Lawrence I. Grossman

Brain ischemia and reperfusion: molecular mechanisms of neuronal injury.

Brain ischemia and reperfusion engage multiple independently-fatal terminal pathways involving loss of membrane integrity in partitioning ions, progressive proteolysis, and inability to check these processes because of loss of general translation competence and reduced survival signal-transduction. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization, which induces release of glutamate and, in selectively vulnerable neurons (SVNs), opening of both voltage-dependent and glutamate-regulated calcium channels. This allows a large increase in cytosolic Ca(2+) associated with activation of mu-calpain, calcineurin, and phospholipases with consequent proteolysis of calpain substrates (including spectrin and eIF4G), activation of NOS and potentially of Bad, and accumulation of free arachidonic acid, which can induce depletion of Ca(2+) from the ER lumen. A kinase that shuts off translation initiation by phosphorylating the alpha-subunit of eukaryotic initiation factor-2 (eIF2alpha) is activated either by adenosine degradation products or depletion of ER lumenal Ca(2+). Early during reperfusion, oxidative metabolism of arachidonate causes a burst of excess oxygen radicals, iron is released from storage proteins by superoxide-mediated reduction, and NO is generated. These events result in peroxynitrite generation, inappropriate protein nitrosylation, and lipid peroxidation, which ultrastructurally appears to principally damage the plasmalemma of SVNs. The initial recovery of ATP supports very rapid eIF2alpha phosphorylation that in SVNs is prolonged and associated with a major reduction in protein synthesis. High catecholamine levels induced by the ischemic episode itself and/or drug administration down-regulate insulin secretion and induce inhibition of growth-factor receptor tyrosine kinase activity, effects associated with down-regulation of survival signal-transduction through the Ras pathway. Caspase activation occurs during the early hours of reperfusion following mitochondrial release of caspase 9 and cytochrome c. The SVNs find themselves with substantial membrane damage, calpain-mediated proteolytic degradation of eIF4G and cytoskeletal proteins, altered translation initiation mechanisms that substantially reduce total protein synthesis and impose major alterations in message selection, down-regulated survival signal-transduction, and caspase activation. This picture argues powerfully that, for therapy of brain ischemia and reperfusion, the concept of single drug intervention (which has characterized the approaches of basic research, the pharmaceutical industry, and clinical trials) cannot be effective. Although rigorous study of multi-drug protocols is very demanding, effective therapy is likely to require (1) peptide growth factors for early activation of survival-signaling pathways and recovery of translation competence, (2) inhibition of lipid peroxidation, (3) inhibition of calpain, and (4) caspase inhibition. Examination of such protocols will require not only characterization of functional and histopathologic outcome, but also study of biochemical markers of the injury processes to establish the role of each drug.

The petite mutation in Yeast: Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide☆

Ethidium bromide is known to convert respiratory sufficient yeast (grandes) to cytoplasmically inherited respiratory deficient mutants (petites) at efficiencies close to 100%. We have investigated the mechanism of this conversion in Saccharomyces cerevisiae by examining the properties of mitochondrial DNA at various times during the mutation process, using cycloheximide to amplify the proportion of radioactivity in mitochondrial DNA. Ethidium bromide is shown to inhibit selectively mitochondrial DNA synthesis; furthermore, in its presence, pre-existing mitochondrial DNA is progressively degraded. In petites made by prolonged treatment with ethidium bromide no mitochondrial DNA could be detected.

Implications of natural selection in shaping 99.4% nonsynonymous DNA identity between humans and chimpanzees: Enlarging genus Homo

What do functionally important DNA sites, those scrutinized and shaped by natural selection, tell us about the place of humans in evolution? Here we compare ≈90 kb of coding DNA nucleotide sequence from 97 human genes to their sequenced chimpanzee counterparts and to available sequenced gorilla, orangutan, and Old World monkey counterparts, and, on a more limited basis, to mouse. The nonsynonymous changes (functionally important), like synonymous changes (functionally much less important), show chimpanzees and humans to be most closely related, sharing 99.4% identity at nonsynonymous sites and 98.4% at synonymous sites. On a time scale, the coding DNA divergencies separate the human–chimpanzee clade from the gorilla clade at between 6 and 7 million years ago and place the most recent common ancestor of humans and chimpanzees at between 5 and 6 million years ago. The evolutionary rate of coding DNA in the catarrhine clade (Old World monkey and ape, including human) is much slower than in the lineage to mouse. Among the genes examined, 30 show evidence of positive selection during descent of catarrhines. Nonsynonymous substitutions by themselves, in this subset of positively selected genes, group humans and chimpanzees closest to each other and have chimpanzees diverge about as much from the common human–chimpanzee ancestor as humans do. This functional DNA evidence supports two previously offered taxonomic proposals: family Hominidae should include all extant apes; and genus Homo should include three extant species and two subgenera, Homo (Homo) sapiens (humankind), Homo (Pan) troglodytes (common chimpanzee), and Homo (Pan) paniscus (bonobo chimpanzee).

Evolution of increased glia–neuron ratios in the human frontal cortex

Evidence from comparative studies of gene expression and evolution suggest that human neocortical neurons may be characterized by unusually high levels of energy metabolism. The current study examined whether there is a disproportionate increase in glial cell density in the human frontal cortex in comparison with other anthropoid primate species (New World monkeys, Old World monkeys, and hominoids) to support greater metabolic demands. Among 18 species of anthropoids, humans displayed the greatest departure from allometric scaling expectations for the density of glia relative to neurons in layer II/III of dorsolateral prefrontal cortex (area 9L). However, the human glia–neuron ratio in this prefrontal region did not differ significantly from allometric predictions based on brain size. Further analyses of glia–neuron ratios across frontal areas 4, 9L, 32, and 44 in a sample of humans, chimpanzees, and macaque monkeys showed that regions involved in specialized human cognitive functions, such as “theory of mind” (area 32) and language (area 44) have not evolved differentially higher requirements for metabolic support. Taken together, these findings suggest that greater metabolic consumption of human neocortical neurons relates to the energetic costs of maintaining expansive dendritic arbors and long-range projecting axons in the context of an enlarged brain.

Starch-gel electrophoresis of malate dehydrogenase

Abstract Malate dehydrogenase preparations were subjected to electrophoresis on starch gel at pH 7.0. Purified preparations of mitochondrial malate dehydrogenase were shown to exist in up to six separable forms on the gel. The distribution pattern of these forms was not influenced by the age of the tissue of origin, the purification procedures used or by a number of degradative treatments, although methyl iodide and urea treatment had some effect on the preparations. After elution from the gel, the components of the mitochondrial enzymes were shown to be relatively similar by some catalytic criteria, but to differ collectively from the “supernatant” (cytoplasmic) enzyme from pig heart.

Genomics, biogeography, and the diversification of placental mammals

Previous molecular analyses of mammalian evolutionary relationships involving a wide range of placental mammalian taxa have been restricted in size from one to two dozen gene loci and have not decisively resolved the basal branching order within Placentalia. Here, on extracting from thousands of gene loci both their coding nucleotide sequences and translated amino acid sequences, we attempt to resolve key uncertainties about the ancient branching pattern of crown placental mammals. Focusing on ≈1,700 conserved gene loci, those that have the more slowly evolving coding sequences, and using maximum-likelihood, Bayesian inference, maximum parsimony, and neighbor-joining (NJ) phylogenetic tree reconstruction methods, we find from almost all results that a clade (the southern Atlantogenata) composed of Afrotheria and Xenarthra is the sister group of all other (the northern Boreoeutheria) crown placental mammals, among boreoeutherians Rodentia groups with Lagomorpha, and the resultant Glires is close to Primates. Only the NJ tree for nucleotide sequences separates Rodentia (murids) first and then Lagomorpha (rabbit) from the other placental mammals. However, this nucleotide NJ tree still depicts Atlantogenata and Boreoeutheria but minus Rodentia and Lagomorpha. Moreover, the NJ tree for amino acid sequences does depict the basal separation to be between Atlantogenata and a Boreoeutheria that includes Rodentia and Lagomorpha. Crown placental mammalian diversification appears to be largely the result of ancient plate tectonic events that allowed time for convergent phenotypes to evolve in the descendant clades.

Mammalian subunit IV isoforms of cytochrome c oxidase.

Cytochrome c oxidase (COX) contains ten nuclear encoded subunits, three of them known to show tissue isoforms in mammals. We have now found a fourth isoform, for subunit IV, in human, rat and mouse (COX IV-2). Comparison of the two human isoform genes shows a similar structural organization, including an overall size of about 8 kb, the presence of five exons, and the initiation of translation in the second exon, consistent with formation by gene duplication. Also consistent is the higher identity of precursor peptides of 78% within the new IV-2 isoform (average in the three species) compared to 44% average identity with the IV-1 isoform. Northern analysis and quantitative PCR with human and rat tissues show high IV-2 expression in adult lung and lower expression in all other tissues investigated, including fetal lung. In contrast, the IV-1 isoform is ubiquitously expressed. In situ hybridizations were performed to localize isoform transcripts in rat lung. Both isoforms are found in similar ratios in most lung cell types except for smooth muscle and respiratory epithelium, which have a IV-2 and a IV-1 preference, respectively. Structural modeling of the IV-2 isoform from human, based on the bovine crystal data, produces a conformation in which two of three conserved cysteine groups, exclusively present in the mammalian IV-2 isoform, are in close proximity. The formation of a cysteine bond and the implications for function of these sequence differences for subunit IV, which plays a pivotal role in COX regulation, are discussed.

Mitochondrial DNA in Yeast and Some Mammalian Species

Yeast DNA, in a cesium chloride density gradient, shows a minor or satellite band with a density lower than that of the main nuclear component. The DNA isolated from purified mitochondria of yeasts corresponds in density to this satellite band. In solution, this DNA more easily undergoes renaturation as compared to DNA from cell nuclei. The ease of this renaturation is presumably due to a homogeneity greater than that of nuclear DNA. Mitochondrial DNA isolated from several mammalian species has the same or higher density than nuclear DNA, but differs in its ready renaturability.

The reaction of formaldehyde with nucleotides and T2 bacteriophage DNA.

The mechanism of ribo- and deoxyribonucleotide reaction with formaldehyde has been examined with respect to kinetics, pH optima and structural specificity. Formaldehyde reacts only With those nucleotides bearing amino groups. The equilibrium constants for the reaction as well as the rate of the forward and reverse directions have been described. The difference between the reactivity of the ribo- and deoxyribo-nucleotides toward formaldehyde is negligible with the exception of the guanine ribo- and deoxyribo-nucleotides. That 5′-dGMP reacts at a faster rate than 5′-GMP has been attributed to a steric interference of the amino group hydroxymethylation and protonation by the 2′-hydroxyl group. Moreover nucleotide hydroxymethylation appears to be pH-dependent since the reaction is base-catalysed. The effect of formaldehyde on DNA thermal transitions was studied using spectrophotometric, viscometric and immunological methods. Hydroxymethylation is preceded by denaturation and lowers the temperature requirements for thermal transition. The depression of the temperature requirement for denaturation of bacteriophage DNA is attributed to the interference by formaldehyde of the equilibrium between native and denatured DNA by hydroxymethylation of the amino nitrogens associated with the purine and pyrimidine rings.

Metabolic costs and evolutionary implications of human brain development

Significance The metabolic costs of brain development are thought to explain the evolution of humans’ exceptionally slow and protracted childhood growth; however, the costs of the human brain during development are unknown. We used existing PET and MRI data to calculate brain glucose use from birth to adulthood. We find that the brain’s metabolic requirements peak in childhood, when it uses glucose at a rate equivalent to 66% of the body’s resting metabolism and 43% of the body’s daily energy requirement, and that brain glucose demand relates inversely to body growth from infancy to puberty. Our findings support the hypothesis that the unusually high costs of human brain development require a compensatory slowing of childhood body growth. The high energetic costs of human brain development have been hypothesized to explain distinctive human traits, including exceptionally slow and protracted preadult growth. Although widely assumed to constrain life-history evolution, the metabolic requirements of the growing human brain are unknown. We combined previously collected PET and MRI data to calculate the human brain’s glucose use from birth to adulthood, which we compare with body growth rate. We evaluate the strength of brain–body metabolic trade-offs using the ratios of brain glucose uptake to the body’s resting metabolic rate (RMR) and daily energy requirements (DER) expressed in glucose-gram equivalents (glucosermr% and glucoseder%). We find that glucosermr% and glucoseder% do not peak at birth (52.5% and 59.8% of RMR, or 35.4% and 38.7% of DER, for males and females, respectively), when relative brain size is largest, but rather in childhood (66.3% and 65.0% of RMR and 43.3% and 43.8% of DER). Body-weight growth (dw/dt) and both glucosermr% and glucoseder% are strongly, inversely related: soon after birth, increases in brain glucose demand are accompanied by proportionate decreases in dw/dt. Ages of peak brain glucose demand and lowest dw/dt co-occur and subsequent developmental declines in brain metabolism are matched by proportionate increases in dw/dt until puberty. The finding that human brain glucose demands peak during childhood, and evidence that brain metabolism and body growth rate covary inversely across development, support the hypothesis that the high costs of human brain development require compensatory slowing of body growth rate.