Taxol maintains organized microtubule patterns in protoplasts which lead to the resynthesis of organized cell wall microfibrils

Summary We have investigated the effects of microtubule stabilizing conditions upon microtubule patterns in protoplasts and developed a new method for producing protoplasts which have non-random cortical microtubule arrays. Segments of elongating pea epicotyl tissue were treated with the microtubule stabilizing drug taxol for 1 h before enzymatic digestion of the cell walls in the presence of the drug. Anti-tubulin immunofluorescence showed that 40 M taxol preserved regions of ordered microtubules. The microtubules in these regions were arranged in parallel arrays, although the arrays did not always show the transverse orientation seen in the intact tissue. Protoplasts prepared without taxol had microtubules which were random in distribution. Addition of taxol to protoplasts with random microtubule arrangements did not result in organized microtubule arrays. Taxol-treated protoplasts were used to determine whether or not organized microtubule arrays would affect the organization of cell wall microfibrils as new walls were regenerated. We found that protoplasts from taxol-treated tissue which were allowed to regenerate cell walls produced organized arrays of microfibrils whose patterns matched those of the underlying microtubules. Protoplasts from untreated tissue synthesized microfibrils which were disordered. The synthesis of organized microfibrils by protoplasts with ordered microtubules arrays shows that microtubule arrangements in protoplasts influence the arrangement of newly synthesized microfibrils.Abbreviations DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - IgG immunoglobulin G - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - PBS phosphate buffered saline     

Beacon interacts with cdc2/cdc28-like kinases

Previously we found elevated beacon gene expression in the hypothalamus of obese Psammomys obesus. Beacon administration into the lateral ventricle of P. obesus stimulated food intake and body weight gain. In the current study we used yeast two-hybrid technology to screen for proteins in the human brain that interact with beacon. CLK4, an isoform of cdc2/cdc28-like kinase family of proteins, was identified as a strong interacting partner for beacon. Using active recombinant proteins and a surface plasmon resonance based detection technique, we demonstrated that the three members of this subfamily of kinases (CLK1, 2, and 4) all interact with beacon. Based on the known sequence and functional properties of beacon and CLKs, we speculate that beacon could either modulate the function of key regulatory molecules such as PTP1B or control the expression patterns of specific genes involved in the central regulation of energy metabolism.     

Homology Modeling of Wild-type,D516V,and H526L Mycobacterium Tuberculosis RNA Polymerase and Their Molecular Docking Study with Inhibitors

Abstract

Rifamicyns (Rifs) are antibiotic widely used for the treatment of tuberculosis (TB); nevertheless, their efficacy has been limited by a high percentage of mutations, principally in the rpoB gene. In this work, the first three-dimensional molecular model of the hypothetical structures for the wild-type and D516V and H526L mutants of Mycobacterium tuberculosis (mtRNAP) were elucidated by a homology modeling method. In addition, the orientations and binding affinities of some Rifs with those new structures were investigated. Our findings could be helpful for the design of new more potent rifamycin analogs.     

Degeneracy and repertoire of the human HIV-1 Gag p17(77-85) CTL response

CD8+ CTL responses are important for the control of HIV-1 infection. The immunodominant HLA-A2-restricted Gag epitope, SLYNTVATL (SL9), is considered to be a poor immunogen because reactivity to it is rare in acute infection despite its paradoxical dominance in patients with chronic infection. We have previously reported SL9 to be a help-independent epitope in that it primes highly activated CTLs ex vivo from CD8+ T cells of seronegative healthy donors. These CTLs produce sufficient cytokines for extended autocrine proliferation but are sensitive to activation-induced cell death, which may cause them to be eliminated by a proinflammatory cytokine storm. Here we identified an agonist variant of the SL9 peptide, p41 (SLYNTVAAL), by screening a large synthetic combinatorial nonapeptide library with ex vivo-primed SL9-specific T cells. p41 invariably immunized SL9-cross-reactive CTLs from other donors ex vivo and H-2Db beta2m double knockout mice expressing a chimeric HLA-A*0201/H2-Db MHC class I molecule. Parallel human T cell cultures showed p41-specific CTLs to be less fastidious than SL9-CTLs in the level of costimulation required from APCs and the need for exogenous IL-2 to proliferate (help dependent). TCR sequencing revealed that the same clonotype can develop into either help-independent or help-dependent CTLs depending on the peptide used to activate the precursor CD8+ T cells. Although Ag-experienced SL9-T cells from two patients were also sensitive to IL-2-mediated cell death upon restimulation in vitro, the loss of SL9 T cells was minimized with p41. This study suggests that agonist sequences can replace aberrantly immunogenic native epitopes for the rational design of vaccines targeting HIV-1.     

Boro-norleucine as a P1 residue for the design of selective and potent DPP7 inhibitors

Dipeptide-based inhibitors with C-substituted (alkyl or aminoalkyl) alpha-amino acids in the P2 position and boro-norleucine (boro-Nle) in the P1 position were synthesized. Relative to boro-proline, boro-Nle as a P1 residue was shown able to significantly dial out DPP4, FAP, DPP8, and DPP9 activity. Dab-boro-Nle (4g) proved to be the most selective and potent DPP7 inhibitor with a DPP7 IC50 value of 480 pM.     

Detection and identification of microorganisms in wine: a review of molecular techniques

The microbial ecology of wine is complex. Microbes can play both positive and negative roles in the quality of the final product. Due to this impact, the microbial ecology of wine has been well studied. Traditional indirect methods, such as plating, have largely been replaced by a number of molecular methods. These methods are typically either indirect methods used for identification of cultured organisms, or direct methods used to profile whole populations or identify specific microbes in a mixed population. These molecular methods offer a number of advantages over traditional methods including speed and precision. This review will examine both direct and indirect molecular methods, provide examples of their impact on the study of the microbial ecology of wine, and also discuss their strengths and limitations.     

Short-term effects of a summer wildfire on a desert grassland arthropod community in New Mexico

Surface-active arthropods were sampled after a lightning-caused wildfire in desert grassland habitat on the Sevilleta National Wildlife Refuge, Socorro County, NM. Pitfall traps (n = 32 per treatment) were used to evaluate species-specific "activity-density" indices after the June wildfire in both burned and unburned areas. In total, 5,302 individuals were collected from 69 taxa. Herbivore activity-densities generally decreased, whereas predators often increased in the burned area; pitfall trap bias likely contributed to this latter observation. Fire caused the virtual extirpation of scaly crickets (Mogoplistidae), field crickets (Gryllidae), and camel crickets (Raphidophoridae), but recolonization began during the first postfire growing season. Several grasshoppers (Acrididae) also exhibited significant postfire declines [Ageneotettix deorum (Scudder), Eritettix simplex (Scudder), Melanoplus bowditchi Scudder, and Amphitornus coloradus (Thomas)]. Some beetles showed lower activity-density, including Pasimachus obsoletus LeConte (Carabidae) and Eleodes extricatus (Say) (Tenebrionidae). Taxa exhibiting significant postfire increases in activity-density included acridid grasshoppers (Aulocara femoratum (Scudder), Hesperotettix viridis (Thomas), Trimerotropis pallidipennis (Burmeis.), and Xanthippus corallipes Haldeman); carabid beetles (Amblycheila picolominii Reiche, Cicindela punctulata Olivier), tenebrionid beetles (Eleodes longicollis LeConte, Edrotes rotundus (Say), Glyptasida sordida (LeConte), Stenomorpha consors (Casey); the centipedes Taiyubius harrietae Chamberlin (Lithobiidae) and Scolopendra polymorpha Wood (Scolopendridae); scorpions (Vaejovis spp.; Vaejovidae); and sun spiders (Eremobates spp.; Eremobatidae). Native sand roaches (Arenivaga erratica Rehn, Eremoblata subdiaphana (Scudder); Polyphagidae) displayed no significant fire response. Overall, arthropod responses to fire in this desert grassland (with comparatively low and patchy fuel loads) were comparable to those in mesic grasslands with much higher and more continuous fuel loads.     

Evidence Against the Involvement of Ionically Bound Cell Wall Proteins in Pea Epicotyl Growth

Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.