Inducible nitric-oxide synthase generates superoxide from the reductase domain

In the absence of L-arginine, the heme center of the oxygenase domain of neuronal nitric-oxide synthase reduces molecular oxygen to superoxide (O-2). Our recent work has provided evidence that inducible NOS (iNOS) may also catalyze O-2 formation in macrophages. However, there has been a lack of direct evidence of superoxide generation from the purified iNOS, and it was previously hypothesized that significant O-2 production does not occur. Moreover, the mechanism and enzyme site responsible for O-2 generation is unknown. To determine whether iNOS produces O-2 and to identify the mechanism of this process, we performed electron paramagnetic resonance measurements on purified iNOS using the spin trap 5,5-dimethyl-1-pyrroline N-oxide. In the presence of NADPH, prominent O-2 adduct signals were detected from iNOS. These signals were totally abolished by superoxide dismutase but not affected by catalase. High concentrations of L-arginine decreased this O-2 formation, whereas its enantiomer D-arginine did not. Pre-incubation of iNOS with the flavoprotein inhibitor diphenyleneiodonium totally blocked these O-2 signals. Conversely, pretreatment of the enzyme with the heme blocker cyanide had no effect on O-2 generation. Furthermore, strong O-2 generation was directly detected from the isolated iNOS reductase domain. Together, these data demonstrate that iNOS does generate O-2, and this mainly occurs at the flavin-binding sites of the reductase domain.     

Cellular localizations of AMPA glutamate receptors within the basal forebrain magnocellular complex of rat and monkey

The cellular distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors within the rodent and nonhuman primate basal forebrain magnocellular complex (BFMC) were demonstrated immunocytochemically using anti-peptide antibodies that recognize glutamate receptor (GluR) subunit proteins (i.e., GluR1, GluR4, and a conserved region of GluR2, GluR3, and GluR4c). In both species, many large GluR1-positive neuronal perikarya and aspiny dendrites are present within the medial septal nucleus, the nucleus of the diagonal band of Broca, and the nucleus basalis of Meynert. In this population of neurons in rat and monkey, GluR2/3/4c and GluR4 immunoreactivities are less abundant than GluR1 immunoreactivity. In rat, GluR1 does not colocalize with ChAT, but, within many neurons, GluR1 does colocalize with GABA, glutamic acid decarboxylase (GAD), and parvalbumin immunoreactivities. GluR1- and GABA/GAD-positive neurons intermingle extensively with ChAT-positive neurons. In monkey, however, most GluR1-immunoreactive neurons express ChAT and calbindin-D28 immunoreactivities. The results reveal that noncholinergic GABAergic neurons, within the BFMC of rat, express AMPA receptors, whereas cholinergic neurons in the BFMC of monkey express AMPA receptors. Thus, the cellular localizations of the AMPA subtype of GluR are different within the BFMC of rat and monkey, suggesting that excitatory synaptic regulation of distinct subsets of BFMC neurons may differ among species. We conclude that, in the rodent, BFMC GABAergic neurons receive glutamatergic inputs, whereas cholinergic neurons either do not receive glutamatergic synapses or utilize GluR subtypes other than AMPA receptors. In contrast, in primate, basal forebrain cholinergic neurons are innervated directly by glutamatergic afferents and utilize AMPA receptors.     

Immunohistochemistry of synapsin I and synaptophysin in human nervous system and neuroendocrine tumors. Applications in diagnostic neuro-oncology

Synapsin I is a phosphoprotein localized to the cytoplasmic surface of synaptic vesicles and is one of the best characterized neuron-specific proteins. Synaptophysin is an integral membrane glycoprotein, also located on presynaptic vesicles, which has been shown to be a useful immunohistochemical marker for neuroendocrine/neuronal differentiation in tumor diagnosis. The sensitivity and specificity of immunohistochemical staining for these two proteins in formalin-fixed, paraffin-embedded tissues was studied in a series of 67 neuroectodermal, neuroendocrine, and non-neural tumors. Intense immunoreactivity for both synapsin I and synaptophysin was observed in tumors containing well-differentiated neurons (gangliocytoma, ganglioglioma, neurocytoma). In these tumors, immunostaining was primarily concentrated along the outer surface of the cell membrane of the neuronal cells. Primitive neuroectodermal tumors (PNETs) (cerebral PNET, medulloblastoma, neuroblastoma) and most neuroendocrine tumors generally showed less intense and more variable immunoreactivity for these proteins. In most cases, immunostaining for synapsin I was sharper and often more intense than for synaptophysin. Some PNETs and neuroendocrine tumors that were immunoreactive for synapsin I did not stain for synaptophysin. We conclude that synapsin I is a reliable, sensitive immunohistochemical marker for neuronal/neuroendocrine differentiation in human neoplasms and may offer some advantages over synaptophysin when applied to formalin-fixed, paraffin-embedded tissues, particularly in the evaluation of primitive neuroectodermal tumors and neuroendocrine tumors.     

Historical perspectives of neonatal transport

A personalized view of the growth and development of neonatal transport is presented. The public display of premature infants in France and Germany and the beginning of the transport incubators in Chicago are explained. How the policy of regionalization was developed and diluted during the readjustment phase is discussed.     

Mitochondrial adenine nucleotide translocase is modified oxidatively during aging

The purpose of this study was to test the hypothesis that elevation in protein oxidative damage during the aging process is a targeted rather than a stochastic phenomenon. Oxidative damage to proteins in mitochondrial membranes in the flight muscles of the housefly, manifested as carbonyl modifications, was detected immunochemically with anti-dinitrophenyl antibodies. Adenine nucleotide translocase (ANT) was found to be the only protein in the mitochondrial membranes exhibiting a detectable age-associated increase in carbonyls. The age-related elevation in ANT carbonyl content was correlated with a corresponding loss in its functional activity. Senescent flies that had lost the ability to fly exhibited a relatively higher degree of ANT oxidation and a greater loss of functional activity than their cohorts of the same age that were still able to fly. Exposure of flies to 100% oxygen resulted in an increase in the level of ANT carbonyl content and a loss in its activity. In vitro treatment of mitochondria with a system that generated hydroxyl free radicals caused an increase in ANT carbonyl level and a decrease in ANT exchange activity. ANT was also the only mitochondrial membrane protein exhibiting adducts of the lipid peroxidation product 4-hydroxynonenal. Results of this study indicate that proteins in mitochondrial membranes are modified selectively during aging.