Functional molecular aspects of the NADH dehydrogenases of plant mitochondria

There are multiple routes of NAD(P)H oxidation associated with the inner membrane of plant mitochondria. These are the phosphorylating NADH dehydrogenase, otherwise known as Complex I, and at least four other nonphosphorylating NAD(P)H dehydrogenases. Complex I has been isolated from beetroot, broad bean, and potato mitochondria. It has at least 32 polypeptides associated with it, contains FMN as its prosthetic group, and the purified enzyme is sensitive to inhibition by rotenone. In terms of subunit complexity it appears similar to the mammalian and fungal enzymes. Some polypeptides display antigenic similarity to subunits fromNeurospora crassa but little cross-reactivity to antisera raised against some beef heart complex I subunits. Plant complex I contains eight mitochondrial encoded subunits with the remainder being nuclear-encoded. Two of these mitochondrial-encoded subunits, nad7 and nad9, show homology to corresponding nuclear-encoded subunits inNeurospora crassa (49 and 30 kDa, respectively) and beef heart CI (49 and 31 kDa, respectively), suggesting a marked difference between the assembly of CI from plants and the fungal and mammalian enzymes. As well as complex I, plant mitochondria contain several type-II NAD(P)H dehydrogenases which mediate rotenone-insensitive oxidation of cytosolic and matrix NADH. We have isolated three of these dehydrogenases from beetroot mitochondria which are similar to enzymes isolated from potato mitochondria. Two of these enzymes are single polypeptides (32 and 55 kDa) and appear similar to those found in maize mitochondria, which have been localized to the outside of the inner membrane. The third enzyme appears to be a dimer comprised of two identical 43-kDa subunits. It is this enzyme that we believe contributes to rotenone-insensitive oxidation of matrix NADH. In addition to this type-II dehydrogenases, several observations suggest the presence of a smaller form of CI present in plant mitochondria which is insensitive to rotenone inhibition. We propose that this represents the peripheral arm of CI in plant mitochondria and may participate in nonphosphorylating matrix NADH oxidation.     

Influence of external factors on secondary embryogenesis and germination in somatic embryos from leaves of Quercus suber

Somatic embryogenesis was obtained in cultures of leaves from young seedlings of Quercus suber L. A two-stage process, in which benzyladenine and naphthaleneacetic acid were added first at high and then at low concentrations, was required to initiate the process. Somatic embryos arose when the explants were subsequently placed on medium lacking plant growth regulators. The embryogenic lines remained productive, by means of secondary embryogenesis, on medium without growth regulators. However, this repetitive induction was influenced by the macronutrient composition of the culture medium. Both low total nitrogen content and high reduced nitrogen concentration decreased the percentage of somatic embryos that showed secondary embryogenesis. Our results suggest that alternate culture on medium that increases embryo proliferation and a low salt medium prohibiting embryo formation will partially synchronize embryo development. Chilling slightly reduced secondary embryogenesis but gave a modest increase in germination. Maturation under light followed by storage at 4 °C for at least 30 days gave the best results in switching embryos from an embryogenic pathway to a germinative one. Under these conditions 15% of embryos showed coordinated root and shoot growth and 35% formed either shoots or mostly roots. These percentages were higher than those of embryos matured in darkness. This result indicates that a specific treatment is required after maturation and before chilling to activate the switch from secondary embryo formation to germination.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA indolebutyric acid - MS Murashige & Skoog (1962) medium - SH Schenk & Hildebrandt (1972) medium - G Gamborg (1966, PRL-4-C) medium (macronutrients in mg l^–1: NaH₂PO₄·H₂O, 90; Na₂HPO₄, 30; KCl, 300; (NH₄)₂SO₄, 200; MgSO₄·7H₂O, 250; KNO₃, 1000, CaCl₂·2H₂O, 150) - PGR plant growth regulator     

Effects of sorbitol addition on the action of free and immobilized hydrolytic enzymes in organic media

The effect of the addition of sorbitol on the activity and stability of enzymes was examined by monitoring transesterification reactions performed in organic media at various water activities (a(w) = 0.08 to 0.97). Lipases from Chromobacterium viscosum and Candida rugosa immobilized on celite, and chymotrypsin, free or immobilized on celite, were used. When the sorbitol-containing enzymes were employed, higher reaction rates and less hydrolysis were observed. Immobilization of chymotrypsin resulted in high activity and operational stability, while the nonimmobilized enzyme was stable only in the presence of sorbitol. The activity of all preparations diminished after washing them with pyridine to remove sorbitol. Furthermore, severe stability problems occurred in the preparations lacking sorbitol. Sorbitol treatment, even after removal of the sorbitol itself, improved the activity of nonimmobilized chymotrypsin relative to the washed control. On the other hand, washing to remove sorbitol had a negative effect on the activity of both coimmobilized lipase and coimmobilized chymotrypsin. Addition of a substrate analogue, N-acetyl-L-phenylalanine, to chymotrypsin yielded a preparation that exhibited higher activity than both the control and its sorbitol-containing counterpart. Differential scanning calorimetry measurements revealed that the chymotrypsin-sorbitol complex was stable against thermal denaturation, undergoing transition at a high temperature (89 degrees C). The transition temperatures of the substrate-containing chymotrypsin and of the control were identical (72 degrees C). (c) 1995 John Wiley & Sons, Inc.     

光合细菌H3菌株色素分析

H3菌株系由盐田微生物层中分离获得的光合细菌株。具有丰富的天然色素。经活细胞色素光谱吸收峰值测定,色素经有机溶剂提取、硅胶薄板层析、SDS－PAGE电泳等,结果表明H3菌株的主要色素包括细菌叶绿素a、细菌脱镁叶绿素（Bacteriophaeophytin）和三种类胡萝卜素。总胡萝卜素含量占细胞于重的0．6％,胡萝卜素蛋白复合体的分子量约11,000.培养条件的差异对色素形成及相对含量有不同程度的影响。     

The effects of domestic compost upon the germination and emergence of barley and six arable weeds

In a bioassay, leachate from composted household waste was found to decrease both cress germination and the germination of barley and six arable weeds, especially in the light. The effect of compost depth upon the emergence of these species was examined in a glasshouse experiment. Compost had no significant effect on barley emergence. However, weed emergence, particularly of those with small seeds, was reduced greatly by the physical effect of compost and, to a lesser extent, by its chemical effect. The emergence of the large-seeded species was relatively unaffected by increasing depths of compost, whereas that of the smaller-seeded weeds steadily decreased, being almost completely prevented by a 3 cm layer. The use of compost to control arable weeds in the field is discussed.     

Comparative Physiology and Behaviour of the Mycoparasites Pythium acanthophoron, P. oligandrum and P. mycoparasiticum

Pythium acanthophoron (two isolates) was as aggressive as P. oligandrum in hyphal interactions with Fusarium oxysporum on water agar films. It caused rapid post-contact lysis or cytoplasmic coagulation of the host, and often branched and penetrated the host at contact points. P. acanthophoron grew across a range of fungi on pre-colonized agar plates; the range was narrower than for P. oligandrum but broader than for P. mycoparasiticum. In chemically defined liquid media, P. acanthophoron was unique among the six known mycoparasitic Pythium spp. in requiring organic nitrogen but not thiamine. It also grew on mannitol as the sole carbon source in the presence of calcium or sterols or both. However, individual isolates of the three mycoparasitic species showed different responses to calcium and the sterols ergosterol, cholesterol and beta-sitosterol when grown on mannitol. All three mycoparasites required components of molasses, carrot extract or sunflower seed extract to produce oogonia in culture; they formed few oogonia on potato extract, with or without dextrose, even when supplied with sterols. The three mycoparasites were less tolerant of high NaCl levels in culture than were three phytopathogens (P. aphanidermatum, P. ultimum, P. graminicola), in contrast to a previous report for P. oligandrum and P. ultimum. These in vitro studies suggest that P. acanthophoron has potential for use as a biocontrol agent instead of, or in addition to, P. oligandrum.     

Growth,sporulation and toxin production byBacillus thuringiensis subsp.israelensis andB. sphaericus in media based on mustard-seed meal

Bacillus thuringiensis subsp.israelensis andB. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towardsCulex pipens quinquefasciatus Say, compared with those of the respective international reference standards, were 46% forB. thuringiensis subsp.israelensis, 62% forB. sphaericus 2362 and 88% forB. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM,B. thuringiensis subsp.israelensis grew better but had undetectable larvicidal activity, whereas theB. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies ofB. sphaericus in medium with 4% MSM were comparable with those of international reference standards.The authors are with the Department of Life Sciences, University of Bombay, Bombay 400 098, India.     

Relationship of alpha MSH-specific neurons to the arcuate opiocortin neuronal system as determined by dual antigen immunocytochemical procedures

The cross-immunoreactivity, topography, and fiber projections of the alpha MSH-immunoreactive specific neurons in the forebrain of the rat appear to be distinctly different from that of the neurons in the hypothalamic arcuate opiocortin system. The cell bodies, immunoreactive only to -MSH, have a specific pattern of distribution in the dorsal and lateral hypothalamic regions from the level of the retrochiasmatic region to the premammillary area of the posterior hypothalamus. Immunoreactive fibers of these cells appear to extend into regions of the cerebral cortex and hippocampus. An antomical relationship between the immunostained fibers and/or terminals of the arcuate opiocortin pool of neurons and the -MSH-immunoreactive perikarya is described utilizing the ABC (Avidin-Biotin-Peroxidase Complex) and ABC-GO (Glucose Oxidase) or glucose oxidase-antiglucose oxidase complex methods of immunocytochemistry in which two tissue antigens with contrasting colors are demonstrated in the same tissue section.     

Roche Susceptibility Test (RST) medium,a defined formulation for susceptibility testing: I. Nutrient interaction and optimization studies

Roche Susceptibility Test (RST) Medium represents the most completely optimized and convenient fully defined medium described. It requires no post-autoclaving supplementation with vitamins, supports good growth of all common aerobic and anaerobic pathogens and may be used as a broth or agar gel on which the swarming of Proteus spp. is virtually eliminated. The broth as a superior buffering capacity to most complex media and an osmolality and pH close to those of human serum. RST is highly satisfactory for the susceptibility testing of commonly used antibiotics and meets the requirements of the National Committe for Clinical Laboratory Standards of the U.S.A. in almost every respect.     

Cultured hepatoma cells for the study of enzyme regulation: Induction of ornithine decarboxylase by insulin and asparagine

Summary The induction and decay of ornithine decarboxylase (ODC) by insulin and asparagine in cultures of H4-II-EC3 (H35) hepatoma cells was studied in a modified Waymouth medium in the presence of fetal bovine serum (FBS) and in serum-free media. The insulin response was enhanced by the presence of asparagine although the effect of asparagine was not so much on the initial increase as it was on a slowing of the decline after the maximum was reached at 6 to 8 h after the supplements were added together with fresh medium. In all cases the initial ODC activity was zero at zero time for addition of media and supplements, and, after reaching the maximum, activity declined to near zero by 24 h. Fetal bovine serum gave induction that followed a similar time course but was inferior to the combintion, of insulin plus asparagine and, in fact, FBS inhibited the latter response. Putrescine (the product formed from ornithine by ODC), at 10⁻⁵ M, markedly inhibited the induction of ODC by insulin or FBS, but the inhibition was less when asparagine was present. This work was supported in part by Grants CA-07175, CA-22484, and CA-17334 from the National Cancer Institute. D. P. G. is a Predoctoral Fellow at the Food Research Institute, supported by a fellowship from the Monsanto Fund and by NIH Grant R01-AI 15693 to Prof. Michael W. Pariza, Food Research Institute, University of Wisconsin, Madison.