Pre-mRNA splicing in yeast

Splicing of introns from nuclear precursor messenger RNAs (pre-mRNAs) occurs in all eukaryotes. Two aspects of the splicing mechanism need to be understood: how intron sequences are recognized and aligned and how splicing is catalysed. Recent genetic and biochemical studies in the simple eukaryote Saccharomyces cerevisiae are revealing some of the features of the splicing mechanism.     

Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase.

The avermectin (Av) polyketide synthase (PKS) and erythromycin (Er) PKS are encoded by modular repeats of DNA, but the genetic organization of the modules encoding Av PKS is more complex than Er PKS. Sequencing of several related DNA fragments from Streptomyces avermitilis that are part of the Av biosynthetic gene cluster, revealed that they encode parts of large multifunctional PKS proteins. The Av PKS proteins show strong similarity to each other, as well as similarity to Er PKS proteins [Donadio et al., Science 252 (1991) 675-679] and fatty acid synthases. Partial DNA sequencing of the 65-kb region containing all the related sequence elements in the avr genes provides evidence for twelve modular repeats encoding FAS-like domains. The genes encoding the Av PKS are organized as two sets of six modular repeats which are convergently transcribed.     

Prey-derived signals regulating duration of the developmental growth phase of Bdellovibrio bacteriovorus.

The filamentous elongation typical of growth-phase cells of the predatory bacterium Bdellovibrio bacteriovorus is mediated by regulatory signals that are derived from the prey cell itself. These signals regulate the differentiation of growth-phase cells into the attack phase and appear to be required for continued filamentous growth by prey-dependent wild-type bdellovibrios and their prey-independent mutant derivatives alike. Using a prey-independent bdellovibrio strain, we have developed an assay for the detection and quantification of the growth-extending signal activity present in extracts of prey cells. This prey-derived regulatory activity was shown to be independent of its nutritional contribution to the bdellovibrios and was found to occur in heat-stable, proteinlike compounds of a variety of native molecular weights within the soluble fraction of extracts from both gram-negative and gram-positive bacteria.     

Complexes of DNA bases and Watson–Crick base pairs interaction with neutral silver Agn (n = 8, 10, 12) clusters: a DFT and TDDFT study

We study the binding of the neutral Ag_n (n = 8, 10, 12) to the DNA base-adenine (A), guanine (G) and Watson–Crick –adenine-thymine, guanine-cytosine pairs. Geometries of complexes were optimized at the DFT level using the hybrid B3LYP functional. LANL2DZ effective core potential was used for silver and 6–31 + G^** was used for all other atoms. NBO charges were analyzed using the Natural population analysis. The absorption properties of Ag_n–A,G/WC complexes were also studied using time-dependent density functional theory. The absorption spectra for these complexes show wavelength in the visible region. It was revealed that silver clusters interact more strongly with WC pairs than with isolated DNA complexes. Furthermore, it was found that the electronic charge transferred from silver to isolated DNA clusters are less than the electronic charge transferred from silver to the Ag_n–WC complexes. The vertical ionization potential, vertical electron affinity, hardness, and electrophilicity index of Ag_n–DNA/WC complexes have also been discussed.     

High-throughput screening of antibody variants for chemical stability: identification of deamidation-resistant mutants

Developability assessment of therapeutic antibody candidates assists drug discovery by enabling early identification of undesirable instabilities. Rapid chemical stability screening of antibody variants can accelerate the identification of potential solutions. We describe here the development of a high-throughput assay to characterize asparagine deamidation. We applied the assay to identify a mutation that unexpectedly stabilizes a critical asparagine. Ninety antibody variants were incubated under thermal stress in order to induce deamidation and screened for both affinity and total binding capacity. Surprisingly, a mutation five residues downstream from the unstable asparagine greatly reduced deamidation. Detailed assessment by LC-MS analysis confirmed the predicted improvement. This work describes both a high-throughput method for antibody stability screening during the early stages of antibody discovery and highlights the value of broad searches of antibody sequence space.     

Intervención domiciliaria por terapia ocupacional postalta para prevenir la rehospitalización de personas mayores: protocolo de estudio de un ensayo clínico aleatorizado

Background and objective

Re-admission to hospital by the elderly is a frequent event that is associated with complications. The aim of this article is to describe a randomised clinical trial protocol which has the aim of describing and comparing the impact of a home-based intervention by Occupational Therapists (OT) in the likelihood of re-admission at 6 months.

Modern pollen deposition in wetlands of Majuli Island and its implication to decipher palaeoflood episodes in northeast India

The present study is the first attempt from any Indian wetland, where palynological data is assessed to understand the flooding histories of the Sakali and Duboi wetlands in the Majuli River Island, a part of the Indo-Burma hotspot. Palynological analyses of surface soil samples from these wetlands were carried out in order to explore the flooding histories of this island. The presence of extra-local taxa such as Rhododendron, Tsuga and Castanopsis, in the Sakali wetland is strongly suggestive of river water transportation and flooding on the Majuli Island in the recent past. In contrast, dominance of local arboreal taxa and low abundance or absence of extra-local pollen taxa in the Duboi wetland does not indicate flooding. Correspondence analysis (CA) was applied on the present dataset to reveal possible correlations between the surface samples of the wetlands and the major group of palynotaxa. The CA successfully discriminated the samples from the two wetlands and also marked strong correlations between flood indicators extra-local taxa with the samples of the Sakali wetland and local arboreal elements with the Duboi samples. The present result will not only provide a baseline for future palaeovegetation and palaeoclimate reconstructions, but will also help in deciphering palaeoflood episodes from these wetlands.     

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in ‘sub G0/G1’ phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.     

N-butyl Methacrylate and Paraffin as an Embedding Medium for Light Microscopy

A method of tissue embedding using n-butyl methacrylate and paraffin is described. Following alcohol dehydration and infiltration with the methacrylate monomer, tissues are embedded in gelatin capsules in a mixture consisting of 3.5 g of paraffin for each 10 ml of methacrylate. Benzoyl peroxide (0.2 g for each 10 ml of monomer) is added as the catalyst and the methacrylate polymerized in a 50 C oven for 18-24 h. Following polymerization the block is trimmed and embedded in paraffin to provide a firm support during sectioning. A water trough attached to the microtome knife is essential to facilitate the handling of sections and ribbons. For serial sections a mixture of equal weights of beeswax and paraffin is used to make the sections adhere to each other. Usual staining procedures can be used since the embedding medium is readily soluble in xylene.     

Film Reviews

Book reviewed in this article:
Daughters of the Nile . 1982. 46 minutes, color. A film by Joop van Wijk and Hillie Molenaar .
Hidden Faces: A Look at the Lives of Egyptian Women . 1990. 52 minutes, color. A film by Claire Hunt and Kim Longinotto .
Marriage Egyptian Style . 1991. 50 minutes, color. A film by Joanna Head .
Permissible Dreams . 1983. 32 minutes, color. A film by Atiat El Abnoudi .