Spectral properties of cobalt carboxypeptidase A. Interaction of the metal atom with anions

At pH greater than 7 the absorption and magnetic circular dichroic spectra of cobalt carboxypeptidase A are insensitive to anions [Latt, S. A., & Vallee, B. L. (1971) Biochemistry 10, 4263-4270], but at pH less than 6 chloride and other anions perturb them in a manner specific for each anion. Lowering of the pH apparently facilitates the entry of an anion into the metal coordination sphere, suggesting that an acidic group normally stabilizes a metal-coordinated water molecule against displacement. The lack of sensitivity to anions at pHs between 7 and 9--when the enzyme is maximally active--and its evident abolition upon protonation of an active-site group are consistent with this interpretation. Selective modification of cobalt carboxypeptidase at Glu-270 using a carbodiimide affinity reagent generates sensitivity to anions at pH 7 very similar to that of the unmodified enzyme at pH approximately 5. This suggests that the group stabilizing the metal-coordinated water is the catalytically essential carboxylate of Glu-270. These and related results provide evidence for a mechanistically important interaction of Glu-270 with a metal-bound water molecule.     

New labdane resin acids from Pinus elliottii

Slash pine needles and cortex oleoresin have been found to contain a new major diterpene constituent, imbricataloic acid. The closely related imbricatoloic acid, previously reported only in Araucaria imbricata, was found to be present in small amounts in slash pine needle extract. Spectral data are given for an unidentified diterpene alcohol isolated from the cortex oleoresin.     

Misincorporation during DNA synthesis, analyzed by gel electrophoresis.

A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by autoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to 'minus' reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme.     

Glycogen debranching enzyme: purification, antibody characterization, and immunoblot analyses of type III glycogen storage disease.

Type III glycogen storage disease is caused by a deficiency of glycogen debranching-enzyme activity. Many patients with this disease have both liver and muscle involvement, whereas others have only liver involvement without clinical or laboratory evidence of myopathy. To improve our understanding of the molecular basis of the disease, debranching enzyme was purified 238-fold from porcine skeletal muscle. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme gave a single band with a relative molecular weight of 160,000 that migrated to the same position as purified rabbit-muscle debranching enzyme. Antiserum against porcine debranching enzyme was prepared in rabbit. The antiserum reacted against porcine debranching enzyme with a single precipitin line and demonstrated a reaction having complete identity to those of both the enzyme present in crude muscle and the enzyme present in liver extracts. Incubation of antiserum with purified porcine debranching enzyme inhibited almost all enzyme activity, whereas such treatment with preimmune serum had little effect. The antiserum also inhibited debranching-enzyme activity in crude liver extracts from both pigs and humans to the same extent as was observed in muscle. Immunoblot analysis probed with anti-porcine-muscle debranching-enzyme antiserum showed that the antiserum can detect debranching enzyme in both human muscle and human liver. The bands detected in human samples by the antiserum were the same size as the one detected in porcine muscle. Five patients with Type III and six patients with other types of glycogen storage disease were subjected to immunoblot analysis. Although anti-porcine antiserum detected specific bands in all liver and muscle samples from patients with other types of glycogen storage disease (Types I, II, and IX), the antiserum detected no cross-reactive material in any of the liver or muscle samples from patients with Type III glycogen storage disease. These data indicate (1) immunochemical similarity of debranching enzyme in liver and muscle and (2) that deficiency of debranching-enzyme activity in Type III glycogen storage disease is due to absence of debrancher protein in the patients that we studied.     

Principles of Physiological Regulation of the Body Functions in Incomplete Adaptation

The main condition of completing the process of adaptation of the body to the effect of an external factor is the return of the homeostatic system parameters to their initial levels or their stabilization at a new level. The article considers the state of incomplete adaptation (IA) based on the process of the stabilization of systemic reactions (respiration and blood circulation) on repeated exposure to extreme environmental factors (hypoxia and cold) associated with the excitation of the central regulatory mechanisms of the respiratory center system performing a compensatory–protective function. It is postulated that a change in the afferent information flows (the thresholds of excitation and reactivity of the peripheral receptor systems) forms the basis of IA. The IA state is supposed to persist for an indefinitely long period of time due to insufficient functional reserves and to be the cause of psychosomatic pathology.     

Obesity-inducing lesions of the central nervous system alter leptin uptake by the blood-brain barrier.

Leptin regulates body adiposity by decreasing feeding and increasing thermogenesis. Obese humans and some obese rodents are resistant to peripherally administered leptin, suggesting a defect in the transport of leptin across the blood-brain barrier (BBB). Defective transport of exogenous leptin occurs in some models of obesity, but in other models transport is normal. This shows that factors other than obesity are associated with impairment of leptin transport across the BBB. In order to further investigate these factors, we determined leptin transport in rats made obese by lesioning of the ventromedial hypothalamus (VMH), paraventricular nucleus (PVN), or posterodorsal amygdala (PDA). These regions all contain leptin receptors and lesions there induce obesity and hyperleptinemia and alter the levels of many feeding hormones which might participate in leptin transporter regulation. We measured the uptake of radioactively labeled leptin by the BBB by multiple-time regression analysis which divides uptake into a reversible phase (Vi, e.g., receptor/transporter binding to the brain endothelial cell) and an irreversible phase (Ki, complete transport across the BBB). Leptin uptake was not affected in rats with VMH lesions. No significant change occurred in the entry rate (Ki) for any group, although Ki declined by over 35% in rats with PVN lesions. Decreased uptake was observed in rats with PVN lesions and with PDA lesions. This was primarily due to a reduced Vi (about 21% for the PDA). This decreased uptake is most likely explained by decreased binding of leptin to the brain endothelial cell, which could be because of decreased binding by either receptors or transporters. This suggests that some of the feeding hormones controlled by the PVN and PDA may participate in regulating leptin uptake by the BBB.     

Revision of Eurudagnostus Lermontova (Agnostida, ?Trilobita)

The validity of Eurudagnostus Lermontova, 1951 was confirmed by reexamination of the collection from the Boshe-Kul’ core. The diagnoses of genera of Agnostidae were reviewed, and Eurudagnostus falls well within the concept of the family Agnostidae. The diagnostic features of Eurudagnostus include a delicate border furrows, the transglabellar furrow bent posteriad or straight, short pygidial axis, incomplete transaxial F1, absent or very poorly developed median preglabellar furrow, and absent lanceolate field on the posteroaxis. Following revision the genus comprises 12 valid species, including some species and varieties previously assigned to other genera. ?Eurudagnostus brevispinus Lermontova, 1951 is synonymized with E. chiushuensis (Kobayashi, 1931). Two species, E. chiushuensis and ?E. intermedius (Palmer, 1968) appeared to have inter-continental distribution in the Cambrian-Ordovician beds, and E. mutabilis (Ergaliev, 1980) is also more widely distributed than previously recognized.