Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.     

Identification and characterization of the ATP·Mg-dependent protein phosphatase activator (FA) as a microtubule protein kinase in the brain

The activating factor of ATP·Mg-dependent protein phosphatase (F _A) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates,F _A could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with aK _m value of 0.4 µM, and tau proteins to 4 moles of phosphates per mole of proteins with aK _m value of about 3 µM. When using microtubules as substrates,F _A could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca⁺²/phospholipid-dependent protein kinase, theF _A-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced byF _A. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed thatF _A could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca⁺²/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due toF _A. Taken together, the results provide initial evidence that the ATP·Mg-dependent protein phosphatase activating factor (F _A) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.     

Role of protein phosphorylation and inositol phospholipid turnover in rat parotid gland proliferation

The involvement of protein phosphorylation in isoproterenol (ISO)-mediated proliferation in the rat parotid gland was investigated by labeling the cells with [³²P] orthophosphate. An increased (4–6 fold) incorporation of the radiolabel was noted in the total parotid gland homogenates of ISO-treated animals when compared to controls. Plasma membrane, nuclear membrane and cytoplasm were isolated, the proteins separated by SDS/PAGE and the phosphoproteins detected by autoradiography. Two phosphoproteins with apparent M_r of 45 and 170 kDa were identified in the cytoplasm while the 170 kDa phosphoprotein also appeared as part of plasma membrane. Transfer of these proteins to nitrocellulose followed by Western blot detection with an antiphosphotyrosine monoclonal antibody showed reactivity with the 170 kDa region of the plasma membrane and cytoplasm. Separate in vitro studies involving incubations of rat parotid slices with 0.2 mM ISO and [³H] myo-inositol for 1 min induced inositol phosphate hydrolysis resulting in a significant increase in inositol-bis and -tris phosphate production. Inositol phosphate production can be blocked by pre-incubation with a mixed -adrenergic receptor antagonist but not with physiological concentrations of - or ₁-specific adrenergic receptor antagonists, indicating the ISO effects are mediated through the ₂-adrenergic receptors. The inclusion of calmodulin antagonists along with ISO prevented the expression of cell-surface galactosyltransferase and retarded gland hypertrophy and hyperplasia. These results suggest that ISO treatment leads to the phosphorylation of target proteins which may be involved in signal transduction pathways leading to cell proliferation.Abbreviations InsP₁, InsP₂, InSP₃ inositol mono-, bis-, and tris-phosphates - UDP Uridine diphosphate - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate - TFP Trifluoperazine - P-tyr phosphotyrosine - Gal Tase galactosyltransferase     

电离辐射引起胃排空延迟机理的初步探讨

本文观察了800rad照射后胃肠肌电的变化及切斷内脏神经与电刺激迷走神经对官的影响,初步分析了电离辐射引起胃排空延迟的机理。结果是:800rad照后大鼠胃肠肌电的峰波平均振幅胃幽部窦部照后1小时至第4天明显降低(P<0.05);幽门括约肌政变不大;十二指肠球部照后第3～7天明显下降(P<0.01)。切断内脏神经后照射大鼠胃肠肌电的峰波平均振幅胃幽门窦部变化不大,幽门括约肌照后1小时至第5天明显升高(P<0.05),十二指肠球部照后第3～7天明显下降(P<0.05)。800rad照后第4天大鼠胃对电刺激迷走神经的反应性明显降低(P<0.01)。上述结果表明:照后胃排空延迟的原因不仅与神经-体液调节紊乱有关,而且与射线损伤了平滑肌,使其兴奋性暂时的功能性降低有关。此外照后由于十二指肠球部运动的变化而使内感受器对胃运动的调节发生紊乱也是值得重视的因素。     

Photoacoustic and fluorescence measurements of the chilling response and their relationship to carbon dioxide uptake in tomato plants

The response of tomato plants to various chilling treatments was studied using two approaches for the measurement of photosynthetic activity. One involved the use of a portable fluorometer for the measurement of in-vivo chlorophyll fluorescence, while the other employed a newly introduced photoacoustic system which allowed changes in oxygen evolution to be followed in a leaf disc. A strong correlation was found between results obtained by each system and those obtained by a conventional open gas-exchange system for the determination of CO₂ uptake. Both systems of measurements could readily distinguish between the effects of chilling in the dark (at 3° C for 18 h) and chilling at high photon flux density (2000 mol m^-2 s^-1 for 5h at 5° C). Chilling in the dark had practically no effect on the quantum yield of oxygen evolution, chlorophyll fluorescence or CO₂ uptake, while chilling at excessively high photon flux density resulted in a sharp reduction (50–70%) in the quantum yields obtained. The results support the view that photosystem II cannot be the primary site of damage by chilling in the dark, although it is significantly affected by chilling at high light intensity.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PA photoacoustic - PFD photon flux density - PSII photosystem II     

Effect of clomiphene citrate on the rabbit ovary

Summary The effect of clomiphene citrate on the rabbit ovary was studied in mature nulliparous rabbits pretreated with three consecutive doses ranging from 0.01–10.0 mg/kg per day. With increasing doses a trend of decrease in mean ovarian weight (mg/kg body weight) is observed 2 days after termination of treatment. Five days later a significant increase occurs, which then subsides again to control values on day 12 after termination of treatment. During this period, no matings or injections of luteinizing hormone were performed to trigger ovulation; consequently no ovulations are observed. Folliculogenesis appears as normally; number and morphology of follicles are within normal ranges. No endogenous, spontaneous gonadotropin surges are detected in blood serum up to the 7th day after termination of treatment (2 and 10 mg doses). The surface epithelium of the ovary resembles normal germinal epithelium; however, after treatment with high doses a secretory-like activity is observed, accompanied with ultrastructural changes.Supported by the Deutsche Forschungsgemeinschaft, Special Program on Biology and Clinics of Reproduction. (Grant Be 524/7-7).Visiting Scientist from the Department of Obstetrics and Gynecology, Hadassah University Hospital, Jerusalem, Israel.     

Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in Percoll,and selective substrate adherence

Summary This paper presents a study on the structure and function of Kupffer cells (KC) and liver endothelial cells (LEC) isolated by a simple and rapid technique involving 1) perfusion of the liver with collagenase; 2) cell separation by means of density centrifugation in Percoll; and 3) cell culture, taking advantage of the fact that KC and LEC differ in their preferences for growth substrate. The KC, which attach and spread under serum-free conditions on surfaces of glass or plastic during the first 15 min in culture exhibit a typical macrophage-like morphology including membrane ruffling and a heterogenous content of vacuoles. Moreover, these cells express (a) Fc receptors (FcR) for binding and phagocytosis of erythrocytes covered with immune globulin G (E-IgG), and (b) complement receptors (CR) for binding and serum dependent phagocytosis of erythrocytes covered with either human C3b or mouse inactivated C3b (iC3b). The cells also bind fluid phase fluoresceinated C3b. Approximately 30% of the KC express immune response-associated (Ia)-antigens.The LEC attach and spread on fibronectin coated surfaces, but not on glass or plastic surfaces, during the first two hours in culture with or without serum, and are morphologically distinct from KC. Cultured LEC are well spread out with no membrane ruffling and with numerous large vesicles surrounding the regularly shaped nucleus. These cells bind, but do not ingest E-IgG via the FcR, but no binding of fluid phase C3b or particle fixed C3b or iC3b can be observed. Incubation of LEC with fluorescein amine conjugates of ovalbumin or formaldehyde treated serum albumin, but not with fluoresceinated native serum albumin, results in accumulation of fluorescence specifically localized in the large perinuclear vesicles. Neither KC nor any other cell types tested have the ability to accumulate fluorescence upon incubation with these compounds. Iaantigens are not present on the LEC.Cytochemical demonstration of unspecific esterase, acid phosphatase, and peroxidase reveals different patterns and intensities of staining in KC as compared to LEC.Abbreviations Used KC Kupffer cells - LEC Liver endothelial cells - C Complement - C3b Major fragment of C3 activation - iC3b C3b that has been cleaved by factor I (C3b inactivator), present in serum - meC3b C3b produced by treating purified human C3 with methyl amine - trC3b C3b produced by treating purified human C3 with trypsin - CR Complement receptors for C3b and iC3b - IgG Immune globulin G - IgM Immune globulin M - E Erythrocytes - E-IgG E covered with anti-E IgG - E-IgM E covered with anti-E IgM - E-C3b(h) E-IgM reacted with purified human C1, C4, oxidized C2 and C3 (E-IgMC14^xyC2C3b) - E-iC3b(m) E-IgM incubated with C5 deficient serum from AKR mice - FcR Receptors for the Fc portion of IgG - FITC Fluorescein isothiocyanate - FITC-meC3b FITC conjugated to meC3b - FITC-trC3b FITC conjugated to trC3b - FA Fluorescein amine - FA-OA Ovalbumin conjugated with FA - FA-SA Serum albumin conjugated with FA - FA-FSA Formaldehyde-treated serum albumin conjugated with FA - Ia Immune response-associated AcE Acid unspecific esterase acting on alpha naphtyl acetate - NASDAE Unspecific esterase acting on naphthol AS-D acetate - NASDCAE Unspecific esterase acting on napthol AS-D chloroacetate     

1-Methylguanine and 7-methylguanine increase cell agglutinability

Summary 1-Methylguanine and 7-methylguanine, both metabolic products of tRNA degradation, are known to induce transformation of Chinese hamster fibroblasts in culture. The effects of these compounds on the cell membrane have been studied by the method of Concanavalin A-mediated hemadsorption. 1-Methylguanine or 7-methylguanine induced a 50% increase of Con A-mediated hemadsorption within 20 hours of exposure of the cells to the agent at a concentration of 10^-5 M. This alteration was reversed within 13 days when the cells were grown in the control medium. Prolonged treatment with 1-methylguanine or 7-methylguanine resulted in changes which were only slowly reversed during growth of the cells in the control medium. The effect of the methylated purines on the cell membrane could be completely inhibited by simultaneous addition of dibutyryl-cAMP at a concentration of 10^-5 M. The possible mechanism of cell membrane alteration by methylated purines and its relevance to transformation in vitro are discussed.     

Detection of an antigenic cell wall layer inHistoplasma capsulatum

Histoplasma capsulatum yeast cells have been studied by immunoelectron microscopy using rabbit polyclonal antisera and a biotin-avidin-peroxidase detection system. An antigenic surface layer has been visualized in the cell wall of immunostained organisms. This layer was not seen in samples prepared by standard electron microscopic methods or in negative controls used with the immunocytochemical technique. Without immunostaining the cell wall ofHistoplasma appeared almost transparent. In contrast, after immunoperoxidase staining the cell wall was conspicuous, bounded by the darkly stained outer layer. This electron dense layer, appeared to be a reservoir of surface antigens that were recognized by anti-Histoplasma antibodies.Abbreviations CHHA Cystine-heart-hemoglobin agar - PBS phosphate buffered saline - Ig immunoglobulin - TBS Tris buffered saline - DAB 3,3-diaminobenzidine tetrachloride - FITC fluorescein isothiocyanate - M199 tissue culture medium 199, according to Morgan et al. (1950)