Nef enhances human immunodeficiency virus type 1 infectivity in the absence of matrix

Nef enhances the serine phosphorylation of the human immunodeficiency virus type 1 matrix (MA) protein, which suggests that MA may be a functional target of Nef. Using mutants that remain infectious despite the absence of most or all of MA, we show in the present study that the ability of Nef to enhance virus infectivity is not compromised even if MA is entirely replaced by a heterologous lipid anchor.     

Partial NMR assignments for uniformly (13C, 15N)-enriched BPTI in the solid state

We demonstrate that high-resolution multidimensional solid state NMR methods can be used to correlate many backbone and side chain chemical shifts for hydrated micro-crystalline U-¹³C,¹⁵N Basic Pancreatic Trypsin Inhibitor (BPTI), using a field strength of 800 MHz for protons, magic angle sample spinning rates of 20 kHz and proton decoupling field strengths of 140 kHz. Results from two homonuclear transfer methods, radio frequency driven dipolar recoupling and spin diffusion, were compared. Typical ¹³C peak line widths are 0.5 ppm, resulting in C-C and C-CO regions that exhibit many resolved peaks. Two-dimensional carbon–carbon correlation spectra of BPTI have sufficient resolution to identify and correlate many of the spin systems associated with the amino acids. As a result, we have been able to assign a large number of the spin systems in this protein. The agreement between shifts measured in the solid state and those in solution is typically very good, although some shifts near the ion binding sites differ by at least 1.5 ppm. These studies were conducted with approximately 0.2 to 0.4 mol of enriched material; the sensitivity of this method is apparently adequate for other biological systems as well.     

Premicellar complexes of sphingomyelinase mediate enzyme exchange for the stationary phase turnover

During the steady state reaction progress in the scooting mode with highly processive turnover, Bacillus cereus sphingomyelinase (SMase) remains tightly bound to sphingomyelin (SM) vesicles (Yu et al., Biochim. Biophys. Acta 1583, 121-131, 2002). In this paper, we analyze the kinetics of SMase-catalyzed hydrolysis of SM dispersed in diheptanoylphosphatidyl-choline (DC7PC) micelles. Results show that the resulting decrease in the turnover processivity induces the stationary phase in the reaction progress. The exchange of the bound enzyme (E*) between the vesicle during such reaction progress is mediated via the premicellar complexes (E(i)#) of SMase with DC7PC. Biophysical studies indicate that in E(i)# monodisperse DC7PC is bound to the interface binding surface (i-face) of SMase that is also involved in its binding to micelles or vesicles. In the presence of magnesium, required for the catalytic turnover, three different complexes of SMase with monodisperse DC7PC (E(i)# with i=1, 2, 3) are sequentially formed with Hill coefficients of 3, 4 and 8, respectively. As a result, during the stationary phase reaction progress, the initial rate is linear for an extended period and all the substrate in the reaction mixture is hydrolyzed at the end of the reaction progress. At low mole fraction (X) of total added SM, exchange is rapid and the processive turnover is limited by the steps of the interfacial turnover cycle without becoming microscopically limited by local substrate depletion or enzyme exchange. At high X, less DC7PC will be monodisperse, E(i)# does not form and the turnover becomes limited by slow enzyme exchange. Transferred NOESY enhancement results show that monomeric DC7PC in solution is in a rapid exchange with that bound to E(i)# at a rate comparable to that in micelles. Significance of the exchange and equilibrium properties of the E(i)# complexes for the interpretation of the stationary phase reaction progress is discussed.     

LIM kinase 1 coordinates microtubule stability and actin polymerization in human endothelial cells

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

Study on interaction between duodenase protease with dual specificity and inhibitors of bowman-birk family

The interaction between duodenase and inhibitors of Bowman-Birk type from soybeans (BBI) and lima beans (LBI) was investigated. Duodenase was shown to interact only with antichymotrypsin site of these inhibitors. The inhibition constants of duodenase by BBI, LBI, BBI-trypsin and LBI trypsin complexes were 4, 23, 400, 600 (n)M respectively.     

Nongastric H-K-ATPase in rodent prostate: lobe-specific expression and apical localization

The molecular basis of active iontransport in secretory glands such as the prostate is not wellcharacterized. Rat nongastric H-K-ATPase is expressed at highlevels in distal colon surface cell apical membranes and thus isreferred to as "colonic." Here we show that the ATPase is expressedin rodent prostate complex in a lobe-specific manner. RT-PCR andWestern blot analyses indicate that rat nongastric H-K-ATPase-subunit (_ng) mRNA and protein are present incoagulating gland (anterior prostate) and lateral and dorsalprostate and absent from ventral lobe, whereas Na-K-ATPase -subunit is present in all lobes. RT-PCR analysis shows that Na-K-ATPase ₄ and ₃ and gastricH-K-ATPase -subunit are not present in significant amounts in allprostate lobes. Relatively low levels of Na-K-ATPase ₂were found in lateral, dorsal, and anterior lobes. _ngprotein expression is anteriodorsolateral: highest in coagulatinggland, somewhat lower in dorsal lobe, and even lower in lateral lobe.Na-K-ATPase protein abundance has the reverse order: expression inventral lobe is higher than in coagulating gland. _ngprotein abundance is higher in coagulating gland than distal colonmembranes. Immunohistochemistry shows that in rat and mouse coagulatinggland epithelium _ng protein has an apical polarizationand Na-K-ATPase ₁ is localized in basolateral membranes.The presence of nongastric H-K-ATPase in rodent prostate apicalmembranes may indicate its involvement in potassium concentrationregulation in secretions of these glands.

     

Revision of the Athyrisininae, Siluro-devonian Brachiopods from China and Russia

Athyrisinine brachiopods from the Upper Silurian of Russia and the Lower–Middle Devonian of China and north Vietnam include over 70 species belonging to Athyrisina and Parathyrisina ; generic and subfamilial diagnoses are emended. Five genera are considered synonyms of Athyrisina and three of Parathyrisina . A neotype is selected and illustrated for Athyrisina squamosa , the type species of Athyrisina . Four nomina nova , Athyrisina xui , Parathyrisina wani , P. minima , and P. cheni , are suggested as new substitute names for Athyrisina tumida Wang, in Xu et al. 1978 (primary homonymy), Athyrisinoides ganxiensis Wan, in Xu et al. 1978, Parathyrisina minor Zhang, in Zhang and Fu 1983, and Athyrisinoides tudilingensis Chen, 1979 (secondary homonyms) respectively. A new genus, Bruntosina , is described from the Emsian to Eifelian of the Qinling region. A new subfamily, Homeathyridinae, is erected within the Athyrididae with Homeathyris , Pseudohomeospira , and Squamathyris included, revised and their diagnoses emended. Homeathyris incisus sp. nov. is described from the Ludfordian of Novaya Zemlya. Ikella is revised and excluded from the athyrisinins. The origin and dispersal of the homeathyridins and the athyrisinins are discussed.     

Historic DNA reveals genetic consequences of fragmentation in an endangered,endemic mustard

Understanding how anthropogenic disturbance affects genetic diversity is essential to appropriately incorporating genetic considerations into conservation plans. Unfortunately, we rarely have information about a population’s genetic diversity before it becomes imperiled. Here we reconstruct the historic range of the naturally rare annual mustard Streptanthus glandulosus subsp. niger (Sgn) and use herbarium specimens to quantify pre-disturbance genetic diversity. We compare this to the genetic diversity in the contemporary plant populations and to plants in the seed bank. We conclude that Sgn was recently a single, panmictic population composed of orders of magnitude more plants than exist today but experienced recent and abrupt declines following housing development. Today Sgn persists as two disjunct populations, the larger of which has retained historic levels of diversity although there is a downward trend in all measures. The smaller population has lost 21–28% of the diversity that was present only 50 years ago with an N_e?~?5–16. The contemporary populations have differentiated from each other due to drift. The seed bank contained no novel alleles and had high levels of homozygosity, indicating that it is incapable of providing genetic rescue. This novel combination of hDNA, the aboveground plant population and the seed bank can be used to design high impact conservation plans that appropriately incorporate genetic diversity for this and other imperiled species.