Amylolytic enzymes from Aspergillus hennebergi (A. niger Group): Purification and characterization of amylases from solid and liquid cultures

Two glucoamylases (I and II) were produced during solid-state culture of Aspergillus hennebergi (A. niger group) on cassava meal, whereas one glucoamylase and one alpha-amylase were synthesized by the mould in liquid culture. These glucoamylases were acidic proteins with thermotolerant activities. Glucoamylase I was not a glycoprotein, but glucoamylase II and the glucoamylase from liquid cultures contained 15% of sugars. The alpha-amylase was significantly less thermotolerant and of smaller molecular weight. The influence of culture conditions on the production of different amylases by the same Aspergillus strain on the same substrate is discussed.     

Synergistic Regulation of Cytosolic Ca2+ Concentration in Mouse Astrocytes by NK1 Tachykinin and Adenosine Agonists

The effects on cytosolic Ca2+ concentration of 2-chloroadenosine and [L-Pro9]-substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L-Pro9]-substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2-chloroadenosine alone occurred. When simultaneously applied, [L-Pro9]-substance P and 2-chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihydropyridines, La3+, and Co2+, excluding the implication of voltage-operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L-Pro9]-substance P and 2-chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L-Pro9]-substance P in potentiating the 2-chloroadenosine-evoked response. Like 2-chloroadenosine, pinacidil, which hyperpolarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L-Pro9]-substance P. Conversely, depolarization with 50 mM KCl canceled the effects of either pinacidil or 2-chloroadenosine applied with [L-Pro9]-substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2-chloroadenosine.     

The use of photosynthesis inhibitor (DCMU) for in situ metabolic and primary production studies on soft bottom benthos

A selective chemical photosynthesis inhibitor, DCMU (Dichorophenyl-dimethylurea), dissolved in DMSO (Dimethyl sulfoxide) was substituted for the dark incubation method commonly used to measure the oxygen consumption in metabolic and primary production studies. We compared oxygen fluxes during light incubations with DCMU and dark incubations procedure, on soft bottom benthos. For this purpose, we studied the effects of different DCMU concentrations. A concentration of 5 · 10^–5 mol l^–1 inside a clear incubation enclosure completely inhibits photosynthesis without affecting the metabolism of soft bottom benthos.     

Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation. Structure/activity studies and mechanistic implications.

Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.0 mol/mol enzyme subunit, interacting with the catalytic iron at the active site. All the isozymes tested were excellent substrates for cAMP-dependent protein kinase (Km = 5 microM, Vmax = 9.5 mumol.min-1.mg kinase-1). The incorporation of about 1.0 mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10. Conversely, the addition of stoichiometric amounts of Fe(II) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by cAMP-dependent protein kinase by 2-3-fold. These data provide evidence for a mutual interaction between the presumed regulatory and catalytic domains of hTH, and show that activation of the enzyme by phosphorylation and inactivation by binding of catecholamines are related events, which probably represent important mechanisms for the regulation of the enzyme activity in vivo.     

Isolation and phenotypic analysis of conditional-lethal, linker-insertion mutations in the gene encoding the largest subunit of RNA polymerase II in Saccharomyces cerevisme

Summary Linker-insertion mutagenesis was used to isolate mutations in the Saccharomyces cerevisiae gene encoding the largest subunit of RNA polymerase II (RP021, also called RPBI). The mutant rpo21 alleles carried on a plamid were introduced into a haploid yeast strain that conditionally expresses RP021 from the inducible promoter pGAL10. Growth of this strain on medium containing glucose is sustained only if the plasmid-borne rpo21 allele encodes a functional protein. Of nineteen linker-insertion alleles tested, five (rpo21-4 to –8) were found that impose a temperature-sensitive (ts) lethal phenotype on yeast cells. Four of these five is alleles encode mutant proteins in which the site of insertion lies near one of the regions of the largest subunit that have been conserved during evolution. Two of the is mutants (rpo21-4 and rpo21-7) display pleiotropic phenotypes, including an auxotrophy for inositol and a decreased proliferation rate at the permissive temperature. The functional relationship between RP021 and RP026, the gene encoding the 17.9 kDa subunit shared by RNA polymerases 1, 11, and III was investigated by determining the ability of increased dosage of RP026 to suppress the is phenotype imposed by rpo21-4 to –8. Suppression of the is defect was specific for the rpo21-4 allele and was accompanied by co-suppression of the inositol auxotrophy. These results suggest that mutations in the largest subunit of RNA polymerase II can have profound effects on the expression of specific subsets of genes, such as those involved in the metabolism of inositol. In the rpo21-4 mutant, these pleiotropic phenotypes can be attributed to a defective interaction between the largest subunit and the RP026 subunit of RNA polymerase II.     

Biomass and composition of size fractionated phytoplankton in the Weddell-Scotia Confluence area

Summary The measurement of Chl a, Chl b and Chl c contents in four size fractions (Nuclepore filters of 10 m, 3m, 1 m and 0.2 m pore-size) together with microscopic examination illustrate the structure and the relative importance of the micro-, nano and pico-phytoplankton in the production system in the Weddell/Scotia Confluence area. In the Scotia Sea, large diatoms were prevalent and their biomass increased during the six week cruise period, exceeding 1 mg Chl a m^–3 at the beginning of January. In contrast, in the Marginal Ice Zone of the Weddell Sea, the biomass remained low, up to 0.3 mg Chl a m^–3. A diversified nanoplankton community accounted for more than 90% of this biomass: small diatoms, naked dinoflagellates, cryptophyceans, prymnesiophytes and green flagellates which increased the Chl b/Chl a ratio to values >0.20. An important trend affected the Confluence area, where a high biomass net-plankton community (4 mg Chl a m^–3) rapidly changed towards a uniform nanoplankton system of the same kind as in the Weddell Sea. At times, autotrophic cryptophyceans were almost dominating (>4.10⁶ cells/l), with a biomass up to 2 mg Chl a m^–3 and a low phaeopytin ratio (<10%). This situation probably arises because of a grazing pressure by krill. However, due to the geographic and oceanographic peculiarities of this area, it is not possible to extrapolate these observations concerning the size structure of the primary producers to the Southern Ocean in general.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Structure and nonlinear optical properties of copper phthalocyanine thin films

This paper describes a joint study of the structure and nonlinear optical properties of vacuum evaporated thin films of copper phthalocyanine (CuPc for brevity). Film thickness ranges from 50 to 500 nm. The anisotropic paramagnetic resonance of Cu⁺⁺ ions reveals that the Pc rings lie almost parallel to the substrate plane with however a large angular distribution (30° FWHM). Third harmonic optical generation measurements performed at 1.064 m and 1.907 m fundamental wavelengths give respectively an average value of the cubic susceptibility ⁽³⁾(-3,)=(4±0.4)·10^–12 e.s.u. and (2.1+-0.2) · 10^-12 These values, although significantly higher than for a common ionic crystal, are about one order of magnitude lower than in conjugated 1-D systems, which shows that the 2-D -electron delocalization is less profitable than the 1-D one. Besides third harmonic, we have also observed second harmonic generation. Its polarization dependence is characteristic of a quadratic susceptibility enhanced in one direction, almost perpendicular to the substrate, withd _eff comprised between 30 and 60 · 10^-9 e.s.u. The possible origins ofd _eff are discussed.