon, D. Johnston, M.V. Robinson, T.E. Statman, J.B. Becker, F. Silverstein, Transient hypoxia alters striatal catecholamine metabolism in immature brain: An in vivo microdialysis study, J. Neurochem., 54 (1990) 605-611 [2]). Whereas recently in newborn piglets, hypoxic hypoxia produced increase of extracellular DA (C.-C. Huang, N.S. Lajevardi, O. Tammela, A. Pastuszko, Relationship of extracellular dopamine in striatum of newborn piglets to cortical oxygen pressure, Neurochem. Res., 19 (1994) 649-655 [6]; Olano, M., Song, D., Murphy, S., Wilson, D. F. and Pastuszko, A., Relationships of dopamine, cortical oxygen pressure, and hydroxyl radicals in brain of newborn piglets during hypoxia and posthypoxic recovery, J. Neurochem., 65 (1995) 1205-1212 [13]). We consider that hypoxic ischemic brain damage of human newborns that we can treat is a damage, which does not show overt neuropathological changes. We therefore tried to show that transient anoxia and hypoxia caused biochemical alteration if the exposure did not produce marked morphological changes. This rodent model is adequate to study perinatal asphyxia and alteration of monoamine level could be useful for evaluation of brain damage, even if it is not detected histologically.

Effect of N-methyl-D-aspartate and potassium on striatal monoamine metabolism in immature rat: an in vivo microdialysis study.

Effects of N-methyl-D-aspartate (NMDA) and potassium on 5-day-old rat's brain were examined. We measured extracellular striatal monoamines such as dopamine (DA), 3,4 dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole-3-acetic acid (5-HIAA) using intracerebral microdialysis. After 3 h stabilization, pups received varying concentrations of NMDA (1-3 mM) and potassium (200-800 mM) by intrastriatal perfusion for 32 minutes. Increasing the concentration of NMDA and potassium induced a dose related DA increase (p < 0.001), whereas DOPAC, HVA, and 5-HIAA decreased significantly. Five days later the same animals were sacrificed and the weight reduction of their cerebral hemispheres was measured. The weight of the drug perfused side was significantly reduced compared with that of the contralateral one. We examined next the relationship between the level of maximum DA and the relative hemisphere weight reduction. The DA peak was highly correlated with the hemisphere weight reduction (r = 0.70, n = 52, p < 0.001 in the NMDA group, r = 0.83, n = 30, p < 0.001 in the potassium group, respectively). These data show that each treatment alter striatal monoamine metabolism in immature rat brain and that the extracellular DA peak is a potential early indicator to estimate brain injury.

Magnesium attenuates a striatal dopamine increase induced by anoxia in the neonatal rat brain: an in vivo microdialysis study.

We evaluated the effects of magnesium on extracellular dopamine (DA) and its metabolites in the striatum of 5-d-old rats submitted to 16 min of anoxia using microdialysis and HPLC. Rat pups were divided into three groups and received either 1) intrastriatal perfusion (IS) of MgSO4, 2) intraperitoneal injection (IP) of MgSO4, and 3) NaCl and Ringer's solution, respectively in place of MgSO4. After stabilization, Mg2+, saline, and Ringer's solution were administered; then, 114 animals were exposed to 100% nitrogen for 16 min. Anoxia induced a DA surge, an acutely marked increase of DA, in both the control and the IP group. In contrast, the DA surge was significantly suppressed in the IS group (p < 0.01, analysis of variance). During anoxia, the plasma Mg2+ in the IP group, but not in the IS group, maintained a significantly higher level compared with the basal level. On the other hand, Mg2+ in the perfusates in the IS group, but not in the IP group, maintained a significantly high level during anoxia. Alterations induced by anoxia in other metabolites, 3,4-dihydroxyphenylacetic acid, homovanillic acid, norepinephrine, and 5-hydroxyindole-3-acetic acid, did not significantly differ among the three groups. We propose that elevated levels of Mg2+ in the striatum had inhibitory effects on the DA surge during anoxia.

Cranial computed tomography scans of premature babies predict their eventual learning disabilities.

It remains difficult to predict, early enough to intervene effectively, the risk of the development of learning disabilities among extremely low birth weight (ELBW) infants (birth weights less than 1,000 g). We prospectively studied the relationship between dilatations of lateral ventricles of the cranial computed tomography (CT) scan taken at the postconceptional age of 40 weeks and learning disabilities in their school age. Using a computer digitizer, we measured the areas of ventricles on cranial CT scans. The mean area of lateral ventricles of the learning disabilities-suspected group was significantly larger than that of the control group (392.9 and 277.4 mm2, respectively; P < .01). There were no significant differences between the two groups in gestation, birth weight, physical measurements, and developmental quotients at early school age. The dilatation of the lateral ventricles assessed by cranial CT at the corrected term may be one of the first predictors of learning disabilities recognizable at early school age.

A case of neonatal choriocarcinoma.

Choriocarcinoma occurring in a placenta and metastasizing to the fetus is quite rare. We describe here a case of such infantile choriocarcinoma, initially appeared as refractory anemia and rapidly metastasized to the liver, lungs, and brain. The placenta looked normal and was not submitted to histological examinations. Neither noninvasive nor invasive diagnostic methods (ultrasonography, computed tomography, magnetic resonance image, scintigraphy, and hepatic arteriography) gave any diagnostic information on the tumor. Liver biopsy was considered too risky due to a possible bleeding. Correct diagnosis was established only after the postmortem examination. Two months after the infant's death, we were informed that the mother was found having hepatic and pulmonary tumors. The importance of the maternal history and measurement of urinary human chorionic gonadotropin is emphasized for a rapid and correct diagnosis of infantile choriocarcinoma.

Short-term fasting alters neonatal rat striatal dopamine levels and serotonin metabolism: an in vivo microdialysis study.

Although frequent feeding is necessary for neonatal brains, rat pups were usually separated from their dams throughout a microdialysis experiment. First, in 5-day-old rats, we examined the effect of probe insertion on initial fluctuation of extracellular striatal monoamines using in vivo microdialysis and subsequent HPLC. Second, fasting effect on monoamine metabolism was examined with or without fasting; the latter was regarded as controls. Extracellular striatal DA in the fasting group decreased promptly to 60% of the basal level in the first 2 h, and reached 50% by the end of the experiment. Dopamine in the fasting group decreased more markedly than in the control group (P < 0.01 by ANOVA) which also decreased to about 80% of the basal level. Extracellular 5-hydroxyindole-3-acetic acid (5-HIAA) continuously increased (P < 0.01), and the serum concentration of tryptophan also increased in the fasting group (P < 0.001). We showed that extracellular striatal monoamine levels fluctuated especially in the first 2 h and fasting altered monoamine metabolism. Therefore, it should take at least 2 h after surgery to stabilize the animals and obtain adequate basal levels. In addition, we should consider that these alterations occur when we use fasting animals as controls in microdialysis studies.

Effect of anoxia on striatal monoamine metabolism in immature rat brain compared with that of hypoxia: an in vivo microdialysis study.

We examined in 5-day-old rats the effects of either anoxia or 8% hypoxia on extracellular monoamines such as dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), norepinephrine (NE), 5-hydroxytryptamine (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) using in vivo microdialysis and subsequent HPLC. After stabilization 64 animals were exposed to 100% nitrogen for 16 min and 40 animals to 8% oxygen for 128 min. Both anoxia and hypoxia produced acute increase in the striatal extracellular DA (anoxia: P < 0.001, hypoxia: P < 0.01). Especially in anoxia, DA levels increased transiently to 2000-times the basal levels and 6-times higher than those in hypoxia. NE also increased in both anoxia and hypoxia. DOPAC and HVA decreased during hypoxia (P < 0.01 and P < 0.001, respectively), while those in anoxia were unchanged. In anoxia, decrease tendency of their levels were in short duration and that of 5-HIAA was followed by gradual increase (P < 0.001). These data demonstrated that brief exposure to anoxia or hypoxia had significant influence on striatal monoamine metabolism in immature brain and the pattern of change of monoamine in anoxia was different from that in hypoxia.

Partial 6p trisomy with abnormal ABR and hypogenesis of the corpus callosum.

A case of 4-month-old male infant with the karyotype 46,XY, -21, +der(21), t(6;21)(p22; p13) mat is reported. His cranial magnetic resonance imaging (MRI) suggested hypoplastic corpus callosum and auditory brain stem response (ABR) revealed brain stem dysfunction.

A preterm infant with secondary carnitine deficiency due to MCT formula--effective treatment of L-carnitine.

We report a preterm infant who was prescribed an MCT formula and subsequently developed carnitine deficiency with liver dysfunction and an elevation of serum CK level. A male infant who had been born at 24 weeks' gestation with birth weight 799 g, was fed with an MCT formula containing 76.8% of all kinds of lipids, because of his steatorrhea after the 30th day. On the 100th day, he was noted hepatomegaly and elevation of serum levels of AST, ALT and CK. The needle biopsy of the liver indicated the existence of the liver damage. He showed low serum carnitine with high urinary loss of acylcarnitine and dicarboxylic aciduria. Administration of L-carnitine was an effective treatment. The carnitine deficiency might be exaggerated by an increased urinary loss of acylcarnitine. We should be cautious of the risk of carnitine deficiency in preterm infants during prolonged use of MCT formula.

Two siblings with partial trisomy 15 and monosomy 21 associated with central nervous system anomalies.

A sister and a brother with 46, XX (46, XY), -21, +der (15) (q22.1; q22.1) mat were reported whose mother had a karyotype of 46, XX, t(15; 21)(q22.1; 22.1) and was phenotypically normal. Both sibs were mentally retarded and dysmorphic. Moreover, the sister had a holoprosencephaly with congenital hydrocephalus, and the brother showed congenital hydrocephalus.