Preparation and characterization of cell-free protein synthesis systems from oocytes and eggs of Xenopus laevis

During the maturation of the oocytes of the frog Xenopus laevis, the rate of protein synthesis shows a twofold increase. Studies of the mechanisms involved in this stimulation have been seriously limited by the lack of an active cell-free translation system. We have now prepared such systems from oocytes, progesterone-matured oocytes and eggs of Xenopus laevis by induction of lysis by centrifugation of whole cells. The extracts are highly active in incorporation of labelled amino acids and, in the progesterone-matured and egg extracts, a substantial proportion of this is due to reinitiation on endogenous mRNA, as shown by the use of inhibitors. The increased rate of protein synthesis previously observed in intact oocytes following progesterone-induced maturation is reflected in the relative activities of the extracts. The difference in activity is not due to the presence of a dominant inhibitor of translation in the extracts from unstimulated oocytes. Labelling studies with initiator tRNA ([35S]Met-tRNAf) indicate a higher concentration of 43S preinitiation complexes in the extracts from unstimulated oocytes, suggesting an impairment of initiation of translation at or after the mRNA-binding step. Extracts from both oocytes and progesterone-matured oocytes translated endogenous mRNAs to give products ranging over a wide spectrum of molecular weight. However, significant translation of exogenous (globin) mRNA required the presence of reticulocyte postribosomal supernatant, suggesting that one or more factors required for mRNA recruitment is limiting in these extracts.     

Perspectives of taste reception

Summary Ion: solute cotransporters frequency are incapable of achieving equilibrium between the solute accumulation and the transmembrane difference of the electrochemical potential of the ion. The presence of uncoupled flows of ion and solutes (leaks) is often advanced as an explanation. Here an alternative is discussed. The net accumulation of solute may be so slow that equilibrium can never be attained at finite times (e.g., several hours). Cotransporters may exhibit strong product inhibition, and the net influx of solute approaches zero far from equilibrium. The inherent slowness of net transport under these conditions is termed catalytic inefficiency. The likelihood that galactoside: H⁺ cotransport inEscherichia coli, hexose: H⁺ cotransport inChlorella vulgaris, andd-glucose: Na⁺ cotransport in brush-border membranes exhibit catalytic inefficiency is examined. The existence of strong product inhibition complicates the determination of the stoichiometry of cotransport and the characterization of chemically modified or mutant cotransporters.     

Molecular genetic study of human arginase deficiency

We have explored the molecular pathology in 28 individuals homozygous or heterozygous for liver arginase deficiency (hyperargininemia) by a combination of Southern analysis, western blotting, DNA sequencing, and PCR. This cohort represents the majority of arginase-deficient individuals worldwide. Only 2 of 15 homozygous patients on whom red blood cells were available had antigenically cross-reacting material as ascertained by western blot analysis using anti-liver arginase antibody. Southern blots of patient genomic DNAs, cut with a variety of restriction enzymes and probed with a near-full-length (1,450-bp) human liver arginase cDNA clone, detected no gross gene deletions. Loss of a TaqI cleavage site was identified in three individuals: in a homozygous state in a Saudi Arabian patient at one site, at a different site in homozygosity in a German patient, and in heterozygosity in a patient from Australia. The changes in the latter two were localized to exon 8, through amplification of this region by PCR and electrophoretic analysis of the amplified fragment after treatment with TaqI; the precise base changes (Arg291X and Thr290Ser) were confirmed by sequencing. It is interesting that the latter nucleotide variant (Thr290Ser) was found to lie adjacent to the TaqI site rather than within it, though whether such a conservative amino acid substitution represents a true pathologic mutation remains to be determined. We conclude that arginase deficiency, though rare, is a heterogeneous disorder at the genotypic level, generally encompassing a variety of point mutations rather than substantial structural gene deletions.     

Secondary sexual traits, parasites, and polygyny in red-winged blackbirds, Agelaius phoeniceus

Data from a 3-year study of red-winged blackbirds (Agelaiusphoeniceus) were used to test the hypothesis that parasites(in this case, haematozoa) reduce male fitness and cause diminishedexpression of secondary sexual traits, which, in turn, are usedby females to select parasite-free males as mates. There wasno evidence indicating a fitness cost to being parasitized becauseparasitized males were as likely as unparasitized males to acquirea territory and to survive from one year to the next. Similarly,parasitized and unparasitized females did not differ with regardto how early they started nesting, how many eggs they laid,or their year-to-year survival. Secondary sexual traits, particularlyintrasexual aggression, did reliably (>80%) reveal the parasitestatus of males. Plumage and morphological traits also alloweddiscrimination of parasitized and unparasitized females. However,apparent mating patterns were unrelated to either the males'or the females' parasite status. Only if genetic analyses revealthat unparasitized males actually realize higher productivesuccess will these results potentially provide support for theparasite hypothesis of sexual selection.     

Effects of abscisic acid on somatic embryo maturation of hybrid larch

Somatic embryos of hybrid larch (Larixleptoeuropaea) whichhad been matured for 4 weeks on maturation medium, developednormally on medium supplemented with 60 µM ABA, but abnormallyon medium with no ABA. A comparative structural and histochemicalinvestigation was carried out on these two types of mature embryos.At the light microscope level, differences between both treatmentswere visible only after 2–3 weeks of maturation. At aroundthis time, abnormal development becomes evident macroscopically:ABA-minus embryos remain rather stubby as opposed to the morecylindrically shaped ABA-plus embryos. Whereas somatic embryosmatured with ABA consist of densely cytoplasmic cells showinga high rate of cell division, ABA-minus embryos are largelymade up of expanded and highly vacuolate cells, indicating thatgrowth in the latter is mainly due to cell expansion and notdivision. After 4 weeks of maturation, ABA-minus embryos beginto elongate in the hypocotyl region, and precocious germinationwas observed frequently. Again, these morphogenetic events werelargely due to abnormal timing of cell expansion. Histochemically,storage proteins were found only in somatic embryos maturedfor 4 weeks with ABA. This observation is in line with resultsobtained by total protein analysis, yielding significantly lowertotal protein contents in ABA-minus embryos both on a freshweight and a per embryo basis after 4–5 weeks of maturation.Deposition of starch grains mainly in the cortex tissue of thehypocotyl region was observed within 2 weeks of maturation invarying amounts regardless of ABA supply. Polyphenols, in particularcatechins and proanthocyanidins, were present in all embryosfrom the very onset of development. They were localized preferentiallyin the proximal suspensor cells and the basal region of theembryo. However, accumulation of polyphenols was generally muchmore pronounced in embryos matured without ABA, indicating alack of biochemical regulatory competence in those embryos. Key words: Abscisic acid, embryonal development, somatic embryo, storage protein, polyphenols     

HOST-PLANT SWITCHES AND THE EVOLUTION OF CHEMICAL DEFENSE AND LIFE HISTORY IN THE LEAF BEETLE GENUS OREINA

Insect-plant interactions have played a prominent role in investigating phylogenetic constraints in the evolution of ecological traits. The patterns of host association among specialized insects have often been described as highly conservative, yet not all specialized herbivorous insect lineages display the same degree of fidelity to their host plants. In this paper, we present an estimate of the evolutionary history of the leaf beetle genus Oreina. This genus displays an amazing flexibility in several aspects of its ecology and life history: (1) host plant switches in Oreina occurred between plant families or distantly related tribes within families and thereby to more distantly related plants than in several model systems that have contributed to the idea of parallel cladogenesis; (2) all species of the genus are chemically defended, but within the genus a transition between autogenous production of defensive toxins and sequestration of secondary plant compounds has occurred; and (3) reproductive strategies in the genus range from oviparity to viviparity including all intermediates that could allow the gradual evolution of viviparity. Cladistic analysis of 18 allozyme loci found two most parsimonious trees that differ only in the branching of one species. According to this phylogeny estimate, Oreina species were originally associated with Asteraceae, with an inclusion of Apiaceae in the diet of one oligophagous species and an independent switch to Apiaceae in a derived clade. The original mode of defense appears to be the autogenous production of cardenolides as previously postulated; the additional sequestration of pyrrolizidine alkaloids could have either originated at the base of the genus or have arisen three times independently in all species that switched to plants containing these compounds. Viviparity apparently evolved twice in the genus, once without matrotrophy, through a retention of the eggs inside the female's oviducts, and once in combination with matrotrophy. We hypothesize that the combination of autogenous defense and a life history that involves mobile externally feeding larvae allowed these beetles to switch host plants more readily than has been reported for highly conservative systems.     

Cloning and characterization of theeapB andeapC genes ofCryphonectria parasitica encoding two new acid proteinases, and disruption ofeapC

Two new proteinases secreted byCryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that theeapB andeapC genes contain three and two introns, respectively. The products of theeapB andeapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. TheeapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of theeapC coding sequence in an endothiapepsin-deficient (EapA⁺)C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.     

Linkage disequilibrium analysis in Machado-Joseph disease patients of different ethnic origins

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration originally described in families of Portuguese-Azorean ancestry. The hypothesis that its present world distribution could result from the spread of an original founder mutation has been raised. To test this possibility we have conducted a linkage disequilibrium study of markers segregating with the MJD1 locus in a total of 64 unrelated families of different geographical origins. Significant association was detected between the MJD1 locus and marker alleles at loci D14S280, D14S1050 and D14S81. All affected individuals, except one Chinese family, had allele 3 (237 bp) at D14S280. This finding is consistent with a founder effect in our MJD population. However, distinct haplotypes were observed in patients originating from the two Azorean islands showing the highest disease prevalence; therefore, the possible existence of more than one founder mutation can not be excluded with the markers currently available. Received: 27 February 1996 / Revised: 4 June 1996     

Dynamics of the sit-to-stand movement

The strategies of the sit-to-stand movement are investigated by describing the movement in terms of the topology of an associated phase diagram. Kinematic constraints are applied to describe movement sequences, thus reducing the dimension of the phase space. This dimensional reduction allows us to apply theorems of topological dynamics for two-dimensional systems to arrive at a classification of six possible movement strategies, distinguished by the topology of their corresponding phase portrait. Since movement is treated in terms of topological structure rather than specific trajectories, individual variations are automatically included, and the approach is by nature model independent. Pathological movement is investigated, and this method clarifies how subtle abnormalities in movement lead to difficulties in achieving a stable stance upon rising from a seated position. This article was processed by the author using the LAT_EX style file pljour2 from Springer-Verlag.