Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with nitrogen-15

A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts [see Delihas, N., Anderson, J., & Singhal, R. P. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31, 161-190] but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a "normal" chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for our understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed.     

Behavior of L-threonine-degrading enzymes during liver regeneration

We examined the effects of a two-thirds hepatectomy in the adult rat on the activities of the three L-threonine-degrading enzymes, L-threonine dehydratase, L-threonine aldolase and L-threonine dehydrogenase. Noticeable variations were observed which did not occur in either sham-operated or turpentine-treated rats and were not linked to food intake. They were considered specific to the regenerating liver. When the reactions were followed in vitro, L-threonine deaminase and L-threonine aldolase were significantly lower for the first 12-24 h: L-threonine dehydrogenase decreased only after 48 h. These results are linked to a decrease in the enzyme concentration in the tissue. L-Serine and L-threonine liver concentrations increased 2-3-fold during the same periods. When the activities were evaluated in vivo, the levels of the first two enzymes remained constant for 24 h, but increased after 48 h; L-threonine dehydrogenase increased between 12 and 48 h. The in vivo activity of the enzymes was reflected by total L-threonine degradation, which had a single sharp peak at 48 h. The asynchronous variations in enzyme activity are related to the differences in protein metabolism which occur in the regenerating liver, and are the consequence of a new transient differential control. The changes observed are significant in liver regeneration; they regulate the consumption and the serum and liver levels of L-serine and L-threonine, setting them aside for protein synthesis. They minutely control the flux of amino acids toward gluconeogenesis, since, during the first 48 h after partial hepatectomy, the production of glucose is ensured principally by lactate; the contribution of L-threonine seems to be more significant only at 48 h. These findings are useful in the study of the regulation of the enzymes involved in amino acid metabolism during liver regeneration.     

Chemical modification of human aldehyde dehydrogenase by physiological substrate

Employing 3,4-dihydroxyphenylacetaldehyde (dopal) as a substrate for human aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) in anaerobic conditions, inactivation of both cytoplasmic E1 and mitochondrial E2 isozymes during catalysis has been observed. Incorporation of 14C-labelled dopal has been demonstrated by retention of label following denaturation and exhaustive dialysis and by peptide mapping following tryptic digestion. Incorporation of label gave linear plots vs. activity remaining with up to two molecules incorporated per molecule of enzyme and 30% activity remaining. Further incorporation (up to 16 molecules) occurred, but was non-linear when plotted vs. activity remaining. Protection against activity loss during incorporation of the first two molecules was afforded by NAD, NADH, chloral, and by chloral and NAD together, the last being the most effective. Saturation kinetics gave y-axis intercepts, suggesting interaction at a specific point on the enzyme surface. The Ki value from saturation kinetics was the same as that from the slope replot in catalytic reaction. Peptide mapping of tryptic digests showed that a single peptide was labelled, confirming specificity of interaction. Even in the absence of complete inactivation, the results suggest that reaction with the first two molecules occurs at some point on the enzyme surface important for enzyme activity. The possibility of such a reaction occurring in vivo is discussed.     

Lipid metabolism in various regions of squid giant nerve fiber

The purpose of this investigation was to compare the incorporation of radioactivity from various precursors into lipids of different regions of squid giant nerve fiber systems including axoplasm, axon sheath, giant fiber lobes which contain stellate ganglion cell bodies, and the remaining ganglion including giant synapses. To identify the labeled lipids, stellate ganglia including giant fiber lobes and the remaining tissue were first incubated separately with [14C]glucose, [32P]phosphate, [14C]serine, [14C]acetate and [3H]myristate. The radioactivity from glucose, after conversion to glycerol and fatty acids, was incorporated into most lipids, including triacylglycerol, free fatty acids, cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, sphingomyelin and ceramide 2-aminoethylphosphanate [corrected]. The radioactivity from serine was largely incorporated into phosphatidylserine and, to a lesser extent, into other phospholipids, mainly as the base component. The sphingoid bases of ceramide and sphingomyelin were also significantly labeled. Saturated and monounsaturated and, to a lesser extent, polyunsaturated fatty acids of these lipids were synthesized from acetate, glucose and myristate. Among the major lipids, cholesterol was not labeled by any of the radioactive compounds used. Ganglion residues incorporated the most radioactivity in total lipids from either [14C]glucose or [14C]serine, followed by giant fiber lobes and then sheath. Axoplasm incorporated the least. Among various lipids, phosphatidylethanolamine with shorter saturated fatty acids and phosphatidylglycerol contained the most radioactivity from glucose in all regions. Axoplasm was characterized by a higher proportion of glucose radioactivity in ceramide, sphingomyelin and phosphatidylglycerol. Axoplasm and sheath contained a higher proportion of serine radioactivity than did the other two regions in ceramide. Essentially no radioactivity from [14C]galactose was incorporated in any region.     

Immunogenicity and antigenicity of immunoglobulins: detection of human immunoglobulin light-chain carbohydrate, using concanavalin A

Concanavalin A binding to glycoprotein bands on nitrocellulose blots was used to detect mannose, sorbose, N-acetylgalactosamine and/or glucose residues on 100% (31/31) of human Bence Jones protein light chains, following sodium dodecyl sulphate-polyacrylamide gel electrophoresis. All (20/20) light chains form IgG myeloma proteins and light chains from a preparation of normal polyclonal human IgG were also bound by concanavalin A. The specificity of concanavalin A for glycoproteins was demonstrated by its binding to human Fc fragments and a human monoclonal anti-Rhesus D antibody (REG-A), but not to human albumin pFc' fragments and aglycosylated REG-A derived from cells grown in the presence of the glycosylation inhibitor tunicamycin. These results suggest that all Bence Jones proteins and light chains from myeloma IgG proteins contain mono- or oligosaccharides linked O-glycosidically to serine or threonine residues.     

Structural and physico-chemical properties of amino acids of functionally important regions of proteins

A correlation of the localization of functionally important regions with places having low and high values on twelve profiles built on a basis of amino acid sequences was analysed using a broad set of proteins. The profiles of hydrophilicity, resemblance to the sequences of human proteins, flexibility, mutability and others were considered. The resemblance profile was plotted by the program fixing short similar fragments in the testing protein and 92 human ones. The active centres were shown to be located in the primary structure regions having relatively low values on the resemblance profiles. Similar effect was observed in the mutability and alpha-helicity profiles. The potential functionally important sites of the human leukocyte interferon and interleukin-2 isolated on the basis of the analysis of this profiles were in accord with the available literary data.     

Endocrine responses in relation to compensatory testicular growth after neonatal hemicastration in boars

Mass (TM) and relative mass (organ mass/body mass; RTM) of the right testis and epididymis (EM and REM, respectively) were determined every 14 days from 10 to 122 days of age for intact boars (I) and boars hemicastrated on Day 10 (HC) in two crossbred herds (Trial 1 and Trial 2). Plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, growth hormone (GH), and testosterone were determined in four blood samples from each pig, three collected 24 h prior to castration and one immediately prior to castration. Values for TM and RTM of HC boars were approximately double (p less than 0.0001) those of I boars by 38 days of age, and these differences were maintained through Day 122. Both EM and REM were greater (p less than 0.05) in HC than in I boars from Day 52 to Day 122. The TM, RTM, EM and REM were greater (p less than 0.05) in Trial 1 than in Trial 2 for both I and HC boars from Day 80 to Day 122, indicating an earlier onset of pubertal testicular growth in the Trial-1 boars. Plasma GH concentration was greater (p less than 0.05) in HC than in I boars from Day 16 to Day 38. A transient increase in plasma FSH (p less than 0.05) was observed from Day 24 to Day 38. After Day 38, there was no difference (p greater than 0.05) in FSH or GH between HC and I boars, or between trials. Plasma LH, prolactin, and testosterone concentrations were also similar in HC and I boars.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tip size of ion-exchanger based K+-selective microelectrodes. I. Effects on selectivity

Double-barreled ion-exchanger based K+-selective microelectrodes (K+ ISMs) of a variety of tip diameters were used to study the dependency of ion selectivity upon tip size. The selectivity of K+ ISMs depended on tip size and barrel configuration. Within the range of tip diameters tested (approximately 0.5-6 micron) all K+ ISMs constructed of two barrels glued side by side ("figure-eight glass") exhibited sensitivity to K+ and NH4+. Figure-eight K+ ISMs with tip diameters less than 1.5 micron were not sensitive to tetramethylammonium, tetraethylammonium, or choline, whereas K+ ISMs with tip diameters greater than or equal to 1.5 micron sensed all of the quaternary amines. Tip size dependent selectivity was not present in K+ ISMs made from thick septum theta glass. The explanation for tip size dependent changes in ion selectivity is unknown but a discussion of theoretical possibilities is given.