Ribonuclease in plant vacuoles: purification and molecular properties of the enzyme from cultured tomato cells

A ribonuclease which was previously shown to be located in isolated vacuoles from suspension-cultured cells of tomato (Lycopersicon esculentum L.; Abel and Glund 1986, Physiol. Plant. 66, 79–86) has been purified to near homogeneity. Purification was up to 55000-fold with a yield of about 20%. The vacuolar origin of the protein was evidenced by comparing its electrophoretic mobility, isoelectric point, pH-optimum for activity and other properties with that of the RNA-degrading activity present in isolated vacuoles. The molecular weight of the native single polypeptide chain was estimated at 17500 and 20300 by gel filtration and sedimentation analysis, respectively. The enzyme hydrolyzed only single-stranded RNA with a mode of action that was endonucleolytic. The vacuolar ribonuclease had no requirement for divalent metal ions, and did not exhibit phosphomonoesterase (EC 3.1.3.1; EC 3.1.3.2) and phosphodiesterase (EC 3.1.15.1; EC 3.1.16.1) activity. The specificity of the enzyme has been studied by using homopolyribonucleotides as substrates. The end-products obtained were the respective nucleoside 2:3-cyclic monophosphates and, to minor extents, the corresponding nucleoside 3(2)-monophosphates. According to these observations, the vacuolar ribonuclease from tomato can be classified as ribonuclease I (EC 3.1.27.1).Abbreviations DEAE diethylaminoethyl - RNase ribonuclease - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The effect of lime on phosphorus adsorption and barley growth in three acid soils

For three acid soils from Santa Catarina, Brazil, lime application and time of incubation with lime had little effect on the adsorption of added phosphorus. In two soils with high contents of exchangeable aluminium, solution P and isotopically exchangeable P were decreased by incubating with lime for 1 month: phosphorus was probably adsorbing on freshly precipitated aluminium hydrous oxides. In one soil with less exchangeable aluminium, P in solution was increased by liming. After 23 months lime increased solution and exchangeable P possibly due to crystallization of aluminium hydrous oxides reducing the number of sites for P adsorption. All these changes were however small. In a pot experiment, lime and phosphorus markedly increased barley shoot and root dry matter and P uptake. Although liming reduced P availability measured by solution P, isotopically exchangeable P and resin extractable P, it increased phosphorus uptake by reducing aluminium toxicity and promoting better root growth. The soil aluminium saturation was reduced by liming, but the concentration of aluminium in roots changed only slightly. The roots accumulated aluminium without apparently being damaged.     

Anaerobic metabolism enzymes as markers of flooding stress in maize seeds

Maize (Zea mays L.) seeds differ in their relative tolerance to the anaerobic environment caused by flooding. Seed tolerance to flooding stress depends on cellular and metabolic processes since gross anatomical responses have not developed at the pre-emergence stage. The study reported here characterizes the activities of four anaerobic respiratory enzymes: pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), and malic enzyme (ME) in the flood-tolerant A632 and floodsusceptible Mo 17 inbred maize seeds during flooding at 10 and 25°C. Each inbred consisted of two seed lots possessing 95% and 75% germination levels. Flooding increased the activities of all four enzymes. However, no consistent correlation between anaerobic enzyme activity and flood tolerance was observed across genotype, seed quality and flooding temperature. The results indicate that it may not be feasible to use whole-seed anaerobic enzyme activities to predict maize seed performance under flooding stress. Contribution from the Soil Drainage Research Unit, USDA-ARS, Columbus, OH, in cooperation with the Ohio Agricultural Research and Development Center, The Ohio State University. OARDC Journal Article No. 66–86.     

NAD(P)H oscillation in relation to the rhythmic contraction in thePhysarum plasmodium

Summary ThePhysarum plasmodium shows rhythmic contractile activities with a period of a few min. Phases of the oscillation in the plasmodium migrating unindirectionally agreed sideways throughout at the frontal part. So, time course of an intracellular chemical component was determined by analyzing small pieces cut off successively from the frontal part of the large plasmodium. Intracellular NAD(P)H concentration oscillated with the same period as the rhythmic contraction but with a different phase advancing about 1/3 of the period. UV irradiation suppressed the rhythmic contraction without affecting the rhythmic variation of NAD(P)H. Thus, the NAD(P)H oscillator works independently of the rhythmic contractile system, but seems entraining with each other.Abbreviations UV ultraviolet - NADH nicotinamide adenine dinucleotide, reduced form - NADPH nicotinamide adenine dinucleotide phosphate, reduced form - ATP adenosine 5-triphosphate - cAMP cyclic adenosine 3, 5-monophosphate - FMNH₂ flavin mononucleotide, reduced form - TCA tricarboxylic acid - BSA bovine serum albumin - DTT dithiothreitol     

Accumulation pattern of amino acid substitutions in protein evolution

Summary A simple method for the evolutionary analysis of amino acid sequence data is presented and used to examine whether the number of variable sites (NVS) of a protein is constant during its evolution. The NVSs for hemoglobin and for mitochondrial cytochrome c are each found to be almost constant, and the ratio between the NVSs is close to the ratio between the unit evolutionary periods. This indicates that the substitution rate per variable site is almost uniform for these proteins, as the neutral theory claims. An advantage of the present analysis is that it can be done without knowledge of paleontological divergence times and can be extended to bacterial proteins such as bacterial c-type cytochromes. It is suggested that the NVS of cytochrome c has been almost constant even over the long period (ca. 3.0 billion years) of bacterial evolution but that at least two different substitution rates are necessary to describe the accumulated changes in the sequence. This two clock interpretation is consistent with fossil evidence for the appearance times of photosynthetic bacteria and eukaryotes.     

A Phorbol Ester-Sensitive Kinase Catalyzes the Phosphorylation of P0 Glycoprotein in Myelin

The proposed structural protein of peripheral nerve myelin, P0, has been shown to have several covalent modifications. In addition to being glycosylated, sulfated, and acylated, P0 is phosphorylated, with the intracellular site of this latter addition being in question. By employing nerve injury models that exhibit different levels of P0 biosynthesis in the absence and presence of myelin assembly, we have examined the cellular location of P0 phosphorylation. It is demonstrated that there is comparable P0 phosphorylation in both normal and crush-injured adult rat sciatic nerves, although the level of biosynthesis of P0 differs between these myelin maintaining and actively myelinating nerve models, respectively. The glycoprotein does not appear to be phosphorylated readily in the transected adult sciatic nerve, a preparation in which P0 biosynthesis is observed but that lacks myelin membrane. These observations suggest that the modification is not associated with the biosynthesis or maturation of P0 in the endoplasmic reticulum or Golgi, but that it instead occurs after myelin assembly. That P0 phosphorylation occurs in the normal nerve even when translation is inhibited by cycloheximide treatment lends further support to this conclusion. P0 is shown to be phosphorylated on one or more serine residues, with all or most of the phosphate group(s) being labile as evidenced by pulse-chase analysis. Addition of a biologically active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate or 4 beta-phorbol 12,13-dibutyrate, substantially increases the extent of [32P]orthophosphate incorporation into the glycoprotein of normal and crushed nerve but not transected nerve. Biologically inactive 4 alpha-phorbol 12,13-didecanoate has no effect on P0 phosphorylation. Similarly, the addition of the cyclic AMP analog 8-bromo-cyclic AMP causes no appreciable changes in P0 labeling. These findings indicate that the phorbol ester-sensitive enzyme, protein kinase C, may be responsible for the phosphorylation of P0 within the myelin membrane.     

Regulation of Dipeptidyl Aminopeptidase I and Angiotensin Converting Enzyme Activities in Cultured Murine Brain Cells by Cortisol and Thyroid Hormone

Cultures of dissociated brain cells from 15-day-old fetal mice were grown in the presence and absence of 20 or 50 nM triiodothyronine (T3), 30 or 300 nM cortisol, and 30 nM cortisol plus 50 nM T3 added to chemically defined media or in media supplemented with 15% serum from control and hypothyroid calves. The specific activities of five lysosomal enzymes--N-acetyl galactosaminidase, beta-glucuronidase, beta-galactosidase, cathepsin B, and dipeptidyl aminopeptidase I (DAP-I)--were higher in cells grown in calf serum than in cells grown in defined media. Of these enzymes, only DAP-I was elevated in activity when the cells were grown in hypothyroid calf serum instead of control calf serum. Elevation of DAP-I activity was reversed by addition of 20 nM T3 to hypothyroid calf serum. The enzymatic properties of DAP-I were similar whether the cells were grown in control or hypothyroid calf serum and were similar to those reported for human fibroblasts and the purified enzyme. When the cells were grown in defined media, cortisol decreased the activities of all lysosomal enzymes, with 300 nM cortisol being more effective than 30 nM cortisol. Addition of 50 nM T3 to 30 nM cortisol decreased DAP-I activity more than 30 nM cortisol alone, but 50 nM T3 alone in defined media did not alter DAP-I levels. The reduction of DAP-I activity in these cells by T3 required cortisol, unidentified components in serum, or both.(ABSTRACT TRUNCATED AT 250 WORDS)     

脑室注射P物质对大鼠血压、心率及交感传出活动的影响

给乌拉坦麻醉六鼠侧脑室注射P物质(SP)10μg,引起动脉血压、心率和内脏交感神经放电增加。同样剂量的SP静脉注射后却引起血压降低。阿托品预处理不影响SP的开心率作用。预先脑室注射0.25,4,64μg阿片受体拮抗剂纳洛酮,对SP的升压效应有剂量依赖式对抗作用。以上说明脑室注射SP 引起的血压升高是交感神经活动增强,导致心率加快及外周血管紧张性增加的结果,并提示SP的中枢升压效应可能与脑内释放内源性阿片样物质有关。     

Experimental evidence on the affinities ofPapaver somniferum (Papaveraceae)

Results obtained from crossing experiments betweenP. somniferum subsp.somniferum (2n = 22) and subsp.setigerum (2n = 44),P. glaucum (2n = 14) andP. gracile (2n = 14) and from the observation of meiotic chromosome pairing in the various hybrids obtained do not provide straightforward evidence for the hypothesis thatP. somniferum originated as a triploid hybrid between taxa similar toP. glaucum andP. gracile (Kadereit 1986a, b).—On the one hand, the pattern of crossability found reflects the closer similarity of subsp.somniferum toP. glaucum and of subsp.setigerum toP. gracile, which was interpreted as segregation of parental characters, and the high frequency of 2n = 28 chromosomes among F₂-progeny from the hybrid subsp.somniferum × subsp.setigerum (2n = 33) might reveal n = 7 as the base number also ofP. somniferum. On the other hand, however, the general difficulty of obtaining hybrids, and the low incidence of bivalent formation in their meiosis, probably indicating a lack of chromosome homology between the different species, do not fit the above hypothesis.—These results are in marked contrast to the morphological similarity between the three species involved.