A Method for Calculating Sucrose Synthesis Rates throughout a Light Period in Sugar Beet Leaves

Sucrose synthesis rate in an exporting sugar beet (Beta vulgaris L.) leaf was calculated from simultaneous measurements of export and changes in leaf sucrose level. The amount of recently fixed carbon exported was determined from net carbon assimilated minus the tracer carbon accumulated in the leaf. The relative amount of ¹⁴C accumulated in the leaf supplied with ¹⁴CO₂ throughout an entire light period was recorded continuously with a Geiger-Mueller detector. To produce a continuous time course for tracer carbon accumulated in the leaf during the light period, the latter curve was superimposed on values for tracer carbon accumulated in leaves sampled at hourly intervals. Validity of the method requires that nearly all of the carbon that is exported be sucrose and that nearly all of the sucrose that is synthesized be either exported or accumulated as sucrose in the exporting leaves. These conditions appeared to be fulfilled in the situations where the method was applied. The method was used to study the effect of increasing atmospheric CO₂ concentration on the rate of sucrose synthesis. Further, the method can be used in conjunction with the gathering of other data such as gas exchange, metabolite levels, and enzyme activities in a set of leaves of a similar age on the same plant. This assemblage of data was found to be useful for understanding how rates of photosynthesis, sucrose synthesis, and translocation are regulated in relation to each other in an intact plant.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Detection and characterization of a glutenin subunit with unusual high Mr at the Glu-A1 locus in hexaploid wheat

A hexaploid wheat landrace collected from the Baluchistan province of Pakistan was found to possess a novel high-molecular-weight glutenin subunit (HMW-GS). The subunit has a very slow electrophoretic mobility as revealed by SDS-PAGE, and its molecular weight is comparable to that of the highest molecular weight glutenin subunit (2.2 encoded in the D-genome) reported so far in hexaploid wheat varieties and landraces of Japanese origin. Evidence obtained from (PCR) gene amplification studies using the primers specific for Glu-1 loci proved that the gene coding for this novel subunit belongs to the Glu-A1 locus located on the long arm of chromosome 1A. Digestion of the amplified gene (PCR product) with restriction enzymes indicated that the novel gene differs from prevailing Glu-A1 alleles (null, 1 and 2^*) by an extra DNA fragment of approximately 600 base pairs. The results also indicated that the novel subunit is most probably a derivative of subunit 2^* that has very likely incorporated the 600-bp fragment following a process of unequal crossing over. The present findings were further substantiated by reserved phase high performance liquid chromatography (RP-HPLC) analysis.     

Comparative Analysis of Short- and Long-Term Changes in Gene Expression Caused by Low Water Potential in Potato (Solanum tuberosum) Cell-Suspension Cultures

To dissect the cellular response to water stress and compare changes induced as a generalized response with those involved in tolerance/acclimation mechanisms, we analyzed changes in two-dimensional electrophoretic patterns of in vivo [35S]methionine-labeled polypeptides of cultured potato (Solanum tuberosum) cells after gradual and long exposure to polyethylene glycol (PEG)- mediated low water potential versus those induced in cells abruptly exposed to the same stress intensity. Protein synthesis was not inhibited by gradual stress imposition, and the expression of 17 proteins was induced in adapted cells. Some polypeptides were inducible under mild stress conditions (5% PEG) and accumulated further when cells were exposed to a higher stress intensity (10 and 20% PEG). The synthesis of another set of polypeptides was up-regulated only when more severe water-stress conditions were applied, suggesting that plant cells were able to monitor different levels of stress intensity and modulate gene expression accordingly. In contrast, in potato cells abruptly exposed to 20% PEG, protein synthesis was strongly inhibited. Nevertheless, a large set of polypeptides was identified whose expression was increased. Most of these polypeptides were not induced in adapted cells, but many of them were common to those observed in abscisic acid (ABA)-treated cells. These data, along with the finding that cellular ABA content increased in PEG-shocked cells but not in PEG-adapted cells, suggested that this hormone is mainly involved in the rapid response to stress rather than long-term adaptation. A further group of proteins included those induced after long exposure to both water stress and shock. Western blot analysis revealed that osmotin was one protein belonging to this common group. This class may represent induced proteins that accumulate specifically in response to low water potential and that are putatively involved in the maintenance of cellular homeostasis under prolonged stress.     

Intergenic spacers of rRNA genes in three species of theCynareae (Asteraceae)

Intergenic spacers of the rRNA genes of three species of theCynareae tribe:Cynara cardunculus subsp.scolymus (artichoke),Onopordum acanthium, andO. illyricum were cloned in the plasmid pGEM-7zf(+). Detailed restriction mapping and partial sequencing of the IGSs were carried out. The structural analysis showed a clear diversity betweenCynara andOnopordum, while a high degree of homology was found between the twoOnopordum spp. In all three species a fragment of about 450 bp from the 5 end of 18S to the Acc I site with a high sequence homology was present. Nucleotide sequences upstream from the above mentioned Acc I site show a gradual decrease of homology betweenCynara andOnopordum.     

Mapping of the mouse Tdgf1 gene and Tdgf pseudogenes

Teratocarcinoma-derived growth factor-1 (Tdgf1), a member of the ``EGF family' of growth factors, is expressed during mouse gastrulation in the forming mesoderm and later in the truncus arteriosus of the developing heart. In humans, TDGF1 is highly expressed in germ cell tumors and in colon and mammary carcinomas. In mouse, one gene (Tdgf1) and two pseudogenes (Tdgf1-ps1 and Tdgf1-ps2) have been isolated and characterized. Tdgf1 corresponds to the gene expressed in F9 teratocarcinoma cells. Tdgf1-ps1 and Tdgf1-ps2 are two intronless sequences with all the characteristics of retroposons. In the present paper, we assign the chromosomal location for Tdgf1, Tdgf1-ps1, and Tdgf1-ps2 sequences to Chromosomes (Chrs) 9, 16, and 17, respectively. Two previously described mouse mutants, scant hair (sch) and fur deficient (fd), map near the Tdgf1 gene. Analysis of their DNA coding region provided no evidence that Tdgf1 could be the responsible gene for these phenotypes. Finally, analysis of the DNA from several Mus musculus strains and from Mus spretus mice revealed a highly variable restriction pattern and the absence of the Tdgf1-ps1 genomic sequence from the Mus spretus genome. Received: 23 November 1996 / Accepted: 17 February 1997     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Effect of an electric field on the motility of Entamoeba histolytica examined by multiple-beam interference microscopy

A recently developed multiple-beam interference microscopic technique has been used to visualize submicroscopic structures of Entamoeba histolytica and their movements in applied external electric fields. The movements were videorecorded and it was found that at low current (120 microA) pseudopods are filled with hyaline ectoplasm. At slightly higher current (about 150 microA), the amoeba stops extending the pseudopods and loosens its attachment to the surface. At higher currents (200 microA), it forms a cyst and remains immobile for a time. Before this stage is reached a narrow ring is formed around the nucleus due to alterations in the proteins to protect it.     

Structure-activity relationships of piperazinebenzylamines as potent and selective agonists of the human melanocortin-4 receptor

SAR studies on a series of piperazinebenzenes directed toward the human melanocortin-4 receptor resulted in potent MC4R agonists. Replacement of the triazole moiety of an initial lead 4 by a basic nitrogen baring a lipophilic side-chain increased the binding affinities of these compounds. Analogs bearing an additional hetero-atom in the side-chain possessed good agonist potency. Thus, 11h had a Ki of 11 nM, and 13g exhibited an EC50 of 3.8 nM and a Ki of 6.4 nM.