Positive and negative control of translation by the leader sequence of cauliflower mosaic virus pregenomic 35S RNA.

We have studied the influence of the 600 nt long leader sequence of cauliflower mosaic virus 35S RNA on downstream translation. Plant protoplasts were transfected with plasmids expressing a CAT reporter gene from a mRNA, containing wild-type or mutant forms of the 35S RNA leader. Deletion analysis revealed the presence of three separate stimulatory sequence regions, S1, S2 and S3. The latter two interact with each other to enhance downstream translation 5- to 10-fold. This enhancement was not observed in protoplasts from a non-host plant. In the absence of either S2 or S3, the region I2, located in between, exerts an inhibitory effect on downstream translation, probably due to the presence of short open reading frames. Expression of a reporter gene inserted into I2 increases 2-fold upon deletion of either S2 or S3. We propose that mRNA regions S2 and S3 form a complex with cellular factors that allows scanning ribosomes to bypass region I2.     

Validation of age readings from otoliths of juvenile roundnose grenadier, Coryphaenoides rupestris, a deep-water macrourid fish

Validation of the ageing of deep-water fish is difficult and there are only a few instances where the rings on the otoliths have been shown to be laid down annually. Roundnose grenadier have been fished commercially in the North Atlantic since the 1960s and the adult fish have frequently been aged by counting the rings in otoliths or scales. All the ageing was done on the assumption that the rings in the otoliths or scales were annual. Between 1975 and 1992, the Scottish Association for Marine Science carried out seasonal trawling surveys in the Rockall Trough using a fine-meshed trawl, and collected otoliths from a wide size range of roundnose grenadier. An examination of the growing edge of otoliths from juvenile fish from these collections suggests that the rings in the otoliths are laid down annually. The broader, opaque zones which represent the growth phase were dominant between September and March. The thinner, hyaline zones were dominant between April and July. The apparent delay in the growth phase compared with most shallow-water species is discussed in relation to the availability of mesopelagic prey.     

Nerve Growth Factor-Stimulated Nuclear S6 Kinase in PC12 Cells

Abstract: Previous work has shown that nerve growth factor (NGF) stimulates the phosphorylation of the ribosomal protein S6 in PC12 cells. In this study, we show that S6 kinase activity is also present in purified PC12 cell nuclei. This activity was increased by treatment of the cells with NGF and, to a lesser extent, by treatment with epidermal growth factor. The NGF-stimulated activity was obtained from nuclear extracts and some of its characteristics described. The increase in activity was prevented by treatment of the cells with rapamycin or with wortmannin, and the overall activity could be precipitated by antibodies directed against the p85^S6K. These data indicate that p85^S6K is the NGF-stimulated S6 kinase in PC12 cell nuclei. The presence of S6 protein in the nucleus of PC12 cells has been confirmed and evidence is presented that suggests that it is identical to a protein called SMP reported some years ago.     

Use of Cellulose Acetate Electrophoresis in the Taxonomy of Steinernematids (Rhabditida,Nematoda)

A steinernematid nematode was isolated from soil samples collected near St. John''s, Newfoundland, Canada. On the basis of its morphometry and RFLPs in ribosomal DNA spacer, it was designated as a new strain, NF, of Steinernema feltiae. Cellulose acetate electrophoresis was used to separate isozymes of eight enzymes in infective juveniles of S. feltiae NF as well as four other isolates: S. feltiae Umeå strain, S. feltiae L1C strain, Steinernema carpocapsae All strain, and Steinernema riobravis TX strain. Based on comparisons of the relative electrophoretic mobilities (μ) of the isozymes, one of the eight enzymes (arginine kinase) yielded zymograms that were distinctive for each of the isolates, except for the Umeå and NF strains of S. feltiae, which had identical banding patterns. Four enzymes (fumarate hydratase, phosphoglucoisomerase, phosphoglucomutase, and 6-phosphogluconate dehydrogenase) yielded isozyme banding patterns that were characteristic for all isolates, except for the L1C and NF strains of S. feltiae, which were identical. Two enzymes (aspartate amino transferase and glycerol-3-phosphate dehydrogenase) yielded zymograms that permitted S. carpocapsae All strain to be discriminated from the other four isolates, while the remaining enzyme (mannose-6-phosphate isomerase) was discriminatory for S. riobravis TX strain. Except for one enzyme, the isozyme banding pattern of the NF isolate of S. feltiae was the same as in the L1C strain, isolated 13 years previously from Newfoundland. Cellulose acetate electrophoresis could prove invaluable for taxonomic identification of isolates of steinernematids, provided that a combination of enzymes is used.     

The other kind of biological NMR—Studies of enzyme-substrate interactions

NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in drug metabolism. Cytochromes P₄₅₀ play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate complexes. The other two enzymes, chloramphenicol acetyltransferase and β-lactamase, are responsible for bacterial resistance to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme A bound to the enzyme, while in the case of β-lactamase the pK of a specific lysine residue at the active site has been determined, providing valuable information on the catalytic mechanism. Special issue dedicated to Dr. Herman Bachelard.     

Rapid reactions of spruce cells to elicitors released from the ectomycorrhizal fungus Hebeloma crustuliniforme, and inactivation of these elicitors by extracellular spruce cell enzymes

Elicitors released from hyphae or cell walls of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. induced in suspension-cultured cells of Picea abies (L.) Karst. a set of fast reactions: (i) an immediate efflux of Cl^– into the medium, followed by a K⁺ efflux; (ii) an influx of Ca²⁺ (measured as accumulation of ⁴⁵Ca²⁺ in the cells); (iii) a phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein (detectable by 4 min after elicitor application); (iv) an alkalinization of the medium, and (v) a transient synthesis of H₂O₂. The removal of extracellular Ca²⁺ by EGTA delayed the elicitor-induced alkalinization. A further reduction of this response could be achieved by TMB-8 an inhibitor of Ca²⁺ release from intracellular stores. Moreover, the inhibition of protein kinase activity by staurosporine prevented the extracellular alkalinization completely. However, the effectiveness of the elicitors in inducing the extracellular alkalinization was strongly impaired by constitutively secreted enzymes of spruce cells which cleaved the elicitors to inactive fragments. It is suggested that in ectomycorrhizae the efficacy of elicitors released from fungal cell walls is controlled by apoplastic enzymes of the host; the plant itself is able to reduce the activity of fungal elicitors on their way through the plant cell wall. But those elicitors which finally reach the plasma membrane of host cells induce reactions that are similar to the early defense reactions in plant-pathogen interactions.Abbreviations DW dry weight - FW fresh weight - TMB-8 3,4,5 trimethoxybenzoic acid 8-(diethylamino)-octyl ester We thank Prof. M. Zenk (Universität München, Germany) for providing spruce cell cultures, and Dr. I. Kottke (Universität Tübingen, Germany) for isolates of Hebeloma crustuliniforme Tü 704. We are also thankful to Dr. W. Mayer (Universität Tübingen) for valuble discussions. This work was supported by Deutsche Forschungsgemeinschaft. B. Zitterell-Haid was financed by Graduiertenkolleg Interaktion in Waldökosystemen (supported by Deutsche Forschungsgemeinschaft) and G. Hebe by a scholarship of the Landesgraduiertenförderungsgesetz.     

An Experimental Test of the Rhizopine Concept in Rhizobium meliloti

In some Rhizobium-legume symbioses, compounds known as rhizopines are synthesized by bacteroids and subsequently catabolized by free-living cells of the producing strain. It has been suggested than rhizopines act as proprietary growth substrates and enhance the competitive ability of the producing strain in its interactions with the diverse microbial community found within the rhizosphere. Wild-type, rhizopine-producing Rhizobium meliloti L5-30 and mutant L5-30 strains deficient for either rhizopine synthesis or catabolism were inoculated onto lucerne host plants in competition experiments. These experiments demonstrated that no apparent advantage resulted from the ability to synthesize a rhizopine, whereas the ability to catabolize rhizopine provided a clear advantage when an organism was in competition with a strain without this ability. The results suggest that when an organism is in competition with a catabolism-deficient mutant, the ability to catabolize rhizopine results in enhanced rates of nodulation. The results of the experiments were not consistent with the hypothesis that the sole role of rhizopines is to act as proprietary growth substrates for the free-living population of the producing strain.     

Differentiation trees,a junk DNA molecular clock,and the evolution of neoteny in salamanders

Obligate neotenic salamanders die if forced to metamorphose. We suggest that this can be explained by assuming: 1) their “excess” DNA is “junk” DNA; 2) the “adult” specifying portion of the DNA becomes junk DNA and is available for repeated duplication. This suggests a “new” junk DNA molecular clock. We obtain remarkable agreement in “predicting” the amount of DNA per nucleus in present day non-obligate neotene salamanders from this molecular clock. These observatons are consistent with the idea that the development of these animals is describable in terms of differentiation trees whose branches (gene cascades) corresponding to adult somatic tissues accumulate deleterious mutations over evolutionary time. We show that the amount of DNA per nucleus increases linearly with the phylogenetic age of salamander families. The lack of constraints by natural selection, on unused adult branches, may account for the large amount of so-called “junk DNA” in obligate neotenic salamanders. The effects of this excess DNA, via increased cell size, suggest a positive feedback, ecophysiological explanation for such junk DNA: adaptation to cool water environments is enhanced by the lower metabolism associated with more DNA, larger cells and slower developmental time.     

Carbohydrate fermentation by three species of polycentric ruminal fungi from cattle and water buffalo in tropical Australia

Fructose, glucose and xylose were the only monosaccharides to be fermented by the polycentric fungi, Orpinomyces joyonii (three cattle isolates) and O. intercalaris (two cattle isolates) and Anaeromyces spp. (four cattle isolates and two water buffalo isolates). Both Orpinomyces spp. utilised a similar range of oligosaccharides and polysaccharides by fermenting cellobiose, gentiobiose, lactose, maltose, sucrose, cellulose, glycogen, starch and xylan. In contrast, there was considerable variation in carbohydrate fermentation amongst Anaeromyces spp., with only cellobiose, gentiobiose and cellulose being fermented by all strains. Formate, acetate and ethanol were the major fermentation end-products formed from glucose by all polycentric fungi. In addition, Anaeromyces spp. produced considerable amounts of lactate, although only small amounts were formed by Orpinomyces spp. This difference was explained by the low specific activity for lactate dehydrogenase in Orpinomyces spp. Several Anaeromyces spp. also produced malate as a significant end-product of glucose fermentation. Fermentation of specifically-labelled Z14C]glucose molecules by polycentric fungi showed that hexose was catabolised by both polycentric and monocentric fungi via the glycolysis pathway with end-products being derived from the following carbon atoms: lactate and malate (C1-C3; C4-C6), acetate and ethanol (C1-C2; C5-C6), CO2 and formate (C3; C4). The results were compared to those obtained for monocentric and polycentric fungi isolated from temperate climate ruminants.     

Sialoadhesin, a macrophage sialic acid binding receptor for haemopoietic cells with 17 immunoglobulin-like domains.

Sialoadhesin is a macrophage-restricted adhesion molecule of 185 kDa that mediates sialic acid-dependent binding to cells. It is expressed strongly by macrophages in lymphoid and haemopoietic tissues where it is likely to mediate cell-cell interactions. Here we report the molecular cloning of murine sialoadhesin and show that it is a new member of the immunoglobulin (Ig) superfamily with 17 Ig-like domains. COS cells transfected with a cDNA encoding full-length sialoadhesin bound mouse bone marrow cells in a sialic acid-dependent manner. Alternatively spliced cDNAs, predicting soluble forms of sialoadhesin containing the first three or 16 Ig-like domains of sialoadhesin, were expressed in COS cells and the respective proteins purified. When immobilized on plastic, the 16-domain form bound cells in a sialic acid-dependent manner, suggesting that sialoadhesin can function in both secreted and membrane-bound forms. The most similar proteins in the database were CD22, myelin-associated glycoprotein, Schwann cell myelin protein and CD33. Like sialoadhesin, CD22 mediates sialic acid-dependent cell adhesion. The sequence similarity of sialoadhesin to CD22 and related members of the Ig superfamily indicates the existence of a novel family of sialic acid binding proteins involved in cell-cell interactions.