Changes in the ultrastructure of mitochondria of roots ofTriticale subjected to anaerobiosis

Summary Anaerobiosis induces the formation of tubular inclusions within the mitochondria ofTriticale roots. These inclusions show a close relationship to the inner membrane of the mitochondrial envelope. Mitochondria of anaerobically cultured roots also become larger and fewer in number. Cristae become less numerous and more dilated in appearance. Despite these alterations the mitochondria retain their metabolic integrity, if the period of anaerobiosis does not exceed 24 hours. Periods of anaerobiosis in excess to 24 hours are increasingly deleterious to the metabolism and structure of the root cells and will eventually lead to their death.     

Dihydroisocoumarins and phthalide from wood samples infested by fungi

Wood samples, infested by fungi during storage, were shown to contain, besides the known 5-methyl-mellein, additional (3R)-8-hydroxy-3-methyl-3,4-dihydroisocoumarins substituted by 7-methyl, 5-formyl, 5-carboxy, 5-hydroxy, 5-methoxy, 6-methoxy-5-methyl and 6,7-dimethoxy-5-methyl groups, as well as 6-formyl-7-hydroxy-5-methoxy-4-methylphthalide. Several 2-methylchromanones were synthesized in order to show that this class of compounds can be distinguished from 3-methyl-3,4-dihydroisocoumarins by MS.     

Mucronulatol,mucroquinone and mucronucarpan,isoflavonoids from Machaerium mucronulatum and M. villosum

Additionally to the cinnamylphenols described in a previous paper, wood samples of Machaerium mucronulatum and M. villosum contain isoflavones, besides (?)-duartin, (?)- and (±)-mucronulatol [(3S)- and rac-7,3′-dihydroxy-2′,4′-dimethoxyisoflavan], (?)-mucroquinone [(3S)-2-methoxy-5-(7-hydroxy-8-methoxychroman-3-yl)-1,4-benzoquinone] and (+)-mucronucarpan [(6aS,11aS)-2,10-dihydroxy-3,9-dimethoxypterocarpan]. The constitutions of mucronulatol, mucroquinone and mucronucarpan were deduced by spectra and degradations, and confirmed by syntheses.     

Immobilization of Lipase from Mucor Miehei Onto Poly (Ethylene) Based Graft Copolymers

Lipase from Mucor miehei was immobilized covalently onto hydrolyzed poly(ethylene)-g.co-hydroxyethyl methacrylate (PE-HEMA). This hydrolysis of the copolymer was achieved using 0.1 M NaOH over different periods of time, under controlled conditions. The graft copolymers and their hydrolyzed equivalents were characterized by scanning electron microscopy (SEM) and by differential scanning calorimetry analysis (DSC). Water sorption studies were undertaken to provide a measure of relative hydrophobicity of the samples.

The lipase immobilization reaction was studied in order to assess the effects of controlling various important parameters. These include the nature of the buffering medium, the time over which the immobilization was allowed to occur, the concentration of the activating and coupling agent used (CMC) and the concentration of enzyme employed during attempts at effective immobilization. The immobilized lipase was used in the hydrolysis of triolein (glycerol trioleate). From this study, the apparent K_M, the optimum pH for hydrolysis and the optimum temperature for hydrolysis were revealed.

The suitability of hydrolyzed poly(ethylene)-g.co-HEMA as a support in the immobilization of lipase was assessed by determination of the amount of lipase coupled to the support and by assessment of the retention of activity of the immobilized lipase after its exposure to the immobilization reagents, procedure and conditions.     

Pollination and Breeding System of Vellozia squamata (Liliales: Velloziaceae): A Species of the Brazilian Cerrados

The pollination biology and breeding system of Vellozia squamata (Velloziaceae), a species of cerrado vegetation in Central Brazil, were studied. V. squamata is unusual in being pollinated by a few, generalist bee species despite having very large flowers, and having a distinctive pulsed flowering phenology. The species is self-incompatible but with a late-acting, post-fertilization rejection mechanism.     

Occurrence of macro B chromosomes in Astyanax scabripinnis paranae (Pisces,Characiformes, Characidae)

B chromosomes occur in several Neotropical fish species. Cytogenetic analysis of 27 specimens (15 females and 12 males) of Astyanax scabripinnis paranae from the Araquá river (a small headwater tributary of the Tietê river) shows that this population has 2n=50 chromosomes (4M+30 SM+4ST+12A), two chromosome pairs with NORs and conspicuous C-band positive blocks in the terminal position of the long arm of four chromosome pairs. In this population, eight females presented 2n=51 chromosomes and the extra chromosome was a large metacentric similar in size and morphology to the first chromosome pair in the karotype. This accessory chromosome is entirely heterochromatic in C-banded metaphases and shows a late replication pattern evidenced by BrdU incorporation. There was no significant correlation between the presence of B chromosomes and increased NOR activity at the P>0.05 level. Some aspects related to these B chromosomes are discussed.     

Epilithic diatom community response to years of P04 fertilization: Kuparuk River,Alaska (68 N Lat.)

An arctic river was fertilized continuously through the ice-free season with phosphoric acid beginning in 1983. The epilithic diatom community increased in biomass in the first two years in response to the added limiting nutrient (Peterson et al., 1983). The diatom community switched from one dominated by Hannea arcus to one dominated by species of Achnanthes and Cymbella. The immediate responses to the P-addition were decreases in both the Shannon diversity and evenness indices. By the second year, the community diversity increased downriver reaching maximal species richness (110–127 spp). In 1985–1987, the epilithic algal biomass decreased an order of magnitude with both whole-river PO₄ (1985, 1987) and PO₄ + NH₄ addition (1986). In the 5th summer of fertilization, the reduction in biomass was clearly caused by a numerical increase of grazing, refugia-building chironomids (Orthocladiinae, primarily) (Gibeau, 1991; Gibeau, Miller, Hershey, in prep.). We assume the algal biomass reduction in the 3rd and 4th years was similarly caused by grazers with a two year time lag in the numerical response of these monovoltine species. The evenness of the community increased in 1986 as if it might have been grazed; however the number of immigrants was reduced. The community became dominated by Eunotia, Cymbella and Achnanthes, species either fast growing or more prostrate, as the erect species of Hannea Diatoma, and Fragillaria declined. A detrended correspondence analysis of the temporal and spatial diatom samples in species space (186 spp.) showed that the largest variation in the community was between years and less variation was associated with river fertilization. Samples from bioassay tubes run by Peterson et al. (1983) in the Kuparuk River showed P and N + P limitation as found in the river in 1983–84. Like the river samples, the largest change in the diatom community occurred between 15 and 25 day samples, more than that induced by fertilization. Diatoms sampled from all treatments taken at day 25 were more similar to one another than those sampled at day 15. Diatoms colonizing glass slides used in the bioassay tubes were dominated by Achnanthes linearis and Cymbella minuta. Of the 84 species found in bioassays, 26 species were present in all river samples for 4 years. Differences in the communities discriminated by multivariate methods were cause by changes in rare species and abundance patterns of common species.     

Application of empirical design methodologies to the study of the influence of reaction conditions and N-alpha-protecting group structure on the enzymatic X-Phe-Leu-NH(2) dipeptide synthesis in buffer/dimethylformamide solvents systems

The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH(2) was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-alpha protection choice. Boc and Z were the most reactive N-alpha groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.     

Coordinate pretranslational control of cAMP-dependent protein kinase subunit expression during development in the water mold Blastocladiella emersonii.

The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella.     

Regulation of aplanospore germination in Vaucheria

Germination of aplanospores in Vaucheria longicaulis Hoppaugh var. macounii Blum proceeds through three stages of development. Stage I begins with the initiation of germination and lasts approx. 2 h. During this stage germinating filaments grow at an accelerated rate (266 ± 12 m · h^–1). Stage II is characterized by a sharp decline in the growth rate of germinating filaments (96 ± 4 m · h^–1) and lasts 4 h. This is followed, during the next 4 h, by a recovery in the growth rate (168 ± 8 m · h^–1) of germinating filaments, stage III. Growth rates stabilize and remain unchanged during subsequent development (Oliveira and Fitch, 1988, J. Submicrosc. Cytol. Pathol. 20, 397–406). The Ca²⁺-influx modulators LaCl₃, nifedipine and methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4 (2-trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K-8644), the ionophore calcimycin (A23187), the intracellular Ca²⁺-release antagonist 8-N-N'-(diethylamino)-octyl-3,4, 5-trimethoxybenzoate (TMB-8), the Ca²⁺-uptake inhibitor ruthenium red and the phosphoinositide-cycle modulators LiCl and myo-inositol show that the events required for the initiation are distinct from those required for the completion of each stage of germination. These studies in conjunction with microinjection of germinating filaments with inositol 1,4,5-trisphosphate, the natural ligand for Ca²⁺ release from Ca-storing organelles (endoplasmic reticulum, vacuole), and treatment with chlorotetracycline (CTC), to visualize the distribution of membrane-bound Ca²⁺ reveal that both the initiation and completion of each stage of germination are controlled by Ca²⁺ signals which are restricted to well-defined time intervals and are modulated by the origin (source) of Ca²⁺.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - Bay K-8644 methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4(2-trifluoromethylphenyl)-pyridine-5-carboxylate - CTC chlorotetracycline - InsP₃ inositol 1,4,5-trisphosphate - RR ruthenium red - TMB-8 8-N-N-(diethylamino)-octyl-3,4,5-trimethoxybenzoate The author wishes to express his gratitude to the technical group of the Immunocytochemistry Unit for their help with the microinjection studies. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (grant A-7844).