Immunogold localization of chymotrypsin inhibitor-2, a lysine-rich protein,in developing barley endosperm

Chymotrypsin inhibitor-2, a lysine-rich protein in the barley endosperm, has been localized at the ultrastructural level by immunocytochemistry in developing barley endosperm cells 14 days post anthesis. The protein is deposited in the protein bodies. Two morphologically distinct types of protein bodies, small spherical and large irregularly shaped, are present. Golgi-apparatus-derived vesicles whose content is labelled by chymotrypsin inhibitor-2 antibody-gold particles are observed at the Golgi complex and around the vacuoles. These observations indicate that the transport of the protein to the site of deposition is mediated by the Golgi apparatus.Abbreviations CI chymotrypsin inhibitor - DPA days post anthesis - ER endoplasmic reticulum The authors wish to thank Dr. V.R. Franceschi (Department of Botany, Washington State University, Pullman, USA) for many helpful discussions and advice during the work, and the staff at the Electron Microscope Center at Washington State University for technical assistance.     

Effect of NaCl and mannitol on plasma membrane proteins in corn roots

Summary The short-term effects of NaCl and mannitol stress on plasma membrane (PM) polypeptides from corn roots (Zea mays L.) were determined using two-dimensional gel electrophoresis following radiolabeled amino acid incorporation. After 2.5 hours, both stress treatments altered synthesis of several polypeptides. Changes included up-regulation of some polypeptides with concomitant down-regulation of others. Some changes were unique to the stress treatment while others were common to both NaCl and mannitol. No new polypeptides appeared in either case. Pulse-chase experiments following 0.5-hours and 2.5-hours incubation periods with radiolabeled amino acids did not reveal differences in turnover of PM polypeptides. The results support the contention that altered synthesis of PM proteins under stress may contribute to the alteration of membrane function.Abbreviations ER endoplasmic reticulum: GA Golgi - PM plasma membrane - PVPP polyvinylpolypyrrolidone     

Quantitative analysis of resistance in cotton to three new isolates of the bacterial blight pathogen

Summary Genetic variability for virulence of the bacterial blight pathogen [Xanthomonas campestris pv malvacearum (Smith) Dye] on cotton (Gossypium hirsutum L.) has been shown by the identification of 19 races of the pathogen based on disease reactions of a set of ten host differentials. This study was conducted to determine the inheritance of host resistance to three recently identified isolates of X. campestris pv malvacearum, which are virulent on the entire set of differentials. True leaves of Tamcot CAMD-E, LEBOCAS-3-80, Stoneville 825, and their f₁, F₂, and backcross progenies were wound-inoculated in the field with separate bacterial suspensions of the virulent HV3, HV7, and Sudan isolates of the pathogen. LEBOCAS-3-80 was replaced with S295, a new immune cultivar, for a greenhouse study in which both cotyledons and true leaves were inoculated. Disease reactions were rated on a scale of 1–10, and genetic models were proposed utilizing generation means analysis. Dominance, when significant, was in the direction of resistance in all but one cross-isolate combination. Digenic interaction components indicated a duplicate type. Narrow-sense heritability for resistance ranged from 0.59 to 0.68; therefore, primarily additive-genetic variability among the selected cutlivars was detected, indicating that breeding for improved resistance to these isolates is a practical goal.Contribution of the Department of Soil and Crop Sciences and the Texas Agricultural Experiment Station     

Relationship between gene expression and hybrid vigor in primary root tips of young maize (Zea mays L.) plantlets

Summary To provide an insight into the molecular basis of heterosis, we investigated gene expression in primary root tips of a heterotic maize hybrid (B73 × Mo17) and its parental lines (B73 and Mo17). This analysis was carried out (i) by differential plaque hybridization of a recombinant cDNA library made to poly(A) RNA isolated from B73 × Mo17 primary root tips, and (ii) by comparing with two-dimensional gel electrophoresis proteins synthesized in vitro in the rabbit reticulocyte system by poly(A) RNA isolated, at different stages of development, from the three genotypes. The results showed that there are sets of proteins and mRNAs that are differentially synthesized and expressed in the F₁ primary root tips in comparison to the parental lines. Moreover, results from the survey of 21 major in-vitrosynthesized polypeptide variants, from mRNAs of primary root tips of the parental lines and their F₁ hybrid, indicated that in seven instances hybrid proteins translated in vitro were more abundant or possibly new. In most of the remaining cases, hybrid spots were similar in intensity to the same protein produced by one of the two parental lines.     

Protein translocationin vitro: Biochemical characterization of genetically defined translocation components

Recent years have seen the convergence of both genetic and biochemical approaches in the study of protein translocation inE. coli. The powerful combination of these approaches is exemplified in the use of anin vitro protein synthesis-protein translocaltion system to analyze the role of genetically defined components of the protein translocation machinery. We describe in this review recent results focusing on the function of thesecA, secB, andsecY gene products and the demonstration of their requirement forin vitro protein translocation. The SecA protein was recently shown to possess ATPase activity and was proposed to be a component of the translocation ATPase. We present a speculative working model whereby the translocator complex is composed of the integral membrane proteins SecY, SecD, SecE, and SecF, forming an aqueous channel in the cytoplasmic membrane, and the tightly associated peripheral membrane protein SecA functioning as the catalytic subunit of the translocator or protein-ATPase.     

On the role of Ca2+-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm ofNeurospora crassa

Summary Pulses of some Ca²⁺ channel blockers (dantrolene, Co²⁺, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca²⁺, or of low Ca²⁺, a Ca²⁺ ionophore (A23187) together with Ca²⁺, and other Ca²⁺ channel blockers (La³⁺, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca²⁺ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca²⁺ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with³⁵S-thio -ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca²⁺ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca²⁺-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca²⁺ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.     

PVA和PEG(6000)预处理对大豆胚轴生长、蛋白质合成和ATP含量的影响

在低温吸胀阶段,经PVA(聚乙烯醇)和PEG(聚乙二醇6000)预处理的大豆胚轴蛋白质合成和ATP含量均比对照高。在萌发阶段,胚轴生长增快,蛋白质合成明显加快,ATP迅速被消耗,而对照胚轴则相反。试验结果表明,预处理大豆种子萌发和生长与其蛋白质合成、ATP水平和消耗能力有密切关系。     

Rat RI beta isoform of type I regulatory subunit of cyclic adenosine monophosphate-dependent protein kinase: cDNA sequence analysis, mRNA tissue specificity, and rat/mouse difference in expression in testis

A rat complementary DNA (cDNA) for the RI beta isoform of type I cyclic adenosine monophosphate (cAMP)-dependent protein kinase regulatory subunit was cloned and sequenced and was found to contain the entire protein coding and 3'-untranslated regions, with a single (ATTAAA) poly-adenylation site. The largest open reading frame was preceded by a short out-of-phase open reading frame, which is not seen in the corresponding mouse RI beta cDNA due to a single base substitution. The rat RI beta cDNA clone was 2,374 bases long and detected a rat mRNA of approximately 2.8 kilobases. Rat RI beta mRNA was abundant in adult rat brain and testis but was undetectable in other rat tissues. The rat RI beta cDNA also detected RI beta mRNA in mouse brain, but not mouse testis, from 10-week-old BALB/c or 10- and 6-week-old Swiss Webster mice. Thus, despite a 96% nucleotide identity in the coding region of RI beta in rat vs. mouse, there are at least two differences in these closely related species. First, there is a short open reading frame, which precedes the coding region in the rat but not the mouse. Second, unlike the mouse testis, the rat testis contains abundant levels of RI beta mRNA.     

Functional Expression of P-Glycoprotein in an Immortalised Cell Line of Rat Brain Endothelial Cells, RBE4

Abstract: The presence of P-glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that P-glycoprotein is expressed as part of the normal blood-brain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P-glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P-glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [³H]colchicine and [³H]vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3'-keto-Bmt¹]-[Val²]-cyclosporin) at 50 or 100 µ M concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P-glycoprotein activity both in the blood-brain barrier and in multidrug resistance in general.     

A Low-Km 5'-Nucleotidase from Rat Brain Cytosolic Fraction: Purification, Kinetic Properties, and Description of Regulation by a Novel Factor that Increases Sensitivity to Inhibition by ATP and ADP

Abstract: A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000- g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low K _m for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by α,β-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of M_r = 54,300 ± 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- K _m"soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000- g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP.