Regeneration in the auditory system of nymphal and adult bush crickets Tettigonia viridissima

Regeneration and reestablishment of synaptic connections is an important topic in neurobiological research. In the present study, the regeneration of auditory afferents and the accompanying effects in the central nervous system are investigated in nymphs and adults of the bush cricket Tettigonia viridissima L. (Orthoptera: Tettigoniidae). In all animals in which the tympanal nerve is crushed, neuronal tracing shows a regrowth of the afferents into the prothoracic ganglion. This regeneration is seen in both adult and nymphal stages and starts 10–15 days after nerve crushing. Physiological recordings from the leg nerve indicate a recovery of tympanal fibres and a formation of functional connections to interneurones in the same time range. Electrophysiological recordings from the neck connective suggest additional contralateral sprouting of interneurones and the formation of aberrant connections. The regeneration processes of the tympanal nerve in nymphal stages and adults appear to be similar.     

Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression.

The regulatory hexokinase PII mutants isolated previously (K.-D. Entian and K.-U. Fröhlich, J. Bacteriol. 158:29-35, 1984) were characterized further. These mutants were defective in glucose repression. The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme. Hence, a regulatory domain for glucose repression was postulated. For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics. None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged. The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D. Entian and E. Kopetzki, Eur. J. Biochem. 146:657-662, 1985), was absent from one of these mutants. It is possible that the PIIM modification is directly connected with the triggering of glucose repression. To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function. Unlike hexokinase PII wild-type transformants, glucose repression was not restored in the hexokinase PII mutant transformants. In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PII structural gene.     

Complete nucleotide sequence of the hexokinase PI gene (HXK1) of Saccharomyces cerevisiae

The nucleotide sequence of the yeast glycolytic hexokinase isoenzyme PI-gene, HXK1, has been determined by sequencing the yeast DNA insert of the previously isolated plasmid HXK1 clone [Entian et al., Mol. Gen. Genet. 198 (1984) 50-54]. The structural gene sequence included 1452 bp coding for 484 amino acid (aa) residues corresponding to the Mr of 153 605 for the HXK1 monomer. Several initiation regions and termination points were located using nuclease S1 mapping. The HXK1 sequence was 76% homologous with that of HXK2, which is responsible for triggering glucose repression in yeasts. Since HXK1 is not involved in this regulatory system, the regulatory function of HXK2 must correspond to one or more of the differences between both isoenzymes. Most changes in the amino acid sequence were statistically distributed; however, four clustered regions with more than five altered aa residues were identified.     

Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Intoxication of rats with CCl4 (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS+) hepatocytes were reestablished. However, while the GS+ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS+ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS+ and GS- hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS+ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS- hepatocytes. Immediately after liver damage by CCl4 and during regeneration small but significant changes in the nuclear pattern were noted for GS- hepatocytes. However, the first GS+ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS- hepatocytes. These results indicate that new GS+ hepatocytes may be derived from formerly GS- cells which are induced to express GS if they have reached the border of the central veins.     

Studies on rapid reversible and non-reversible inactivation of fructose-1,6-bisphosphatase and malate dehydrogenase in wild-type and glycolytic block mutants of Saccharomyces cerevisiae

Experimental conditions have been elaborated to test for reversibility of the malate dehydrogenase inactivation (E.C.1.1.1.37) after addition of glucose to derepressed yeast cells. Malate dehydrogenase inactivation was shown to be irreversible at all stages of inactivation. In contrast fructose-1,6-bisphosphatase inactivation (E.C.3.1.11) remained reversible for at least 30 min after addition of glucose. Rapid reversible inactivation of fructose-1,6-bisphosphatase and irreversible inactivation of malate dehydrogenase were additionally investigated in glycolytic block mutants. Normal inactivation kinetics were observed in mutants without catalytic activity of phosphoglucose isomerase (E.C.5.3.1.9), phosphofructokinase (E.C.2.7.1.11), triosephosphate isomerase (E.C.5.3.1.1) and phosphoglycerate kinase (E.C.2.7.2.3). Hence, neither type of inactivation depended on the accumulation of any glucose metabolite beyond glucose-6-phosphate. Under anaerobic conditions irreversible inactivation was completely abolished in glycolytic block mutants. In contrast rapid reversible inactivation was independent of energy provided by respiration or fermentation. Reversibility of fructose-1,6-bisphosphatase inactivation was tested under conditions which prevented irreversible malate dehydrogenase inactivation. In these experiments, fructose-1,6-bisphosphatase inactivation remained reversible for at least 120 min, whereas reversibility was normally restricted to about 30 min. This indicated a common mechanism between the irreversible part of fructose-1,6-bisphosphatase inactivation and irreversible malate dehydrogenase inactivation.     

Photophobic responses of the cyanobacterium Anabaena variabilis

The photophobic responses in the Cyanobacterium Anabaena variabilis which belongs to the Nostocaceae have been studied with aid of a population method as well as by single trichome observations. In white light experiments both step-up and step-down photophobic responses were observed. The wavelength dependence was examined at a constant fluence rate. The photophobically active light is absorbed by the photosynthetic pigments, mainly by the phycobiliproteins and chlorohyll a. Above 690 nm only negative reactions were observed, i.e. the trichomes left the light trap. In white light experiments DCMU strongly inhibited the photophobic responses, whereas photokinesis was not affected to the same extent indicating that the reaction is coupled with the non cyclic photosynthetic electron transport. DBMIB impaired the photophobic behaviour only slightly. It seems that the photophobic responses of A. variabilis are controlled by a similar mechanism as in Phormidium uncinatum (Oscillatoriaceae) although the two families and, hence, the two species differ in their movement mechanism as well as in their photoactic behaviour.     

Pollination strategies in Cretan Arum lilies

Flowers of the genus Arum are known to attract dung‐breeding flies and beetles through olfactory deceit. In addition to this strategy, the genus has evolved several other pollination mechanisms. The present study aimed to characterize the pollination strategies of the Cretan Arum species by investigating the flowering phenology, thermogeny, inflorescence odours, and the pollinating fauna. The results obtained show that Arum cyrenaicum and Arum concinnatum emit a strong dung smell and exhibit the distinctive features associated with this pollination syndrome. Both species are highly thermogenic, have a similar odour profile and attract small‐bodied Diptera. Although sharing the same habitat, these two plant species are never found growing sympatrically as a result of the early blooming period of A. cyrenaicum. By contrast, Arum creticum and Arum idaeum have evolved a more traditional and mutually beneficial pollination mechanism. The stinking smell has been replaced by a more flower‐like odour that attracts bees (Lasioglossum sp.) and, occasionally, bugs (Dionconotus cruentatus). Although attracting the same pollinator, the main compound present in the odour of A. creticum is different from that of A. idaeum. Principal component analysis (PCA), based on physiologically active components of the flower odours determined by testing on the antenna of the Lasioglossum bee, revealed two different clusters, indicating that pollinators can potentially discriminate between the odours of the two species. A further PCA on the main floral odour volatiles as identified by gas chroatography‐mass spectroscopy from all the Arum species under investigation displayed odour‐based similarities and differences among the species. The PCA‐gas chomotography‐electroantennographic detection active peaks analysis showed that the two species, A. creticum and A. idaeum, form two groups and are clearly separated from A. cyrenaicum and A. concinnatum, which, conversely, cluster together. The evolutionary forces and selective pressures leading to diversification of pollination mechanisms in the Cretan Arum spp. are discussed. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 991–1001.     

Dynamics of glial fibrillary acidic protein distribution in cultured glomerular podocytes and mesangial cells of the rat kidney

Glial fibrillary acidic protein (GFAP) has recently been shown to be expressed in the glomerular podocytes and mesangial cells (MC) of kidney (Buniatian et al (1998) Biol Cell 90, 53-61). The different localization of GFAP in podocytes and MC has raised the question whether this might reflect specific cellular functions. To address this question, in the present study podocytes and MC in early (2, 3 day-old), prolonged (5, 7 day-old) and late (14, 21 day-old) primary cultures from out-growths of glomerular explants were used. Double-immunolabeling studies demonstrated that podocytes transiently acquire myofibroblastic features, characterized by the expression of smooth muscle alpha-actin (SMAA) and increased perinuclear reaction of GFAP in prolonged cultures. The morphological differentiation of cobblestone-like podocytes into process-bearing cells was followed by loss of the myofibroblastic marker, SMAA, de novo expression of desmin, and distribution of GFAP, vimentin and desmin into the processes. In late culture, GFAP and SMAA were nearly absent from the podocytes which maintained the cobblestone-like morphology. By contrast, the myofibroblastic features gained by MC during prolonged culturing increased with time. A myofibroblast-like cytoskeleton of podocytes and MC similar to that of healthy astrocytes suggest an increased spectrum of functional activities of these cells during the acquisition of myofibroblastic features. In addition, the present study provides a new combination of biochemical and biological features by which podocytes and MC can be distinguished in culture.