A role for Rev in the association of HIV-1 gag mRNA with cytoskeletal β-actin and viral protein expression

Human immunodeficiency virus type-1 (HIV-1) Rev acts by inducing the specific nucleocytoplasmic transport of a class of incompletely spliced RNAs that encodes the viral structural proteins. The transfection of HeLA cells with a rev-defective HIV-1 expression plasmid, however, resulted in the export of overexpressed, intron-containing species of viral RNAs, possibly through a default process of nuclear retention. Thus, this system enabled us to directly compare Rev⁺ and Rev⁻ cells as to the usage of RRE-containing mRNAs by the cellular translational machinery. Biochemical examination of the transfected cells revealed that although significant levels of gag and env mRNAs were detected in both the presence and absence of Rev, efficient production of viral proteins was strictly dependent on the presence of Rev. A fluoroscence in situ hybridisation assay confirmed these findings and provided further evidence that even in the presence of Rev, not all of the viral mRNA was equally translated. At the early phase of RNA export in Rev⁺ cells, gag mRNA was observed throughout both the cytoplasm and nucleoplasm as uniform fine stippling. In addition, the mRNA formed clusters mainly in the perinuclear region, which were not observed in Rev⁻ cells. In the presence of Rev, expression of the gag protein was limited to these perinuclear sites where the mRNA accumulated. Subsequent staining of the cytoskeletal proteins demonstrated that in Rev⁺ cells gag mRNA is colocalized with β-actin in the sites where the RNA formed clusters. In the absence of Rev, in contrast, the gag mRNA failed to associate with the cytoskeletal proteins. These results suggest that in addition to promoting the emergence of intron-containing RNA from the nucleus, Rev plays an important role in the compartmentation of translation by directing RRE-containing mRNAs to the β-actin to form the perinuclear clusters at which the synthesis of viral structural proteins begins.     

Die Entwicklung der Zellforschung

Obwohl erst im 17. Jahrhundert begonnen, hat die Zellforschung doch bald zur Formulierung allgemein-biologischer Theorien geführt und trägt auch heute ständig weiter zu fundamentalen Theorienbildungen bei. Die Entwicklung der Zellforschung verdient daher als Paradigma für die Gewinnung naturwissenschaftlicher Einsichten besonderes Interesse: An ihr können Grundbedingungen wissenschaftlichen Erkenntnisfortschrittes gut verdeutlicht werden als Wechselspiel von Methodenerweiterung und Konzeptualisierung des Neuentdeckten. Erst eineinhalb Jahrhunderte nach der Erstbeobachtung von Zellen gelang die Formulierung der “Zellentheorie”. Auf der durch sie geschaffenen, konzeptionellen Grundlage war es vielen biologischen und medizinischen Disziplinen möglich, von einem beobachtend-beschreibenden (“induktiven”) zu einem experimentierenderklärenden (“deduktiven”) Status vorzustoßen. Ein außergewöhnlicher Erfolg war dabei der Cytogenetik beschieden. Dagegen war der Versuch, in den subzellulären und submikroskopischen Bereich vorzudringen, nur teilweise erfolgreich (Polarisationsmikroskopie, Nägelis Micellartheorie). Ein breiter Durchbruch gelang in diesem Sektor erst um 1950 durch eine beinahe einmalige zeitliche Kumulierung methodischer Fortschritte, wobei vor allem Zellfraktionierung und Elektronenmikroskopie eine entscheidende Rolle spielten. Zusammen mit der vorausgegangenen Entwicklung “flankierender” Wissenschaften (insbesondere der Makromolekularen Chemie) wurde so die Etablierung der modernen Zellbiologie möglich. Einige ihrer bedeutendsten Aussagen werden diskutiert (Zellkompartimentierung und genetische Kontinuität ohne Nucleinsäuren; Zweiteilung der Organismenwelt nach der Zellorganisation; Evolution zellulärer Organismen und Symbiontentheorie). Abschließend wird versucht, Bedingungen und Erfordernisse von wissenschaftlichem Fortschritt allgemein zu formulieren und daran die heutige Wissenschaftswirklichkeit kritisch zu messen.     

Receptor-associated independent cAMP nanodomains mediate spatiotemporal specificity of GPCR signaling

1. Download : Download high-res image (164KB)
2. Download : Download full-size image

     

Micrometric segregation of fluorescent membrane lipids: relevance for endogenous lipids and biogenesis in erythrocytes

Micrometric membrane lipid segregation is controversial. We addressed this issue in attached erythrocytes and found that fluorescent boron dipyrromethene (BODIPY) analogs of glycosphingolipids (GSLs) [glucosylceramide (BODIPY-GlcCer) and monosialotetrahexosylganglioside (GM1BODIPY)], sphingomyelin (BODIPY-SM), and phosphatidylcholine (BODIPY-PC inserted into the plasma membrane spontaneously gathered into distinct submicrometric domains. GM1BODIPY domains colocalized with endogenous GM1 labeled by cholera toxin. All BODIPY-lipid domains disappeared upon erythrocyte stretching, indicating control by membrane tension. Minor cholesterol depletion suppressed BODIPY-SM and BODIPY-PC but preserved BODIPY-GlcCer domains. Each type of domain exchanged constituents but assumed fixed positions, suggesting self-clustering and anchorage to spectrin. Domains showed differential association with 4.1R versus ankyrin complexes upon antibody patching. BODIPY-lipid domains also responded differentially to uncoupling at 4.1R complexes [protein kinase C (PKC) activation] and ankyrin complexes (in spherocytosis, a membrane fragility disease). These data point to micrometric compartmentation of polar BODIPY-lipids modulated by membrane tension, cholesterol, and differential association to the two nonredundant membrane:spectrin anchorage complexes. Micrometric compartmentation might play a role in erythrocyte membrane deformability and fragility.     

An evaluation of the evidence for, and implications of, cytoplasmic nitrate homeostasis

A review of literature, reporting values of cytoplasmic/cytosolic [NO₃^–] in plant cells, identified two major areas of disagreement: (1) disparity in the absolute values within the same system, and (2) constancy versus variability in cytoplasmic/cytosolic [NO₃^–] with varying [NO₃^–]_o. These differences are related to the techniques used by the different authors. Estimates of cytoplasmic [NO₃^–] by compartmental analysis and by cell fractionation were consistently higher than the estimates by NO₃^–selective microelectrodes and by techniques based upon in vivo and in vitro nitrate reductase activity (NRA). A model recognizing more than one cytoplasmic ionic pool would satisfactorily reconcile the differences in both aspects, i.e. absolute values and constancy. Compartmental analysis and cell fractionation techniques may measure the amount of NO₃^– in the cytoplasm as a whole (including organelles); by contrast, NO₃^– selective microelectrodes and NRA estimate only the cytosolic NO₃^– and, hence, may result in lower estimates. Thus, variable organellar pool(s) may maintain a constant cytosolic pool as estimated by microelectrodes. However, certain observations remain at odds with the notion of a constant cytosolic [NO₃^–].     

Exaggerated root respiration accounts for growth retardation in a starchless mutant of Arabidopsis thaliana

The knock‐out mutation of plastidial phosphoglucomutase (pgm) causes a starchless phenotype in Arabidopsis thaliana, and results in a severe growth reduction of plants cultivated under diurnal conditions. It has been speculated that high soluble sugar levels accumulating during the light phase in leaf mesophyll might cause a reduction of photosynthetic activity or that shortage of reduced carbon during the night is the reason for the slow biomass gain of pgm. Separate simultaneous measurements of leaf net photosynthesis and root respiration demonstrate that photosynthetic activity per unit fresh weight is not reduced in pgm, whereas root respiration is strongly elevated. Comparison with a mutant defective in the dominating vacuolar invertase (AtβFruct4) revealed that high sucrose concentration in the cytosol, but not in the vacuole, of leaf cells is responsible for elevated assimilate transport to the root. Increased sugar supply to the root, as observed in pgm mutants, forces substantial respiratory losses. Because root respiration accounts for 80% of total plant respiration under long‐day conditions, this gives rise to retarded biomass formation. In contrast, reduced vacuolar invertase activity leads to reduced net photosynthesis in the shoot and lowered root respiration, and affords an increased root/shoot ratio. The results demonstrate that roots have very limited capacity for carbon storage but exert rigid control of supply for their maintenance metabolism.     

莳萝蒿适应盐渍环境的Na~+区域化方式和生理特征

莳萝蒿是广泛分布在我国北方的一种特殊类型的菊科盐生植物,阐明莳萝蒿特殊的耐盐机制和生理特征有助于丰富植物抗盐性研究的内容。用0、100、200、300、400 mmol/L Na Cl处理莳萝蒿7 d后,比较莳萝蒿盐处理植株与对照植株在生长和生理方面的差异,并详细分析了Na+在莳萝蒿体内的积累水平和区域化方式。结果显示:莳萝蒿虽然能够耐受400 mmol/L Na Cl,但盐处理显著抑制了莳萝蒿的生长,整株鲜重随着盐处理浓度的升高逐渐减小。在水分生理方面,随着盐处理浓度的升高,莳萝蒿叶片细胞的渗透调节能力逐渐增强,其叶片肉质化程度却呈逐渐降低的趋势。分析盐处理对光合作用的影响发现,盐处理后莳萝蒿叶片光合速率与气孔导度显著下降,而其PSⅡ光化学活性并未受到抑制,叶绿素含量甚至逐渐增大,说明盐处理后莳萝蒿叶片光合速率的降低主要是由于气孔因素造成的,而不是由于光合结构被破坏。莳萝蒿体内的Na+含量随着盐处理浓度的升高显著增加,400 mmol/L Na Cl条件下叶、茎、根中的Na+含量分别高达321.4、242.1和182.3μmol/g鲜重;莳萝蒿体内的Na+70%以上积累在叶片内,而叶片内98%左右的Na+积累在叶片原生质体中,叶片原生质体中的Na+平均浓度是质外体1.2—1.8倍,推测其叶片细胞内存在着有效的Na+区域化机制。盐处理后莳萝蒿叶片液泡膜V-H+-ATPase的质子泵活性比对照增加了30%—50%,液泡膜Na+/H+逆向转运活性则增加至对照的4—7倍,进一步证实莳萝蒿叶片具有较强的液泡Na+区域化能力。随着盐处理浓度的升高,Na+在叶片中的分布比例相对减少,V-H+-ATPase的质子泵活性和Na+/H+逆向转运活性增幅也减缓。这种Na+区域化能力使莳萝蒿获得了较强的耐盐性,有效保护了其光系统,降低了细胞汁液渗透势。但是盐处理后这种耐盐方式并不能阻止莳萝蒿叶片肉质化程度和光合活性下降,莳萝蒿生长仍然受盐抑制,说明Na+区域化是莳萝蒿适应盐渍环境的必要条件而非充分条件。     

The utilization of tyrosine for mescaline and protein biosynthesis in Lophophora williamsii

Studies on the comparative utilization of tyrosine for protein and alkaloid biosynthesis indicate that this amino acid is incorporated into peyote alkaloids at three times the rate at which it is incorporated into protein. In addition, the biosynthetic pathway for tyrosine formation appears to be compartmented into two channels; one supplying the needs for alkaloid biosynthesis and the other providing tyrosine for protein biosynthesis. The latter compartment is possibly under a negative feedback control mechanism.