Weak field influence on biomolecular changes

Model equations for the kinetics of the synthesis and decay of molecular aggregates are used to show the high sensitivity of equilibrium concentrations of high-molecular aggregates to external radiation. This phenomenon is used to explain the effects of low-intensity microwave fields on the functioning of biological systems. The experimental results on the influence of SHF-radiation on ferricyanide reduction by erythrocytes are interpreted in detail.     

Effect of applied and internal hormones on vitrification and apical necrosis of different plants cultured in vitro

Development of vitrification and apical necrosis was followed in Camellia sinensis, Gerbera jamesonii, Malus domestica and hybrid Populus tremula x P. alba shoots cultured in vitro on Murashige & Skoog (MS) medium with different concentrations of growth regulators. High humidity in the culture vessels and excess of BA in the medium were found to be the major factors influencing vitrification. Lack of exogenous cytokinin in the medium during successive subcultures induced apical necrosis in poor-rooting species (Malus domestica, Camellia sinensis). The level of internal phytohormones (ABA, IAA, IPA, 2iP, Z, ZR) was determined in the apple shoots by means of ELISA. The content of internal cytokinins in the vitrified apple shoots was several times greater than in normal ones, which supports the hypothesis that excess of cytokinins, inducing rapid divisions of cells in meristems in the atmosphere with high humidity, is responsible for vitrification. Apical necrosis of the plantlets that appeared after cultivation on cytokinin-free medium is the result of deficiency in endogenous hormones in apple shoots and this being confirmed by analysis of endogenous hormones in apple shoots.Abbreviations BA benzyladenine - BHT butylated hydroxy-toluene - ABA abscisic acid - IAA indole-3-acetic acid - ELISA enzyme-linked immunosorbent assay - IPA isopentenyladenosine - 2iP isopentenyladenine - NAA naphthyl-3-acetic acid - TBS trishydroxymethylaminomethane buffered saline - TLC thin layer chromatography - Z zeatin - ZR zeatin riboside     

Liquefaction of dulse (Palmaria palmata (L.) Kuntze) by a commercial enzyme preparation and a purified endo ,β-1,4-D-xylanase

Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo--1,4-D-xylanase was studied.The endo--1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl--D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a K_m of 2.2 and 2.8 mg ml^-1 and V_max of 3600 and 3900 nkat mg^-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater.     

Nuclear genome characterization of the carrageenophyteAgardhiella subulata (Rhodophyta)

DNA reassociation kinetics were used to determine nuclear genome organization and complexity inAgardhiella subulata (Gigartinales, Rhodophyta). Results indicate the presence of three second-order components corresponding to fast (22%), intermediate (68%) and slow (10%) fractions. Thus, the genome consists of 90% repetitive sequences. Microspectrophotoometry with the DNA-localizing fluorochrome DAPI was used to confirm ploidy level differences in the gametophytic and tetrasporophytic phases. Results indicate that meiosis occurs during tetrasporogenesis. Comparison of mean nuclear DNA (I_f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.9 pg/2C genome forAgardhiella. Karyological studies using aceto-orcein revealed a chromosome complement of 2N = 44 in carposporangia and the presence of 22 bivalents during diakinesis of tetraspore mother cells.     

Restriction enzyme digestion of heterochromatin inDrosophila nasuta

In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types ofDrosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while withTaqI, no such undigested band was seen. TheAluI resistant 23 kb DNA hybridized insitu specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome ofDrosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.     

The appearance of an enzyme activity catalysing the conversion of norsolorinic acid to averantin in Aspergillus parasiticus cultures

The activity of the enzyme responsible for the conversion of norsolorinic acid to averantin was studied in two strains of Aspergillus parasiticus. Cell-free extracts of the enzyme were purified from different aged mycelia and little activity was found prior to 24 hours after inoculation but this quickly reached a maximum at 48 hours and declined thereafter. Both strains of A. parasiticus, one in aflatoxin producing strain, the other a versicolorin A accumulating mutant, showed this trend. It was concluded that the enzyme responsible for this conversion was a secondary metabolic enzyme and was distinct from alcohol and mannitol dehydrogenases.     

Genetical and biochemical comparisons of alcohol dehydrogenase isozymes from Anastrepha fraterculus and A. obliqua (Diptera: Tephritidae): Evidence for gene duplication

The electrophoretic patterns of the enzyme alcohol dehydrogenase (ADH) from Anastrepha fraterculus and A. obliqua were studied. Two loci were found to code for the enzyme in A. fraterculus, and three in A. obliqua. In both species, all isozymes were active in third-instar larvae. A cationic isozyme (Adh-1) was active mainly in the visceral fat body of both species. In A. fraterculus, the locus had an anionic polymorphic isozyme (Adh-3) that was detected in the parietal fat body. In addition to these two loci, a third locus for an anionic isozyme (Adh-2), which was active in the digestive tube of larvae, was present in A. obliqua and probably resulted from gene duplication. For both species, multiple forms of the isozymes are formed by binding of an NAD-carbonyl compound, as in Drosophila melanogaster. Both larvae and early pupae of A. obliqua had almost twice the specific ADH activity as A. fraterculus. The ethanol content of the host fruit infested with A. obliqua (red mombim) was also higher than that of the host fruit infested with A. fraterculus (guava).This research was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq-PIG 40.2486/82).     

Restriction endonuclease mapping of ribosomal RNA genes: Sequence divergence and the origin of the tetraploid treefrog Hyla versicolor

Hyla chrysoscelis (2n=24) and H. versicolor (2n=48) are a diploid-tetraploid species pair of treefrogs. Restriction endonuclease mapping of ribosomal RNA (rRNA) gene repeat units of diploids collected from eastern and western populations reveals no differences within rRNA gene coding regions but distinctive differences within the nontranscribed spacers. A minimum of two physical maps is required to construct an rRNA gene map for the tetraploid, whose repeat units appear to be a composite, with about 50% of the elements resembling the western diploid population and about 50% resembling the eastern population. These results imply that this population of the tetraploid species may have arisen from a genetically hybrid diploid. Alternatively, the dual level of sequence heterogeneity in H. versicolor may reflect some type of gene flow between the two species. The coding region of the rRNA genes in the tetraploid differs from that in either diploid in about 20% of all repeat units, as exemplified by a BamHI site located near the 5 terminus of the 28 S rRNA gene. If the 20% variant class of 28 S rRNA gene coding sequences is expressed, then there must be two structural classes of ribosomes; if only the 80% sequence class is expressed, then a genetic control mechanism must be capable of distinguishing between the two different sequence variants. It is postulated that the 20% variant sequence class may be correlated with a partial functional diploidization of rRNA genes in the tetraploid species.This research was supported, in part, by NSF Grants CDP-8002341 and PRM-8106947 and by faculty research grants from Miami University to J.C.V.     

Characterization of a Ca-dependent maxi K channel in the apical membrane of a cultured renal epithelium (JTC-12.P3)

Summary A Ca and potential-dependent K channel of large unit conductance was detected in the apical membrane of JTC-12.P3 cells, a continuous epithelial cell line of renal origin. The open probability of the channel is dependent on membrane potential and cytoplasmic-free Ca concentration. At cell-free configuration of the membrane patch, the open probability shows a bell-shaped behavior as function of membrane potential, which decreases at larger depolarization. With increasing Ca concentration, the width of the bell-shaped curve increases and the maximum shifts into the hyperpolarizing direction. For the first time the kinetics of this channel was analyzed under cell-attached conditions. In this case the kinetics could sufficiently be described by a simple open-closed behavior. The channel has an extremely small open probability at resting potential, which increases exponentially with depolarization. The low probability induces an uncertainty about the actual number of channels in the membrane patch. The number of channels is estimated by kinetic analysis. It is discussed that this K channel is essential for the repolarization of the membrane potential during electrogenic sodium-solute cotransport across the apical membrane.     

A minimum mechanism for Na+−Ca++ exchange: Net and unidirectional Ca++ fluxes as functions of ion composition and membrane potential

Summary Both simultaneous and consecutive mechanisms for Na⁺–Ca⁺⁺ exchange are formulated and the associated systems of steady-state equations are solved numerically, and the net and unidirectional Ca⁺⁺ fluxes computed for a variety of ionic and electrical boundary conditions. A simultaneous mechanism is shown to be consistent with a broad range of experimental data from the squid giant axon, cardiac muscle and isolated sarcolemmal vesicles. In this mechanism, random binding of three Na⁺ ions and one Ca⁺⁺ on apposing sides of a membrane are required before a conformational change can occur, translocating the binding sites to the opposite sides of the membranes. A similar (return) translocation step is also permitted if all the sites are empty. None of the other states of binding can undergo such translocating conformational changes. The resulting reaction scheme has 22 reaction steps involving 16 ion-binding intermediates. The voltage dependence of the equilibrium constant for the overall reaction, required by the 31 Na⁺Ca⁺⁺ stoichiometry was obtained by multiplying and dividing, respectively, the forward and reverse rate constants of one of the translocational steps by exp(–FV/2RT). With reasonable values for the membrane density of the enzyme (120 sites m²) and an upper limit for the rate constants of both translocational steps of 10⁵·sec^–1, satisfactory behavior was obtainable with identical binding constants for Ca⁺⁺ on the two sides of the membrane (10⁶ m ^–1), similar symmetry also being assumed for the Na⁺ binding constant (12 to 60m ^–1). Introduction of order into the ion-binding process eliminates behavior that is consistent with experimental findings.