Feedback inhibition of homoserine dehydrogenase and threonine deaminase in the genusBifidobacterium

Feedback inhibition of crude and purified extracts of homoserine dehydrogenase and threonine deaminase activities in the genusBifidobacterium was studied. Homoserine dehydrogenase was partially or completely inhibited byl-threonine, and a marked inhibitory effect byl-isoleucine on threonine deaminase was observed. In the speciesBifidobacterium cuniculi high levels ofl-valine reversed the inhibitory effect ofl-isoleucine. The -aminobutyric acid-resistant mutant Ru 326/106 of the speciesB. ruminale, overproducer ofl-isoleucine, had a derepressed homoserine dehydrogenase and a lesser feedback inhibition byl-threonine. Homoserine dehydrogenase appeared to be in bifids specifically NAD dependent. The regulatory mechanisms of aspartate family amino acid biosynthesis in bifidobacteria was discussed.     

Supra-additive stimulation of adenylate cyclase activity by prostaglandin E2 andD-Ala2-met-enkephalinamide in the guinea-pig superior cervical ganglion: Role of Mg2+ ions

Agonists modulation of Mg²⁺-dependent adenylate cyclase activity has been studied in guinea-pig superior cervical ganglion crude membrane preparations. In the absence of receptors ligands, Mg²⁺ stimulates the enzyme in a concentration-dependent manner. The dose-activation curve shows heterogeneity and two components with higher and lower apparent affinity states, are extrapolated. In the presence ofD-Ala²-met-enkephalinamide only one component is present and the apparent affinity of the ganglionic adenylate cyclase system for the divalent cation as well as Vmax are inhibited. On the contrary, prostaglandin E₂ increases affinity and Vmax values of the lower and, to a lesser extent, of the higher Km component. When the two drugs are tested in combination, not only the inhibitory effect of the opiate is overcome, but a large increase of the apparent affinities and Vmax values for both components is obtained, suggesting the involvement of the Mg²⁺-regulated subunits of the adenylate cyclase system in the supra-additive stimulation mechanism of the enzyme.     

Arabidopsis: Sensitivity of growth to high temperature

Arabidopsis thaliana seedlings as measured by an electrolyte leakage assay, have been found to be extremely sensitive to high temperature stress as compared to a high temperature tolerant variety (Tracy) of soybean. Over 50% ion leakage occurred in Arabidopsis leaves during a 15-minute exposure to 50°C, indicating a heat killing time of less than 15 minutes. In contrast, the heat killing time for soybean at 50°C was over five times longer. When soybean or Arabidopsis seedlings in culture plates were exposed to 37°C for 2 hours and then returned to 23°C, they suffered no apparent short-term or long-term damage. Soybean seedlings given a 42°C, treatment for 2 hours also showed no damage. Arabidopsis seedlings after a 42°C treatment for 2 hours showed no apparent immediate damage, but 48 hours after return to 23°C severe damage symptoms were visible and after 96 hours all the seedlings were dead. Both soybean and Arabidopsis seedlings synthesize heat shock proteins (hsps) when exposed to 42°C for 2 hours. The hsps synthesized are of similar molecular weights, although the relative abundances of the different size classes are very different in the two plants. Even though hsps are produced in Arabidopsis seedlings after a 2 hour exposure to 42°C their presence is not sufficient for the seedlings to recover from the effects of rhe heat shock when returned to 23°C. Our results show that Arabidopsis has a heat sensitive genotype. This along with its other characteristics should make it a good model system in which to assay in transgenic plants, the functions of homologous and heterologous genes that might be candidates for determining heat tolerance in plants.     

Minor pollinosis fromCryptomeria japonica in Varese,northern Italy. Aerobiological monitoring and main clinical features

In the last 2 years we have conducted an aerobiological monitoring program ofCryptomeria japonica, a plant belonging to the family of Taxodiaceae that sometimes causes pollinosis in the period from February to April. Throughout 1994, we checked the incidence of its sensitization and the clinical effects in 85 subjects with correlated seasonal symptoms, who gave a positive skin prick test (SPT) for Betulaceae and/or Corylaceae. Twenty-five patients (29.4%; 19 M; 6 F; mean age, 38.8 years) all with oculorhinitis, were SPT positive to an allergenic extract ofCryptomeria. RAST confirmed this positivity in 44% of the cases. No patients showed monosensitization forCryptomeria to either SPT or RAST. Two subjects gave a positive result on specific nasal provocation. RAST inhibition showed no cross-reaction betweenCryptomeria and birch pollen. During the pollen season each patient made a list, scoring symptoms and specifying any drugs used, so we could correlate these elements with aerobiological observations. The pollen concentration probably exceeded the allergizing threshold forCryptomeria on 8 days during 3 months of recording. The intensity and duration of symptoms seemed to be mainly influenced by sensitization to Betulaceae and Corylaceae. It is thus possible that a combination of minor pollinosis may produce seasonal symptoms in allergic patients.     

Early degradation of photosynthetic membranes in carob and sunflower cotyledons

The assimilatory activity of cotyledons can play an essential role in the survival of seedlings with a slow and delayed development of primary leaves. Changes in the photosynthetic activity of the cotyledon, from the onset of greening through senescence, were studied in two such plants, carob and sunflower, in order to determine its efficiency and duration, also in connection with the achievement of assimilatory autonomy by the plantlet. Chlorophyll analyses showed that the cotyledon's chloroplasts reached maximal greening in plantlets with a pair of expanded leaves. In contrast, the cotyledon's photosynthetic activity, measured as the rate of oxygen release, started to decrease early, before expansion of primary leaves. The decrease was due to the inactivation of a number of photosystem II (PSII) units, as revealed by immunodetection of breackdown products of the reaction centre's D1 and D2 thylakoid proteins. No signals of PSII alteration were noticed in the primary leaf chloroplasts that differentiated under the same environmental conditions. The damage to the cotyledon PSII, occurring in a non-photoinhibitory situation, might be due to a slower rate of turnover of D1 polypeptide than in the leaf thylakoids. The differential turnover of this protein in cotyledons and in leaves might represent an organ-specific regulation of the photosynthetic activity. The peculiarity of the cotyledon thylakoids make these organs useful objects for studying the metabolic cycle of both D1 and D2 proteins in vivo, under non-photoinhibiting conditions.     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.     

The isochore patterns of mammalian genomes and their phylogenetic implications

The compositional distributions of high molecular weight DNA fragments from 20 species belonging to 9 out of the 17 eutherian orders were investigated by analytical CsCl density gradient centrifugation and by preparative fractionation in Cs₂SO₄/BAMD density gradients followed by analysis of the fractions in CsCl. These compositional distributions reflect those of the isochores making up the corresponding genomes. A “general distribution” was found in species belonging to eight mammalian orders. A “myomorph distribution” was found in Myomorpha, but not in the other rodent infraorders Sciuromorpha and Histricomorpha, which share the general distribution. Two other distributions were found in a megachiropteran (but not in microchiropteran, which, again, shares the general distribution) and in pangolin (a species from the only genus of the order Pholidota), respectively. The main difference between the general distribution and all other distributions is that the former contains sizable amounts (6–10%) of GC-rich isochores (detected as DNA fragments equal to, or higher than, 1.710 g/cm³ in modal buoyant density), which are scarce, or absent, in the other distributions. This difference is remarkable because gene concentrations in mammalian genomes are paralleled by GC levels, the highest gene concentrations being present in the GC-richest isochores. The compositional distributions of mammalian genomes reported here shed light on mammalian phylogeny. Indeed, all orders investigated, with the exception of Pholidota, seem to share a common ancestor. The compositional patterns of the megachiropteran and of Myomorpha may be derived from the general pattern or have independent origins.     

Presence of telomeric and subtelomeric sequences at the fusion points of ring chromosomes indicates that the ring syndrome is caused by ring instability

In situ hybridization of a telomeric (TTA-GGG) _n sequence to metaphases from three cases of ring chromosome, involving respectively chromosomes 4, 16, and 20, showed the presence of the cognate sequences in all three rings. To investigate whether these ring chromosomes originated by telomere-telomere fusion, we determined, by in situ hybridization, whether telomere-associated sequences and/or specific distal sequences were still present in the ring chromosomes. The finding that these sequences were preserved in all the ring chromosomes strongly indicates that they originated by telomere-telomere fusion. All three subjects carrying the ring chromosomes are affected by the so-called ring syndrome, with failure to thrive, minor dysmorphic signs and no major anomalies. The r(4) patient has the ring in mosaic form with a normal cell line and has normal intelligence. The r(16) and the r(20) patients have moderate mental retardation and suffer from seizures. We conclude that the ring syndrome, even in its more severe manifestation, is caused by ring chromosome instability.