Structure, synthesis and orientation of microfibrils. VI. The role of ions in microfibril deposition in Oocystis solitaria.

The action of a series of chelators, cryptates and ionophores on microfibril deposition and the presence of cortical microtubules in Oocystis solitaria has been investigated. Having a potent inhibitory effect on cellulose synthesis were the calcium ionophores A 23187 and X-537A, the flourescent calcium chelator chlorotetracycline and the cryptates 211 and 221. Only at unphysiological concentrations, e.g. 100 mM or more, were the chelators EDTA and EGTA effective in completely inhibitory cellulose synthesis. A reduction in the rate of cellulose synthesis was observed with the sodium-selectiv ionophore dianemycin. This partial inhibition was independent of the ionophore concentration. Without effect on microfibril deposition were the potassium-selective ionophore valinomycin and cryptate 222. In all cases, whether cellulose synthesis is blocked or not, none of the above agents prevented the reassembly of cortical microtubules when applied during the recovery from colchicine treatment. Destruction of cortical microtubules through the calcium ionophore may be achieved, but only when additional (2 mM) calcium is added to the culture medium.     

单生卵囊藻(Oocystis solitaria)和月牙藻(Selenastrumsp.)的培养条件及其细胞组成

从海滨的淡水池沼中分离得到单生卵囊藻(Oocystis solitaria)和月牙藻(Selenastrum sp.),分别研究了温度、光照强度、盐度对2种微藻生长繁殖的影响,分析了2种微藻的细胞组成、脂肪酸组成及盐度对2种微藻脂肪积累的影响.结果表明:单生卵囊藻和月牙藻的适宜生长温度分别为35.9℃～40.5℃和29.7℃～32.8℃;单生卵囊藻和月牙藻的适宜光照强度分别为46～70μmol · m-2·s-1和17 ～54 μmol·m-2·s-1;单生卵囊藻在淡水培养液中生长最好,月牙藻在盐度为2的半成水培养液中生长最好;在适宜培养条件下,单生卵囊藻细胞蛋白、总糖和总脂肪分别为27.61％、22.00％和3.84％;月牙藻细胞的蛋白、总糖和总脂肪分别为28.06％、21.99％和12.53％;单生卵囊藻的脂肪酸组成中含有丰富的18∶3n3;月牙藻的脂肪酸组成中含有DHA;盐度影响2种微藻的总脂肪含量;单生卵囊藻和月牙藻分别在盐度4和10的半咸水培养液中细胞总脂含量最高.     

Cell wall biogenesis in Oocystis: experimental alteration of microfibril assembly and orientation.

Cell wall biogenesis in the unicellular green alga Oocystis apiculata has been studied. Under normal growth conditions, a cell wall with ordered microfibrils is synthesized. In each layer there are rows of parallel microfibrils. Layers are nearly perpendicular to each other. Terminal linear synthesizing complexes are located in the plasma membrane, and they are capable of bidirectional synthesis of cellulose microfibrils. Granule bands associated with the inner leaflet of the plasma membrane appear to control the orientation of newly synthesized microfibrils. Subcortical microtubules also are present during wall synthesis. Patterns of cell wall synthesis were studied after treatment with EDTA and EGTA as well as divalent cations (MgSO4, CaSO4, Cacl2). 0.1 M EDTA treatment for 15 min results in the disassociation of the terminal complexes from the ends of microfibrils. EDTA-treated cells followed by 15 min treatment with MgSO4 results in reaggregation of the linear complexes into a paired state, remote from the original ends to which they were associated. After 90 min treatment with MgSO4, normal synthesis resumes. EGTA and calcium salts do not affect the linear complexes or microfibril orientation. Treatments with colchicine and vinblastine sulphate do not depolymerize the microtubles, but the wall microfibril orientation is altered. With colchicine or vinblastine, the change in orientation from layer to layer is inhibited. The process is reversible upon removal of the drugs. Lumicolchicine has no effect upon microfibril orientation, but granule bands are disorganized. Treatment with coumarin, a known inhibitor of cellulose synthesis, causes the loss of visualization of subunits of the terminal complexes. The possibility of the existence of a membrane-associated colchicine-sensitive orientation protein for cellulose microfibrils is discussed. Transmembrane modulation of microfibril synthesis and orientation is presented.     

Structure of rigid-layer of Rhizobium cell wall. I. Purification on the peptidoglycan from the cellulose microfibrils

The bag shaped peptidoglycan layer of Rhizobium cell wall was isolated from intact cells after treatment with sodium dodecylsulfate and trypsin, chymotrypsin or pepsin digestion. Results of chemical analysis of acid hydrolyzed peptidoglycan revealed beside two amino sugars: glucosamine and muramic acid, three major amino acids; alanine, glutamic acid and 2,6-diaminopimelic acid and also significant amount of glucose. Evidence were provided that the polyglucose found in peptidoglycan preparations of three strains of Rhizobium trifolii, one of Rhizobium leguminosarum and one of Rhizobium meliloti consist of cellulose microfibrils. The content of cellulose present in Rhizobium peptidoglycans ranged from 60 to 80%. Methods of peptidoglycan purification from the cellulose microfibrils are described.     

Structure and synthesis of the growth inhibitor batatasin I from Dioscorea batatas

The synthesis of 6-hydroxy-2,4,7-trimethoxyphenanthrene is described and its identity with the growth inhibitor, batatasin I, is confirmed.     

Selective synthesis of plasmid-coded proteins by Escherichia coli during recovery from chloramphenicol treatment.

Protein products of recombinant ColE1 plasmids are preferentially synthesized and can easily be identified in Escherichia coli cells recovering from prolonged treatment with chloramphenicol.     

Polarity orientation and assembly process of microtubule bundles in nocodazole-treated, MAP2c-transfected COS cells.

Microtubule bundles reminiscent of those found in neuronal processes are formed in fibroblasts and Sf9 cells that are transfected with the microtubule-associated proteins tau, MAP2, or MAP2c. To analyze the assembly process of these bundles and its relation to the microtubule polarity, we depolymerized the bundles formed in MAP2c-transfected COS cells using nocodazole, and observed the process of assembly of microtubule bundles after removal of the drug in cells microinjected with rhodamine-labeled tubulin. Within minutes of its removal, numerous short microtubule fragments were observed throughout the cytoplasm. These short fragments were randomly oriented and were already bundled. Somewhat longer, but still short bundles, were then found in the peripheral cytoplasm. These bundles became the primordium of the larger bundles, and gradually grew in length and width. The polarity orientation of microtubules in the reformed bundle as determined by "hook" procedure using electron microscope was uniform with the plus end distal to the cell nucleus. The results suggest that some mechanism(s) exists to orient the polarity of microtubules, which are not in direct continuity with the centrosome, during the formation of large bundles. The observed process presents a useful model system for studying the organization of microtubules that are not directly associated with the centrosomes, such as those observed in axons.     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro. V. Inhibition of macromolecular synthesis and of microfilament and microtubule function.

T lymphocytes from immune guinea pigs produce clusters in vitro with macrophages exposed to soluble protein antigens. The formation of clusters is antigen specific. Cultures containing macrophage-lymphocyte clusters produced in response to purified protein derivative of tuberculin were treated with inhibitors of macromolecular synthesis and of microfilament and microtubule systems. Cytosine arabinoside, puromycin, actinomycin D, cycloheximide, cytochalasin B, and colcemide were used. The formation of clusters is independent of DNA synthesis. Microfilaments and microtubules are needed in the formation but not in the maintenance of clusters. Protein or RNA synthesis or both are needed in the formation of clusters. Finally, continuous protein synthesis is necessary for the maintenance of clusters.     

Structure and dynamics of the G121V dihydrofolate reductase mutant: lessons from a transition-state inhibitor complex

It is well known that enzyme flexibility is critical for function. This is due to the observation that the rates of intramolecular enzyme motions are often matched to the rates of intermolecular events such as substrate binding and product release. Beyond this role in progression through the reaction cycle, it has been suggested that enzyme dynamics may also promote the chemical step itself. Dihydrofolate reductase (DHFR) is a model enzyme for which dynamics have been proposed to aid in both substrate flux and catalysis. The G121V mutant of DHFR is a well studied form that exhibits a severe reduction in the rate of hydride transfer yet there remains dispute as to whether this defect is caused by altered structure, dynamics, or both. Here we address this by presenting an NMR study of the G121V mutant bound to reduced cofactor and the transition state inhibitor, methotrexate. NMR chemical shift markers demonstrate that this form predominantly adopts the closed conformation thereby allowing us to provide the first glimpse into the dynamics of a catalytically relevant complex. Based on (15)N and (2)H NMR spin relaxation, we find that the mutant complex has modest changes in ps-ns flexibility with most affected residues residing in the distal adenosine binding domain rather than the active site. Thus, aberrant ps-ns dynamics are likely not the main contributor to the decreased catalytic rate. The most dramatic effect of the mutation involves changes in μs-ms dynamics of the F-G and Met20 loops. Whereas loop motion is quenched in the wild type transition state inhibitor complex, the F-G and Met20 loops undergo excursions from the closed conformation in the mutant complex. These excursions serve to decrease the population of conformers having the correct active site configuration, thus providing an explanation for the G121V catalytic defect.     

Structure of lactate dehydrogenase inhibitor generated from coenzyme.

Two inhibitors of lactate dehydrogenase generated during NADH storage have been isolated by chromatography. One is a dimer of the dinucleotide where the AMP moiety is unmodified. The other is also generated from NAD+ in the presence of a high concentration of phosphate ions at alkaline pH. This inhibitor was proved to be the addition compound of one phosphate group to position C-4 of the nicotinamide ring of NAD+ by NMR spectroscopy, enzymatic cleavage, and dissociation to NAD+ at neutral pH. This compound is a competitive inhibitor with respect to NAD+ in the presence of the lactate dehydrogenase with a Ki of 2 X 10(-7) M. The interaction of this inhibitor with lactate dehydrogenase is discussed relative to the structure of this enzyme.     

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. V. Inability of inhibitor to repress 5S RNA synthesis.

A specific inhibitor of ribosomal RNA (rRNA) synthesis was partially purified from an acid-soluble fraction of Xenopus laevis blastulae. Effects of this inhibitor on 5S rRNA synthesis of isolated neurula cells of the same species were investigated. The results show that the synthesis of both 5S rRNA and 4S RNA proceeds normally when both 18 and 28S rRNA are almost completely inhibited. Failure of the inhibitor to suppress 5S rRNA synthesis suggests that it plays an important role in the regulation of 18 and 28S rRNA synthesis during development and that the synthesis of 5S rRNA is not coordinated to that of 18 and 28S rRNA.     

Callus induction from protoplasts of V. unguiculata,V. sublobata and V. mungo

Summary Protoplasts were isolated from hypocotyl of V. mungo (L.) Hepper or hypocotyl-derived callus of V. sublobata (Phaseolus sublobata Roxb.) and V. unguiculata (L.) Walp (syn. V. sinensis (L.) Saviex Hassk) using an enzyme solution comprising Cellulase 2.5%, Macerozyme, Hemicellulase and Driselase each at a 0.5% level in 0.5 M sorbitol. Isolated protoplasts were cultured in Murashige and Skoog's (1962) basal liquid medium supplemented with BA, NAA, 2,4-D (1 mg/l each) and sucrose (14%). After four weeks, protoplast colonies were transferred to the same medium with a reduced level of sucrose (7%). Colonies proliferated into actively growing calli. Further attempts to regenerate plants from such calli were not successful. However, protoclones of V. unguiculata differentiated roots on auxin/cytokinin supplemented media. Alternative methods for shoot differentiation from protoplastderived cultures were tried by the use of Agrobacterium tumefaciens shooter strains pGV 2215 or pGV 2298 or wild type strain B6S3.     

Solubilization of a protein synthesis inhibitor from vaccinia virions.

The protein synthesis inhibitor previously demonstrated to be associated with vaccinia cores was quantitatively solubilized from vaccinia virions or cores after an endogenous protein kinase reaction at pH 10. The presence of the inhibitor in the soluble extract correlated with the presence of soluble virion proteins phosphorylated in vitro. Partially purified inhibitor blocked methionyl-tRNAfMet-40S initiation complex formation, as was the case in cell-free extracts derived from vaccinia virus-infected cells.