Identification of nuclear proteins that specifically interact with adeno-associated virus type 2 inverted terminal repeat hairpin DNA.

A palindromic hairpin duplex containing the inverted terminal repeat sequence of adeno-associated virus type 2 (AAV) DNA was used as a substrate in gel retardation assays to detect putative proteins that specifically interact with the AAV hairpin DNA structures. Nuclear proteins were detected in extracts prepared from human KB cells coinfected with AAV and adenovirus type 2 that interacted with the hairpin duplex but not in nuclear extracts prepared from uninfected, AAV-infected, or adenovirus type 2-infected KB cells. The binding was specific for the hairpin duplex, since no binding occurred with a double-stranded DNA duplex with the identical nucleotide sequence. Furthermore, in competition experiments, the binding could be reduced with increasing concentrations of the hairpin duplex but not with the double-stranded duplex DNA with the identical nucleotide sequence. S1 nuclease assays revealed that the binding was sensitive to digestion with the enzyme, whereas the protein-bound hairpin duplex was resistant to digestion with S1 nuclease. The nucleotide sequence involved in the protein binding was localized within the inverted terminal repeat of the AAV genome by methylation interference assays. These nuclear proteins may be likely candidates for the pivotal enzyme nickase required for replication or resolution (or both) of single-stranded palindromic hairpin termini of the AAV genome.     

A new family of dispersed repeats from Brassica nigra: characterization and localization

The 459-bp HindIII (pBN-4) and the 1732-bp Eco RI (pBNE8) fragments from the Brassica nigra genome were cloned and shown to be members of a dispersed repeat family. Of the three major diploid Brassica species, the repeat pBN-4 was found to be highly specific for the B. nigra genome. The family also hybridized to Sinapis arvensis showing that B. nigra had a closer relationship with the S. arvensis genome than with B. oleracea or B. campestris. The clone pBNE8 showed homology to a number of tRNA species indicating that this family of repeats may have originated from a tRNA sequence. The species-specific 459-bp repeat pBN-4 was localized on the B. nigra chromosomes using monosomic addition lines. In addition to the localization of pBN-4, the chromosomal distribution of two other species-specific repeats, pBN34 and pBNBH35 (reported earlier), was studied. The dispersed repeats pBN-4 and pBNBH35 were found to be present on all of the chromosomes, whereas the tandem repeat pBN34 was localized on two chromosomes.     

PCR-based DNA markers linked to a gall midge resistance gene, Gm4t, has potential for marker-aided selection in rice

Rice DNAs from a gall midge resistant variety, Abhaya, a susceptible variety, Tulsi and their F₃ progeny were screened using 500 random primers in conjunction with bulked-segregant analysis in a polymerase chain reaction (PCR) with a view to detecting random amplified polymorphic DNAs (RAPDs) linked to the gene, Gm4t, which confers resistance to gall midge, a dipteran insect pest of rice. A total of 454 primers were able to produce a distinct amplification pattern, and 3695 bands/loci were amplified between the phenotypically different parents. Of these, 304 bands were polymorphic between the parents, with 19 being phenotypespecific. One of these primers, E20, amplified 2 bands, E20₅₇₀ and E20₅₈₃, which are tightly linked to resistance and susceptibility, respectively. These specific bands were cloned and sequenced, and a 94% sequence homology was found between the two fragments. Two specific 20-mer oligonucleotides were synthesized, based on the sequence information of E20₅₈₃, for use in PCR amplification directly from genomic DNAs. These PCR primers were able to amplify phenotype-specific bands, a 583-bp fragment in susceptible F₃ lines and a 570-bp fragment in resistant F₃ lines that had been derived from a cross between the parents, indicating their potential and utility for marker-aided selection of the Gm4t gene in rice. Its use would facilitate the early and efficient selection of resistant genes in plant breeding programmes and even in those areas where the insect is not known to occur. These phenotype-specific bands are single-copy sequences and are being mapped to ascertain their chromosomal location in rice.     

Chemical and biological removal of cyanides from aqueous and soil-containing systems

A study was conducted to determine the effect of various factors on the rate and extent of potassium cyanide and potassium hexacyanoferrate (II, complex form) removal from aqueous and soil-containing systems. In a sterile aqueous system at neutral pH, the concentration of free cyanide was reduced by 42% in 334 h as a result of the protonation of CN^– and the volatilization of the HCN formed. In the presence of aerobic mixed consortium of the Institute of Gas Technology and a methylotrophic culture, Isolate 3, the concentration of free cyanide was reduced by 59% and 66% in 357 h, respectively, as a result of combined chemical conversion and microbial degradation. In the sterile aqueous system amended initially with the complex form of cyanide, a less-than-20% reduction in cyanide occured. The sorption equilibria for free and complex cyanides in slurries of the topsoil and manufactured gas plant (MGP) soil was reached in less than 22 and 4 days, respectively. The extent of desorption of cyanides from topsoil and MGP soil into water decreased with time. In sterile systems containing topsoil and MGP soil that were previously equilibrated to cyanides, only a 2% reduction in cyanide concentration occurred in 336 h due to chemical conversion. In the presence of microbial cultures, the concentration of cyanide was reduced by less than 15% and 7% in the slurries of topsoil and MGP soil, respectively. The comparison of the rate and extent of cyanide removal from the aqueous and soil-containing systems in the presence of micro-organisms suggests that cyanides were retained by the solid phase of the soil-containing systems and therefore were less available for biodegradation.     

Utilization of cuticular components ofAntheraea mylitta Drury larvae byPenicillium citrinum Thom.

The major cuticular components of Indian tasar silkworm,Antheraea mylitta Drury, were sequentially extracted and estimated to ascertain preferential utilization of these components for growth by the entomopathogenic fungusPenicillium citrinum Thom. Proteins which constituted 61.64% dry weight of cuticule were found to play a key role in the growth ofP. citrinum whereas lipids (7.15%) and chitin (30.02%) were least involved. Also, this study suggests absence of any mycocidal substance in the cuticle ofA. mylitta.     

Weaving a tight social net: Allogrooming in free-ranging female langurs (Presbytis entellus)

We studied grooming among adults of a one-male multifemale troop of free-ranging Hanuman langurs (Presbytis entellus)living near Jodhpur, India, for 9 years. The 11–13 females devoted about 6% of their day to allogrooming. Adult males, whose tenures averaged 2.2 years, were transient figures in the troop's history, as reflected by their rather peripheral role in the grooming network. Females groomed males 4–40 times more frequently (1006 episodes) than vice versa- (176 episodes). Adult females received 97% of all grooming from other adult females (6655 episodes). Although females exhibited an age- inversed dominance hierarchy, they did not compete for grooming access to particular troop mates. Dyads of all possible rank differences occurred as frequently as expected: 51% of grooming was directed up the hierarchy and 49% down it. Young, high- ranking individuals gave and received significantly more grooming than the oldest, low- ranking females did. The pattern seemed to be influenced by kin selection because of the presumably high degree of female relatedness. They invested most in troopmates with the highest reproductive value, i.e., the youngest individuals. This trend was coupled with a preference of closest kin (mothers and daughters). Reciprocity was the outstanding feature since all adult females groomed and were groomed by all others. Such a tight social net might establish the necessary cohesion during frequent territorial disputes with neighboring troops.     

Removal of endotoxin from antibody preparations for clinical use

Cell Biochemistry and Biophysics - Despite attempts to maintain asepsis, good manufacturing practices, and the use of terminal sterilization by millipore filtration, the nuclear practitioner is...     

Pentosan polysulfate,a potent anti HIV and anti tumor agent,inhibits protein serine/threonine and tyrosine kinases

The effect of nicotinamide-adenine dinucleotides (NAD⁺ and NADP⁺) on Ca²⁺ transport in rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺ uptake was dependent on adenosine triphosphate (ATP; 2mM). The presence of NAD⁺ (2mM) or NADP⁺ (1 and 2mM) caused a significant inhibition of Ca²⁺ uptake following addition of 2mM ATP. Ca²⁺, which accumulated in the nuclei during 6 min after ATP addition, was significantly released by the addition of NAD⁺ (0.5–2mM) or NADP⁺ (0.1–2mM). However, the effect of NADH (2mM) or NADPH (2mM) on Ca²⁺ uptake and release clearly weakened in comparison with the effects of NAD⁺ and NADP⁺. Meanwhile, ryanodine (10M), thapsigargin (10M) or oxalate (0.5mM) had no effect on Ca²⁺ uptake and release in rat liver nuclei. These reagents did not significantly alter the effects of 2mM NAD⁺ on Ca²⁺ uptake and release. Thus, NAD⁺ and NADP⁺ had a potent effect on Ca²⁺ transport in rat liver nuclei. The present findings suggest that the liver cytosolic NAD⁺ (NADP⁺) is a factor in the regulation of the nuclear Ca²⁺ concentration. (Mol Cell Biochem121: 127–133, 1993)     

Facile technique of protein precipitation by application of electric current

Summary Precipitation of proteins has been achieved following passage of direct electric current in various protein solutions. Application of as low as 3 V of electric current showed precipitation but the rate increased with increase in electric current. With 9 V there was more than 85% precipitation of protein within 15 min. Precipitation occurred at a wide range of pH and temperature. Electrophoretic analysis of precipitated proteins show that they are not denatured by application of electric current. Proteins thus precipitated can be easily recovered by centrifugation.     

Properties of thermostable hemicellulolytic enzymes from Thermomonospora strain 29 grown in solid state fermentation on coffee processing solid waste

During decaffeination of Coffee Processing Plant Solid Wastes (CPSW) by actinomycetes, Thermomonospora, Strain 29 exhibited high titers of cellulase and xylanase. This organism, originally isolated on soybean seed coat was grown in solid state fermentation on CPSW supplemented with mineral salts. Enzymes recovered were arabinosidase, xylanase, and beta-D-xylosidase. Higher activity of the former two enzymes was in the extracellular broth, whereas the beta-D-xylosidase activity was highest in the cell fraction. The enzymes were characterized after precipitation with (NH(4))(2)SO(4), dialysis, and gel filtration. Production of all three enzymes was inhibited by monomeric sugars and sugar alcohols but not by arabinoxylan, xylans, or xylan containing water insoluble carbohydrates. The optimum pH for the activity was 6.5, 7.0, and 7.5 for beta-xylosidase, xylanase and arabinosidase (alpha-L-arabinofuranosidase, alpha-arabinosidase, alpha-L-arabinosidase) respectively. These enzymes were stable in the pH range of 6.5 to 8.0. All three enzymes were thermostable up to 80 degrees C. At 55 degrees C, arabinosidase had the longest half life of 120 h. However, at 40 degrees C, xylanase had the longest half life (504 h). At either temperature, beta-D-xylosidase had the shortest half life. The molecular weights (kDa), and Kms (mM) were estimated to be 95, 0.27; 45, 12.4; and 106, 0.67 for arbinosidase, xylanase, and beta-xylosidase respectively. Step wise addition of the three enzymes showed higher saccharification of lignocellulosics.