The carbamate reaction of glycylglycine, plasma, and tissue extracts evaluated by a pH stopped flow apparatus.

We have used a stopped flow rapid reaction pH apparatus to investigate the carbamate equilibrium in glycylglycine solutions and in three biological tissues, human plasma, sheep muscle, and sheep brain, as well as to investigate the kinetics of carbamate formation in glyclyglycine solution and in human plasma. The rapid reaction apparatus was equipped with a pH sensitive glass electrode in order to follow the time course of pH from 0.005 to 100 s after rapid mixing of a solution of amine or protein and CO2. Two phases of the pH curve were observed: a fast phase representing carbamate formation, and a slow phase due to the hydration of CO2 which was uncatalyzed since a carbonic anhydrase inhibitor was added to the biological solutions. From the time course of pH change during the fast phase K2, the R-NH2 ionization constant, and Kc, the carbamate equilibrium constant as well as the velocity constant for the formation of carbamate, ka could be calculated from data at different pH and pCO2. The carbamate formed in glycylglycine solutions over a wide range of pH and pCO2 was found consistent with the theory of carbamate formation and with published data. At ionic strength 0.16 and 37 degrees pK is 7.67. pKc 4.58. The heat of the carbamate reaction (deltaH) was calculated to be -3.2 kcal/mol between 20 degrees and 37 degrees. Kt of glycylglycine depends quantitatively on ionic strength as predicted by the Debye-Huckel theory. With ionic strength 0.16 ku was found to be 2,500 M1 S1 at 37 degrees. The activation energy of carbamate formation is 6.7 kcal/mol. Carbamate measurements in human plasma at pCO2 from 38 to 359 Torr. pH from 6.9 to 8.3, temperature 37 degrees, and ionic strength 0.15 provided evidence that two kinds of amino groups participate in carbamate formation. From the equilibrium constants computed for the two species they could be identified as alpha- and epsilon-amino groups. On the basis of a protein molecular weight of 69.000. 0.6 alpha-amino groups/molecule with pKz=7.0 and pKc=4.2, and 5.9 epsilon-amino groups/molecule with pKz=9.0 and pKc=4.3 contribute to carbamate formation. The velocity constant ka was estimated to be 4,950 M1 S1 for the alpha-amino groups and 13,800 M1 S1 for the epsilon-amino groups. Under physiological conditions (pCO2=40 Torr. pH=7.4). The concentration of carbamate in plasma is 0.6 mM and the half-time of carbamate formation is 0.05 s. In extracts prepared from sheep brain at 37 degrees pH=7 and pCO2=35 Torr. the carbamate formation was estimated to be 0.8 mM. With pCO2=70 Torr and the same pH and temperature the carbamate concentration in muscle approximates 0.3 mM and increases to 7 mM as pH rises to 8. It is concluded that, as in plasma, a considerable number of epsilon-amino groups appear to be available for carbamate formation in these tissues.     

Effects of temperature on diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart. Aspects of the kinetics, stability, and quarternary structure of the enzyme.

A temperature-dependent conformational change of the active DPN-linked isocitrate dehydrogenase was observed. When initial reaction kinetic data were examined between 35 and 5 degrees, the Hill number (n) varied from 2 at higher to n approaching unity at lower temperatures, with an inflection point at 17 degrees. The presence of manganous isocitrate in the incubation media shifted the transition temperature for enzyme inactivation by 5,5'-dithiobis(2-nitrobenzoate) from 8-16 degrees. These temperature-dependent transitions were paralleled by progressive changes in sedimentation velocities from s20, w of 10.4 at 25 degrees to 7.3 at 10 degrees as measured by active band centrifugation. The linear Arrhenius plot for apparent V max and the constancy of S0.5 for the substrate manganous isocitrate between 35 and 5 degrees suggest that this temperature-dependent conformational change may not be solely related to manganous isocitrate. Further indications of equilibria between different species of enzyme in solution and effects of substrates and cofactors on conformation came from studies of specific activity of enzyme diluted into buffers at 3 and 25 degrees. Dilution to concentrations between 10 and 25 mum enzyme resulted in relatively rapid protein concentration-dependent inactivation which could be prevented and fully reversed by manganous isocitrate. No further substantial inactivation was found subsequent to this phase at 25 degrees. Lowering the temperature of the dilution buffer to 3 degrees favored formation of enzyme species exhibiting a further time and pH-dependent loss of activity which became independent of protein concentration below 7 mum enzyme. The rate of cold inactivation was reduced by raising the ionic strength of the buffer and its progress could be arrested by manganous isocitrate; however, the substrate did not restore the original activity.     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Participation of lipopolysaccharide in hyperplasic adipose expansion: Involvement of NADPH oxidase/ROS/p42/p44 MAPK‐dependent Cyclooxygenase‐2

Obesity is a world‐wide problem, especially the child obesity, with the complication of various metabolic diseases. Child obesity can be developed as early as the age between 2 and 6. The expansion of fat mass in child age includes both hyperplasia and hypertrophy of adipose tissue, suggesting the importance of proliferation and adipogenesis of preadipocytes. The changed composition of gut microbiota is associated with obesity, revealing the roles of lipopolysaccharide (LPS) on manipulating adipose tissue development. Studies suggest that LPS enters the circulation and acts as a pro‐inflammatory regulator to facilitate pathologies. Nevertheless, the underlying mechanisms behind LPS‐modulated obesity are yet clearly elucidated. This study showed that LPS enhanced the expression of cyclooxygenase‐2 (COX‐2), an inflammatory regulator of obesity, in preadipocytes. Pretreating preadipocytes with the scavenger of reactive oxygen species (ROS) or the inhibitors of NADPH oxidase or p42/p44 MAPK markedly decreased LPS‐stimulated gene expression of COX‐2 together with the phosphorylation of p47^phox and p42/p44 MAPK, separately. LPS activated p42/p44 MAPK via NADPH oxidase‐dependent ROS accumulation in preadipocytes. Reduction of intracellular ROS or attenuation of p42/p44 MAPK activation both reduced LPS‐mediated COX‐2 expression and preadipocyte proliferation. Moreover, LPS‐induced preadipocyte proliferation and adipogenesis were abolished by the inhibition of COX‐2 or PEG₂ receptors. Taken together, our results suggested that LPS enhanced the proliferation and adipogenesis of preadipocytes via NADPH oxidase/ROS/p42/p44 MAPK‐dependent COX‐2 expression.     

Sensitivity of the S1 neuronal calcium network to insulin and Bay‐K 8644 in vivo: Relationship to gait,motivation, and aging processes

Neuronal hippocampal Ca²⁺ dysregulation is a critical component of cognitive decline in brain aging and Alzheimer''s disease and is suggested to impact communication and excitability through the activation of a larger after hyperpolarization. However, few studies have tested for the presence of Ca²⁺ dysregulation in vivo, how it manifests, and whether it impacts network function across hundreds of neurons. Here, we tested for neuronal Ca²⁺ network dysregulation in vivo in the primary somatosensory cortex (S1) of anesthetized young and aged male Fisher 344 rats using single‐cell resolution techniques. Because S1 is involved in sensory discrimination and proprioception, we tested for alterations in ambulatory performance in the aged animal and investigated two potential pathways underlying these central aging‐ and Ca²⁺‐dependent changes. Compared to young, aged animals displayed increased overall activity and connectivity of the network as well as decreased ambulatory speed. In aged animals, intranasal insulin (INI) increased network synchronicity and ambulatory speed. Importantly, in young animals, delivery of the L‐type voltage‐gated Ca²⁺ channel modifier Bay‐K 8644 altered network properties, replicating some of the changes seen in the older animal. These results suggest that hippocampal Ca²⁺ dysregulation may be generalizable to other areas, such as S1, and might engage modalities that are associated with locomotor stability and motivation to ambulate. Further, given the safety profile of INI in the clinic and the evidence presented here showing that this central dysregulation is sensitive to insulin, we suggest that these processes can be targeted to potentially increase motivation and coordination while also reducing fall frequency with age.     

Staining of Some Specific Regions of Human Chromosomes,particularly the Secondary Constriction of No. 9

SEVERAL procedures have been described recently which produce specific patterns of differential staining in human chromosomes^1–9. Techniques which involve DNA denaturation and reannealing reveal deeply stained areas on centromere and secondary constriction regions which have been equated with constitutive heterochromatin⁹.