Effect of pH and bicarbonate on phosphoenolpyruvate carboxykinase and glutaminase mRNA levels in cultured renal epithelial cells.

A gluconeogenic strain of renal epithelial cells (LLC-PK1-F+) was used to characterize the effect of pH and bicarbonate concentration on the levels of phosphoenolpyruvate carboxykinase (PCK) and glutaminase (GA) mRNAs. The levels of both mRNAs are markedly dependent upon medium glucose concentration. The level of PCK mRNA is increased with increasing glucose concentration from 0 to 40 mM, whereas the level of GA mRNA is maximal between 3 and 5 mM glucose. When LLC-PK1-F+ cells are grown with 5 mM glucose and then subjected to an acute decrease in pH (from 7.4 to 6.9) and bicarbonate concentration (from 25 to 10 mM), the level of PCK mRNA exhibits a biphasic response. The PCK mRNA is initially increased 4-fold within 3 h, then decreases slightly and subsequently increases between 10 and 20 h to a level that is 17-fold greater than normal. Only the initial increase parallels the changes observed in vivo. In contrast, after onset of acidosis, the level of GA mRNA initially remains unchanged, is then increased 8-fold between 10 and 16 h, and then decreases slightly. This response closely mimics the results obtained in vivo. A decrease in media pH at constant bicarbonate causes a marked increase in both mRNAs. However, the levels of the two mRNAs are also elevated by decreasing bicarbonate at a constant pH. Thus, both parameters independently affect the level of the two mRNAs. The use of actinomycin D to measure the half-lives of PCK and GA mRNAs at pH 7.4 and 6.9 indicates that stabilization may fully account for the induction of GA mRNA and contributes to the inductive effects of decreased pH and/or bicarbonate on PCK mRNA. Following recovery from acidic conditions, the two mRNAs exhibit a rapid and coordinate decrease (t1/2 approximately 20 min). Dexamethasone had no effect on the level of either mRNA, whereas cAMP increased only PCK mRNA. The latter effect was additive with the increase caused by decreased pH and/or bicarbonate and was reversed by incubating in alkalotic media. Thus, the induction of PCK and GA mRNAs during acidosis is initiated in direct response to a decrease in extracellular pH and/or bicarbonate.     

An axial binding site in the Tetrahymena precursor RNA.

Previous studies allow the construction of three distinct models of the binding of G and arginine within the active site of the Tetrahymena self-splicing preribosomal precursor RNA. These models (base triple, axial I and axial II) are now distinguished by measurements on the specificity of RNAs with nucleotide substitutions at positions spanning the site. Because the semi-conserved unpaired nucleotide 263 has no effect on substrate or inhibitor selection by the Tetrahymena RNA we conclude that the axial I model is improbable. In contrast, data with substituted RNAs and nucleoside analogs suggest that nucleotide 265 makes a hydrogen bond with the substrate. Accordingly the active site appears axial because substrate contacts exist at more than one nucleotide on the 5' side of the P7 helix. The effects of this hydrogen bond are observable in cases where the donor or acceptor is on the RNA, whether nucleotide 265 is a purine or pyrimidine, or whether nucleotide 265 is mispaired, wobble paired or normally paired. This pattern is consistent with the axial II model. Molecular dynamics and energy minimization calculations lead to the same conclusions as these site-directed substitutions; the base triple and axial I models are unstable dynamically. Under thermal agitation, the third model site (axial II) is transformed to a related, but more stable structure, axial III. The axial III active site is characterized by the extrusion of the conserved bulged base 263 from the P7 helix, a semi-pocket for G base formed by stacking of nucleotide 262, and formation of all bonds to the G base originally proposed for both the base triple and axial II sites. Because of these hydrogen bonds the axial III site is also consistent with data on enzymatic specificity. The axial III model indicates an unforeseen capacity for pocket formation within the groove of an RNA helix, suggests that the site may be unusually flexible, and bears on a hypothesis concerning the origin of the genetic code.     

Kann die ?spezifisch dynamische Wirkung“ einen Beitrag zur Thermoregulation k?rnerfressender Singv?gel leisten?

Zusammenfassung 1. Respirometrische Messungen der spezifisch-dynamischen Wirkung (SDW) bei körnerfressenden Singvögeln zeigen, daß die SDW mit sinkenden Umgebungstemperaturen relativ kleiner wird. 2. Der Existenzstoffwechsel körnerfressender Singvögel weist nahrungsspezifische Unterschiede auf. Diese Unterschiede sind durch den unterschiedlichen SDW-Effekt verschiedener Nährstoffzusammensetzungen verursacht und sind von der Umgebungstemperatur abhängig: Sinken die Umgebungstemperaturen, werden die Unterschiede relativ kleiner. Diese Ergebnisse weisen darauf hin, daß körnerfressende Singvögel in der Lage sind, mit der SDW die Thermoregulation zu unterstützen.

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Can the specific dynamic action (SDA) contribute to the thermoregulation of granivorous song birds?

Summary 1. Respirometric measurements of the specific dynamic action (SDA) of granivorous song birds show that the SDA decreases relatively with dropping temperatures. 2. The existence metabolic rates of granivorous song birds show diet-related differences. These differences are caused by the SDA of different food types and are depend on the ambient temperature: With dropping ambient temperature differences become relatively smaller. These results indicate that granivorous song birds are able to use the SDA in thermoregulation.

     

Pollen and seed morphology of Nordic Draba (Brassicaceae): phylogenetic and ecological implications

Pollen and seed morphology were examined in 54 diploid and allopolyploid populations representing 15 Nordic species of the taxonomically complex genus Draba. The pollen size was strongly correlated with chromosome number, but it was unreliable for inferring the exact ploidal level of individual populations. Five main pollen types were recognized based on sculpturing of the exine. Two populations of D. lactea had conspicuously different exine sculpturing, supporting a previous hypothesis based on molecular data that this hexaploid is polyphyletic and has been derived from various combinations of diploid species. The pollen morphological data are also consistent with the hypotheses that the 16-ploid D. corymbosa is an intersectional allopolyploid derived from the sections Draba and Chrysodraba , that the hexaploid D. cinerea is a progenitor of the decaploid D. arctica , and that D. crassifolia and D. adamsii represent isolated allopolyploid lineages. The seed size and weight were only weakly correlated with chromosome number, but showed a close relationship to habitat ecology. The largest seeds were observed in species typical of closed habitats, in which seedling establishment probably is limited by competition with mosses. The seed coat surface had a characteristic verrucate reticulum, but the differentiation among species was vague or absent. The seed size and colour, however, distinguish some species, e.g., in the D. alpina complex, which otherwise are morphologically very similar.     

Physical and chemical scavenging of singlet molecular oxygen by tocopherols

Singlet molecular oxygen (1O2) arising from the thermal decomposition of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate was used to assess the effectiveness of alpha-, beta-, gamma-, and delta-tocopherol in the physical quenching as well as the chemical reaction of 1O2. The relative physical quenching efficiencies of the tocopherol homologs were found to decrease in the order of alpha greater than or equal to beta greater than gamma greater than delta-tocopherol. The ability of physical quenching depends on a free hydroxyl group in position 6 of the chromane ring. Chemical reactivity of the tocopherol homologs with 1O2 was low, accounting for 0.1-1.5% of physical quenching with beta-tocopherol showing particularly low reactivity, resulting in the sequence alpha greater than gamma greater than delta greater than beta-tocopherol. Tocopheryl quinones were products of all tocopherol homologs, and in addition a quinone epoxide was a major product from gamma-tocopherol. This quinone epoxide was not cleaved by rat liver microsomal epoxide hydrolase; however, it reacted further with 1O2. It is concluded that methylation in position 5 of the chromane ring enhances physical quenching of 1O2, whereas chemical reactivity is favored by a methylated position 7. In view of the fact that beta-tocopherol is as effective as alpha-tocopherol in physical quenching of 1O2 but shows very low chemical reactivity, this tocopherol homolog might be particularly suitable for biological conditions in which an accumulation of oxidation products might weaken the antioxidant defense.     

Avian alcohol dehydrogenase. Characterization of the quail enzyme, functional interpretations, and relationships to the different classes of mammalian alcohol dehydrogenase

The primary structure of the major quail liver alcohol dehydrogenase was determined. It is a long-chain, zinc-containing alcohol dehydrogenase of the type occurring also in mammals and hence allows judgement of the gene duplications giving rise to the classes of the human alcohol dehydrogenase system. The avian form is most closely related to the class I mammalian enzyme (72-75% residue identity), least related to class II (60% identity), and intermediately related to class III (64-65% identity). This pattern distinguishes the mammalian enzyme classes and separates classes I and II in particular. In addition to the generally larger similarities with class I, the avian enzyme exhibits certain residue patterns otherwise typical of the other classes, including an extra Trp residue, present in both class II and III but not in class I, with a corresponding increase in the UV absorbance. The avian enzyme further shows that a Gly residue at position 260 previously considered strictly conserved in alcohol dehydrogenases can be exchanged with Lys. However, zinc-binding residues, coenzyme-binding residues, and to a large extent substrate-binding residues are unchanged in the avian enzyme, suggesting its functional properties to be related to those of the class I mammalian alcohol dehydrogenases. In contrast, the areas of subunit interactions in the dimers differ substantially. These results show that (a) the vertebrate enzyme classes are of distant origin, (b) the submammalian enzyme exhibits partly mixed properties in relation to the classes, and (c) the three mammalian enzyme classes are not as equidistantly related as initially apparent but suggest origins from two sublevels.     

Determination of plasma free fatty acids, free cholesterol, cholesteryl esters, and triacylglycerols directly from total lipid extract by capillary gas chromatography

An accurate capillary gas chromatographic method using different internal standards for determining free fatty acids, cholesterol, cholesteryl esters, and triacylglycerols in plasma and other biological sources is described. It is designed to give information about species composition and, consequently, more detailed information about changes in lipid metabolism of patients suffering from metabolic disorders. After plasma extraction the lipids, except phospholipids, are directly examined without any further derivatization. For free fatty acid determination the programmed temperature vaporizer (PTV) injector was heated from 40 degrees C (sample introduction) to 190 degrees C. In a second gas chromatographic run the PTV-injector system was heated from 60 degrees C (sample introduction) to 400 degrees C, enabling the determination of free cholesterol, cholesteryl esters, and triacylglycerol species, differing in the number of carbon atoms. Evaluation of the values obtained resulted in coefficients of variation (%) of 1.0-2.8, 2.0, 1.29-2.24, and 2.8, for free fatty acid standards, plasma free fatty acids, cholesterol and cholesteryl ester standards, and plasma total cholesterol, respectively. Free fatty acids, cholesterol, and cholesteryl esters were not influenced by storage of plasma at -24 degrees C up to 4 days prior to extraction. The results of the gas chromatographic method and the enzymatic methods correlated well. Determination by gas chromatography yielded higher total cholesterol and lower triacylglycerol values than those values obtained by enzymatic methods.     

Continuous production of L-alanine from fumarate in a two-stage membrane reactor

Summary L-alanine was produced continuously from fumaric acid by means of soluble aspartase and L-aspartate--decarboxylase. The two reaction steps were carried out in two membrane reactors in series at different pH and temperature. The retention of the soluble enzymes within the reactor vessels was achieved by means of ultrafiltration membranes.