Thin-layer separation and quantification of bile acids

A method has been developed for quantification of total free and conjugated bile acids separated on silica gel HR coated thin-layer chromatography plates. Aliquots of bile acid solutions are applied to channeled plates which are developed with either ethyl acetate: isooctane: glacial acetic acid 10:10:2 v/v for free bile acid separation, or chloroform:methanol:glacial acetic acid:water 130:50:4:8 v/v for conjugated bile acid separation. Bile acids are determined directly in serial areas of silica gel by treating gel areas suspended in tris buffer with resazurin reagent. The method is quantitative and as little as 0.1 μg of bile acid is readily determined. Application of the method to determinations of bile acids in crude fecal extracts is described.     

Source-sink relations in maize mutants with starch-deficient endosperms

Partitioning and translocation of photosynthates were compared between a nonmutant genotype (Oh 43) of corn (Zea mays L.) and two starch-deficient endosperm mutants, shruken-2 (sh2) and brittle-1 (bt1), with similar genetic backgrounds. Steady-state levels of ¹⁴CO₂ were supplied to source leaf blades for 2-hour periods, followed by separation and identification of ¹⁴C-assimilates in the leaf, kernel, and along the translocation path. An average of 14.1% of the total ¹⁴C assimilated was translocated to normal kernels, versus 0.9% in sh2 kernels and 2.6% in btl kernels. Over 98% of the kernel ¹⁴C was in free sugars, and further analysis of nonmutant kernels showed 46% of this label in glucose and fructose. Source leaves of mutant plants exported significantly less total photosynthate (24.0% and 36.3% in sh2 and bt1 compared to 48.0% in the normal plants) and accumulated greater portions of label in the insoluble (starch) fraction. Mutant plants also showed lower percentages of photosynthate in the leaf blade and sheath below the exposed blade area. The starch-deficient endosperm mutants influence the partitioning and translocation of photosynthates and provide a valuable tool for the study of source-sink relations.     

Analysis in vivo of translational mutants of the rIIB cistron of bacteriophage T4

We have mapped the mutants isolated by Nelson et al. (1981) that reduce the amount of rIIB protein synthesized during bacteriophage T4 infection of Escherichia coli B and characterized their rIIB expression in vivo. These mutants fall into four distinct groups in terms of mapping and phenotype. We have located the probable site of each mutation on the DNA sequence. We have also analyzed a number of other mutations near the initiating AUG of rIIB with respect to their rIIB expression. In some of these mutants, ribosomal recognition of the wild-type initiating AUG is precluded and so initiation occurs at a different AUG, which, in some instances, we have identified.     

Degradation of Arachidonoyl-Labeled Phosphatidylinositols by Brain Synaptosomes

Abstract: Incubation of synaptosomes together with 1-acyl-2-[¹⁴C]arachi-donoyl-sn-glycerophosphoinositols (GPI) and sodium deoxycholate yielded diacylglycerols and free arachidonic acid. Diacylglycerol formation is attributed to hydrolysis by the diacyl-GPI-specific phospholipase C (EC 3.1.4.10), and this reaction requires sodium deoxycholate for optimal activity. The free arachidonic acid formed is attributed to hydrolysis of diacyl-GPI by phospholipase A (EC 3.1.1.5). Free fatty acid release was observed during incubation, even in the absence of bile salts, but this process was preferentially stimulated by sodium taurocholate. The release of fatty acids was not specific for diacyl-GPI, as similar release was obtained during incubation with other phosphoglycerides. In the presence of deoxycholate (2 mg/ml), the release of diacylglycerols was maximal at a diacyl-GPI concentration around 1.0 mM. However, the free fatty acid release was linear with respect to the substrate at least up to 1.4 mM. The rate of diacylglycerol release from diacyl-GPI was more rapid in the initial 30 min, whereas the free fatty acid release was linear with time up to 2 h. Under this incubation condition, calcium was found to stimulate both types of hydrolytic action, although the concentration needed to achieve this stimulation was rather high. This type of labeled precursor is potentially useful for studies of the different modes of diacyl-GPI degradation by enzymes in brain subcellular membranes.     

Ultrastructural changes in the endoplasmic reticulum during dormancy release of apple embryos (Pyrus malus L.)

Apple embryos were treated by cold (0°C) within the fruits, to break their dormancy; the controls were treated at 12°C or at 20°C. Ultrastructural features of meristematic cells in the embryonic axis were compared for each treatment. The organization of the cells of dormant embryos was described: Endoplasmic reticulum consisted in some short rough cisternae; lipid droplets regularly arranged near the plasmalemma constituted a kind of shell; mitochondria had a few cristae; and dictyosomes were rarely observed. All these features are typical of dry seeds. After cold treatments, the only evolution observed was in the endoplasmic reticulum, where highly organized stacks appeared progressively as a function of time at 0°C. An intermediate temperature (12°C) induced similar formations in the reticulum but they were rarely observed and their degree of organization was lower than that obtained at 0°C. At 20°C, endoplasmic reticulum resembled that of the dormant embryo cells. The relation between the appearance of these structures in the reticulum and the disappearance of dormancy induced by cold is discussed.Abbreviations ER endoplasmic reticulum - RER rough endoplasmic reticulum     

Linkage of the HMP pathway to ATP generation by the proline cycle

The reactions catalyzed by proline oxidase and pyrroline-5-carboxylate reductase form a catalytic cycle linking the hexose-monophosphate pentose (HMP) pathway to mitochondrial ATP generation. The cycling of proline and pyrroline-5-carboxylate couples glucose oxidation to ATP generation by a mechanism independent of the Embden-Meyerhof pathway and the tricarboxylic acid cycle.     

Head-tail connector of bacteriophage lambda

The head-tail connector of phage λ, a protein knob inside the head shell to which the tail attaches, is composed primarily of head protein gpB ⁴ and its cleaved form gpB¹. All of the gpB and gpB¹ in the virion is located in the connector. gpFII, the protein that is thought to form the site on the head to which the tail binds, is also located in the connector. Head proteins gpE, gpD, X1 and X2 are not components of the connector. These assignments were made by disrupting virions with guanidine hydrochloride, in such a way that heads and tails separate with the connectors attached to the tails, and determining which head proteins co-purify with the tails.We find that lysates from a λE^? infection contain a high proportion of tails with connectors attached. (Gene E codes for the major component of the head shell.) Connectors are also present on tails from a λE^?C^? infection, arguing that gpE, gpC, and their processed forms, X1 and X2, are all unnecessary for assembly of biologically competent connectors. The gpB in the connectors on E^? and E^?C^? tails is in the uncleaved form. Connectors are not seen on tails from infections by λE^?B^?, λE^?FII^?, or λE^? in a groE^? host.     

Identification of the diol associated with variations in wax ultrastructure on Rhus cotinus leaves

The diol constituent of Rhus cotinus leaf epicuticular wax has been identified as nonacosane-5,10-diol from chemical investigations of the free compound, the TMSi ether and the nonacosane-5,10-dione prepared from the diol by oxidation. The form and distribution of the crystalline waxes changed as the leaves expanded, dense clusters of short tubes covering the thin ribbons formed during the initial stages of growth. The diol content of the wax decreased by more than 50% over the same period.     

Inhibition of the (Na+ + K+)-dependent ATPase by inorganic phosphate

Inhibition of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase by inorganic phosphate, P_i, was examined in terms of product inhibition of the various activities catalyzed by an enzyme preparation from rat brain, and considered in terms of the specific transport processes of the membrane Na⁺,K⁺-pump that these activities reflect. The K⁺-dependent phosphatase activity of the enzyme was most sensitive to P_i, and inhibition was competitive toward the substrate, nitrophenyl phosphate, as would be expected if P_i were released from the same enzyme form that bound substrate. However, this enzymatic activity does not seem to represent a transport process, and thus a cyclical discharge of K⁺ may not be involved. The Na⁺-dependent exchange activity was unaffected by P_i, in accord with the absence of P_i release in the reaction sequence. For the corresponding Na⁺/Na⁺ exchange function of the pump, which reportedly does not involve ATP hydrolysis either, prior release of P_i obviously cannot be required for Na⁺ discharge. With the Na⁺-dependent ATPase activity, measured using micromolar concentrations of ATP, P_i inhibited, but far less than with the phosphatase activity, and inhibition was not competitive toward ATP. Moreover, inhibition decreased as the Na⁺ concentration was raised from 10 to 100 mM. This elevated concentration of Na⁺ also led to substrate inhibition. For this ATPase activity, and the corresponding transport process, uncoupled Na⁺ efflux, the findings suggest that Na⁺ discharge follows P_i release, in contrast to Na⁺/Na⁺ exchange. The $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase activity, measured with millimolar concentrations of ATP and reflecting the coupled Na⁺,K⁺-transport function, was similarly sensitive to P_i, and again inhibition was not competitive toward ATP. However, in this case inhibition did not increase as the Na⁺ concentration was lowered. For this activity, and the associated transport process, the site of Na⁺ discharge in the overall reaction sequence remains unresolved.