Lactate and pH in the Brain: Association and Dissociation in Different Pathophysiological States

Brain tissue pH and lactate content were measured in rats under three different experimental conditions, namely: during complete global cerebral ischemia; after reversible near-complete cerebral ischemia; and in experimental brain tumors. At the end of the experiments brains were frozen with liquid nitrogen. A series of 20-microns thick coronal sections was prepared in a cryostat and then used for the regional determination of tissue pH (umbelliferone technique) and tissue lactate (bioluminescent technique). In addition, tissue samples were taken for the quantitative measurement of brain lactate (enzymatic fluorometric technique). The relationship between lactate content and tissue pH was different for each of the three experimental models studied: only after short-term global cerebral ischemia did an increase in the lactate content correlate with a decrease in tissue pH (r = 0.94; p less than 0.001). A highly significant increase in the lactate content (p less than 0.001) was accompanied by physiological pH values (6.96 +/- 0.08 in comparison to 6.97 +/- 0.04 in controls) during recirculation after transient cerebral ischemia and in brain tumors even by an alkaline pH shift. In view of these observations the term "lactacidosis" should not be used without measuring both the lactate content and the pH. The observed dissociation between pH and lactate is due to the fact that both parameters are regulated independently. During anaerobiosis the main source of proton production is ATP hydrolysis rather than glycolysis. It is, therefore, suggested that the terms "acidosis" and "lactosis" should be used instead of "lactacidosis."     

Identification of replication and stability functions in the complete nucleotide sequence of plasmid pUH24 from the cyanobacterium Synechococcus sp. PCC 7942

The complete nucleotide sequence is presented for pUH24, the small plasmid of Synechococcus sp. PCC 7942. pUH24 consists of 7835bp and has a G+C content of 59%. The distribution of translation start and stop codons in the sequence allows 36 open reading frames that potentially encode polypeptides of 50 or more amino acids. We postulate that eight of these open reading frames are actual coding sequences. A region has been identified, by experiment, that contains two functions, designated pmaA and pmaB, involved in the segregational stability of the plasmid. The minimal region of pUH24 fully capable of supporting autonomous replication consists of a 3.6kb DNA fragment, which is almost entirely occupied by two overlapping genes most likely coding for essential replication proteins (repA and repB).     

SAR, species specificity, and cellular activity of cyclopentene dicarboxylic acid amides as DHODH inhibitors

Novel DHODH inhibitors were developed based on a previously described series by introduction of heteroatoms into the cyclopentene ring and hydroxyl groups attached to it. Also, the hydrophobic biphenyl side chain was replaced with benzyloxy phenyl groups. Activities on human, rat, and mouse enzymes indicate a species specificity of these inhibitors.     

Phosphorylation State,Solubility, and Activity of Calcium/Calmodulin-Dependent Protein Kinase IIα in Transient Focal Ischemia in Mouse Brain

During and after middle cerebral artery occlusion in mice, CaMKII protein was irreversibly translocated from the soluble to the Triton X-100–nonsoluble fraction. This decrease in solubility had a strong effect on activity: CaMKII was almost completely inactivated after being translocated. Results from solubilization experiments suggest that different mechanisms underlie the conversion of CaMKII protein from a soluble to a detergent nonsoluble form in ischemic as opposite to nonischemic tissue. Analysis of the phosphorylation state of CaMKII revealed that in the total homogenate and the Triton X-100–nonsoluble fraction, CaMKII phosphorylated at only one site was the dominant phosphorylated form, whereas in the soluble fraction CaMKII phosphorylated at two sites was the predominant phosphorylated species. Investigation of the mechanisms underlying ischemia-induced changes in the solubility of CaMKII could help to elucidate processes triggered by transient focal cerebral ischemia that lead to neuronal cell injury.     

Comparison of Freeze-Blowing and Funnel-Freezing of Rat Brain for the Measurement of Cerebral Glucose Concentration In Vivo

The efficacy of funnel-freezing of rat brain to inactivate metabolic processes and preserve in vivo tissue glucose concentration was validated by comparing the results obtained by funnel-freezing with those obtained with freeze-blowing of brain. The arterial plasma glucose level was clamped at 9 mM in halothane-anesthetized rats to produce identical glucose levels in brain tissue prior to freeze fixation. In funnel-frozen and freeze-blown brains, tissue glucose concentrations were 2.47 +/- 0.05 and 2.47 +/- 0.06 mumol/g (means +/- SEM), respectively. Lactate levels in funnel-frozen brains were slightly but significantly higher than those in freeze-blown brains, i.e., 1.56 +/- 0.05 mumol/g versus 1.30 +/- 0.05 mumol/g (means +/- SEM; p less than 0.05). Regional analysis in funnel-frozen brains revealed that glucose concentrations in superficial and basal brain areas remained approximately equal at 2.30 +/- 0.1 mumol/g and 2.31 +/- 0.09 mumol/g (means +/- SEM), respectively. Our findings indicate that in the anesthetized rat, funnel-freezing of brain is suitable for the measurement of regional in vivo glucose concentrations.