Two-dimensional NMR spectroscopy of connected transitions by linear combination of flip angle dependent cosy: An alternative scheme for E.COSY

An alternative pulse scheme which simplifies and improves the recently proposed P.E.COSY experiment is suggested for the retention of connected or unconnected transitions in a coupled spin system. An important feature of the proposed pulse scheme is the improved phase characteristics of the diagonal peaks. A comparison of various experiments designed for this purpose, namely COSY-45, E.COSY, P.E.COSY and the present scheme (A.E.COSY), is also presented. The suppression of unconnected transitions and the measurement of scalar coupling constants and their relative signs are illustrated from A.E.COSY spectra of 2,3-dibromopropionic acid and 2-(2-thienyl)pyridine.

Reinstate hydrogen peroxide as the product of alternative oxidase of plant mitochondria

Chill treatment of potato tubers for 8 days induced mitochondrial O-2 consumption by cyanide-insensitive alternative oxidase (AOX). About half of the total O-2 consumption in such mitochondria was found to be sensitive to salicylhydroxamate (SHAM), a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive O-2 consumption by nearly half, and addition at the end of the reaction released half of the O-2 consumed by AOX, both typical of catalase action on H2O2. This reaffirmed that the product of reduction of O-2 by plant AOX was H2O2 as found earlier and not H2O as reported in some recent reviews.

Post-transcriptional Repair of a Split Heat Shock Protein 90 Gene by mRNA trans-Splicing

Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 ( glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the ``intron'' regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing.

Saturated liquid viscosity correlations for alternative refrigerants

This article presents dimensionless equations for the temperature dependence of the saturated liquid viscosity of R32, R123, R124, R125, R134a, R141b, and R152a valid over a temperature range of engineering interest. The correlation has the form Phi(D)(n)=A+BTD where Phi(D) is the dimensionless fluidity (1/eta(D)) and T-D is a dimensionless temperature. n, A, and B are evaluated for each of the above refrigerants based on a least-squares fit to experimental data. This equation is found to provide an improved fit over those existing in the literature up to T-D=0.8.

Two regiospecific alternative strategies in the synthesis of (-)-valeranone

Two regiospecific modifications have been developed in the synthesis of valeranone. The first one is based on the regiospecific protection of a diol and the second is based on the Wittig reaction of a hemiacetal.

Variable-speed wind power generation using doubly fed wound rotor induction machine - A comparison with alternative schemes

In this paper, a wind energy conversion system (WECS) using grid-connected wound rotor induction machine controlled from the rotor side is compared with both fixed speed and variable speed systems using cage rotor induction machine. The comparison is done on the basis of (I) major hardware components required, (II) operating region, and (III) energy output due to a defined wind function using the characteristics of a practical wind turbine. Although a fixed speed system is more simple and reliable, it severely limits the energy output of a wind turbine. In case of variable speed systems, comparison shows that using a wound rotor induction machine of similar rating can significantly enhance energy capture. This comes about due to the ability to operate with rated torque even at supersynchronous speeds; power is then generated out of the rotor as well as the stator. Moreover, with rotor side control, the voltage rating of the power devices and dc bus capacitor bank is reduced. The size of the line side inductor also decreasesd. Results are presented to show the substantial advantages of the doubly fed system.

Identification of a core promoter and a novel isoform of the human TSCI gene transcript and structural comparison with mouse homolog

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with loci on chromosome 9q34.12 (TSC1) and chromosome 16p13.3 (TSC2). Genes for both loci have been isolated and characterized. The promoters of both genes have not been characterized so far and little is known about the regulation of these genes. This study reports the characterization of the human TSC1 promoter region for the first time. We have identified a novel alternative isoform in the 5' untranslated region (UTR) of the TSC1 gene transcript involving exon 1. Alternative isoforms in the 5' UTR of the mouse Tsc1 gene transcript involving exon I and exon 2 have also been identified. We have identified three upstream open reading frames (uORFs) in the 5' UTR of the TSC1/Tsc1 gene. A comparative study of the 5' UTR of TSC1/Tsc1 gene has revealed that there is a high degree of similarity not only in the sequence but also in the splicing pattern of both human and mouse TSC1 genes. We have used PCR methodology to isolate approximately 1.6 kb genomic DNA 5' to the TSC1 cDNA. This sequence has directed a high level of expression of luciferase activity in both HeLa and HepG2 cells. Successive 5' and 3' deletion analysis has suggested that a -587 bp region, from position +77 to -510 from the transcription start site (TSS), contains the promoter activity. Interestingly, this region contains no consensus TATA box or CAAT box. However, a 521-bp fragment surrounding the TSS exhibits the characteristics of a CpG island which overlaps with the promoter region. The identification of the TSC1 promoter region will help in designing a suitable strategy to identify mutations in this region in patients who do not show any mutations in the coding regions. It will also help to study the regulation of the TSC1 gene and its role in tumorigenesis. (C) 2003 Elsevier B.V. All rights reserved.

The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function

In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160–455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the [beta]-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between [beta] strands in the WD repeats, we have identified four amino acids, 235TETG238, that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing.

Zircon Sand--a Viable Alternative Moulding System for Titanium Castings

The experimental observations of casting titanium in sodium silicate bonded zircon sand mould are presented in this paper. Metal-mould reactions, in general, involved dissolution of oxides in liquid titanium resulting in contamination of the casting. Minimal metal-mould reactions occurred when titanium was cast in zircon sand mould containing about 7.5 wt% of ZrO2. It has been further shown that the metal-mould reaction is considerably reduced if moulds were fired at high temperatures (> 1273K). This ensured elimination of moisture from the mould and also resulted in some beneficial changes in the mould chemistry. The reduction in metal-mould reaction is reflected in the decrease in oxygen and hydrogen contamination and decrease in hardness. Thus microhardness profile and oxygen analysis seems to provide a good index for evaluation of severity of metal-mould reaction. The method has been demonstrated to be satisfactory for casting titanium components.

Saccharomyces cerevisiae NineTeen Complex (NTC)-associated Factor Bud31/Ycr063w Assembles on Precatalytic Spliceosomes and Improves First and Second Step Pre-mRNA Splicing Efficiency

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a similar to 25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.