Studies on lactic dehydrogenase and sorbitol dehydrogenase release in relation to deep freezing of buffalo semen in certain extenders

Forty seminal ejaculates from five mature buffalo bulls (n=8 from each bull), exhibiting more than 70% initial sperm motility, were frozen in the following three extenders: egg-yolk sodium citrate glycerol (EYCG); tris-egg yolk glycerol (TYG); and citric acid whey glycerol (CAWG). The extenders were evaluated for the release of intracellular enzymes, lactic dehydrogenase (LDH) and sorbitol dehydrogenase (SODH) from the spermatozoa during freezing (in fresh semen, after dilution and after equilibration) and post freezing (24 h and 7 d after freezing. It was found that the release of LDH and SODH enzymes was significantly lower in TYG than in EYCG and CAWG extenders. The most critical stage, at which the enzyme release was maximal, was between equilibration and 24 h post freezing in all three extenders.     

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.     

Physiological and enzymatic characterization of a novel pullulan-degrading thermophilic Bacillus strain 3183

Summary A new thermophilic Bacillus strain 3183 (ATCC 49341) was isolated from hot-spring sediments. The organism grew on pullulan as a carbon source and showed optimum pH and temperature at pH 5.5 and 62° C, respectively, for growth. The strain reduced nitrate to nitrite both aerobically and anaerobically. It produced extracellular thermostable pullulanase and saccharidase activities which degraded pullulan and starch into maltotriose, maltose, and glucose. Medium growth conditions for pullulanase production were optimized. The optimum pH and temperature for pullulanase activity were at pH 6.0 and 75° C, respectively. The enzyme was stable at pH 5.5-7.0 and temperature up to 70° C in the absence of substrate. The K _m for pullulan at pH 6.0 and 75° C was 0.4 mg/ml. The pullulanase activity was stimulated and stabilized by Ca²⁺. It was inhibited by ethylenediaminetetraacetate (EDTA), beta and gamma-cyclodextrins but not by alpha-cyclodextrin and reagents that inhibit essential enzyme SH-groups. Offprint requests to: B. C. Saha     

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.     

Inhibition of rabbit sperm acrosomal enzymes by gossypol

The effect of gossypol on the activities of 10 acrosomal enzymes of the rabbit sperm was evaluated. Acrosin, Azocoll proteinase, neuraminidase, and arylsulfatase were significantly inhibited or completely inactivated by 12–76 μM gossypol. Hyaluronidase, β-glucuronidase, and acid phosphatase were inhibited only at a higher concentration of gossypol (380 μM). Phospholipase C, alkaline phosphatase, and β-N-Acetyl glucosaminidase were not inhibited even at 380 μM gossypol. Gossypol was found to be a noncompetitive inhibitor of arylsulfatase with a Ki of 120 μM. The inhibition was reversible and dose-dependent. As the acrosomal enzymes were more sensitive to the inhibition by gossypol compared to sperm enzymes involved in glycolysis or energy production, these assays may serve as a more reliable indicator for monitoring the occurence of gossypol-induced sterility. © 1995 Wiley-Liss, Inc.     

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.     

Quantitative analysis of the effect of selection history on sugar yield adaptation of sugarcane clones

An objective of the CSR sugarcane breeding programme in Australia was to assess the scope for broadening the genetic base of the commercial sugarcane germ plasm through interspecific hybridization with Saccharum spontaneum clones. The contribution of both selection history and S. spontaneum to sugar yield and its components was investigated in the germ plasm pool assembled. The analysis was conducted on a data-set of 256 clones, consisting of parents and full-sib families generated from 32 biparental crosses, tested in six environments. The minimum number of generations back to S. spontaneum ancestor in the clone's pedigree was used as a germ plasm score. The geographical origin and selection history of each parent and their use in the biparental crosses were used to develop a selection history score for parents and offspring. The variation for seven attributes, cane yield, commercial cane sugar %, sugar yield, stalk number per stool, stalk weight, fibre % and ash % juice was partitioned according to the germ plasm and selection history scores. Significant (P<0.05) clone variation and clone x environment interaction for all attributes was present. The germ plasm scores accounted for a significant (P<0.05) component of the clone variation for all of the attributes except cane yield. There was an increase in sugar yield with an increase in the minimum number of generations back to a S. spontaneum clone. The selection history groups accounted for a high proportion of the variation among parental clones for all of the attributes except cane yield. This suggested that parents were the outcome of strong selection pressure for the commercial cane attributes. However, the selection history groups for the offspring produced by random mating of parents did not account for a high proportion of the variation for the attributes. Using the mixture method of classification we partitioned the 256 clones into five groups for patterns of performance for the seven attributes across the six environments. The five groups emphasized major differences in the patterns of performance for the seven attributes across environments. The distribution of germ plasm and selection history scores in each of the five groups indicated that their patterns of performance were associated with selection history and minimum generations to S. spontaneum. Therefore, both the analysis on selection history and germ plasm scores (extrinsic classification) and the analysis on the mixture method of classification (intrinsic classification) emphasized the influence of selection history on the sugar yield of sugarcane.