Purification and properties of xylanase from the thermophilic fungus, Humicola lanuginosa (Griffon and Maublanc) Bunce

An extracellular xylanase was purified to homogeneity from the culture filtrate of the thermophilic fungus, Humicola lanuginosa (Griffon and Maublanc) Bunce and its properties were studied. A fourfold purification and a yield of 8% were achieved. The molecular-weight of the protein was found to be 22,500 based on electrophoretic mobility and 29,000 by gel filtration behavior. The protein is rich in acidic amino acids, glycine and tyrosine, and poor in sulfur-containing amino acids. The kinetic properties of the enzyme are similar to those of other fungal xylanases. The enzyme shows high affinity toward larchwood xylan (Km = 0.91 mg/ml) and hydrolyzes only xylan. The enzyme becomes inactivated when stored for more than 2 months at −20 °C in the dry state. Such an inactivation has not been reported so far for any xylanase. Using chromatographic techniques, one species of protein differing from the native protein in charge but enzymatically active was isolated in low yields. However, a large molecular-weight species of the protein devoid of enzyme activity was isolated in substantial quantities and further characterized. Based on ultracentrifugation and gel electrophoretic studies, it was concluded that this species may be an aggregate of the native protein and that such an aggregation might be taking place on storage in the dry state at −20 °C, leading to loss in activity.

DNA Polymerase4 from the Silk Glands of Bombyx mori

The silk gland of Bombyx mori is a terminally differentiated tissue in which DNA replication continues without cell or nuclear division during larval development. DNA polymerase-delta activity increases in the posterior and middle silk glands during the development period, reaching maximal levels in the middle of the fifth instar larvae. The enzyme has been purified to homogeneity by a series of column chromatographic and affinity purification steps. It is a multimer comprising of three heterogeneous subunits, M(r) 170,000, 70,000, and 42,000. An auxiliary protein from B. mori silk glands, analogous to the proliferating cell nuclear antigen, enhances the processivity of the enzyme and stimulates catalytic activity by 3-fold. This auxiliary protein has also been purified to homogeneity. It is a dimer comprised of a single type M(r) 40,000 subunit. Polymerase-delta possesses an intrinsic 3' --> 5' exonuclease activity which participates in proofreading by mismatch match repair during DNA synthesis and is devoid of any primase activity. DNA polymerase-delta activity could be further distinguished from polymerase-alpha from the same tissue based on its sensitivity to various inhibitors and polyclonal antibodies to the individual enzymes. Like DNA polymerase-alpha, polymerase-delta is also tightly associated with the nuclear matrix. The polymerase alpha-primase complex could be readily separated from polymerase-delta (exonuclease) in the purification protocol adopted. DNA polymerase-delta from B. mori silk glands resembles the mammalian delta-polymerases. Considering that both DNA polymerase-delta and -alpha are present in nearly equal amounts in this highly replicative tissue and their close association with the nuclear matrix, the involvement of both the enzymes in the chromosomal endoreplication process in B. mori is strongly implicated.

Crystal and Molecular Structure of Sclerophytin F Methyl Ether from the Soft Coral Cladiella krempfi

new cembranoid diterpene was isolated from the soft coral Ckdiella h p f ifrom Minicoy Island (India), and its structure was established by X-ray crystallography to be sclerophytin F methyl ether (21 with the R absolute configuration at all six epimeric centers,assuming a configuration similar to that of sclerophytin C. Compound 2 may be an artifact of the isolation process.

DNA Polymerase-β from the pupal ovaries of bombyx mori

The silk glands of Bombyx mori, a highly replicative tissue contains high levels of DNA polymerases α, σ and epsilon (Porson) but not DNA polymerase-β. However, we detected the latter activity in the gonadal tissues, viz. the pupal ovaries and testes of B. mori. The enzyme has been purified to homogeneity from the pupal ovaries by a series of column chromatographic and affinity purification steps. The enzyme satisfied the criteria to be designated as DNA polymerase-β based on its small size, requirement for high concentration of monovalent cations for catalytic activity, sensitivity to ddTTP and insensitivity to aphidicolin. It is a monomeric polypeptide of Mr 40 kDa, and the Km for dNTPs ranges between 8–20 μM. DNA polymerase-β is biochemically and immunologically distinct from DNA polymerase-α from the silk glands of B. mori. The enzyme showed a preference for gapped DNA, and could not elongate ultraviolet irradiated template beyond the pyrimidine dimers. The absence of any associated primase and exonuclease activities from this enzyme, and its conspicuous absence in the highly replicative tissue, imply that it is unlikely to participate in the DNA endoreplication process.

Isolation and characterization of DNA polymerase epsilon from the silk glands of Bombyx mori

The silk gland of Bombyx mori, an endomitotically replicative tissue shows high levels of DNA polymerases alpha, delta, and epsilon activities. The ratio of polymerase alpha to that of delta plus epsilon is maintained at 1.1 to 1.3 in both the posterior and middle silk glands for the entire duration of late larval development. The three activities copurify in the initial stages of fractionation through phosphocellulose and DE52 but polymerase alpha gets resolved from the others on hydroxylapatite column. Separation between polymerase delta and epsilon is achieved by chromatography on QAE-Sephadex. DNA polymerase epsilon is a heterodimer comprising of 215- and 42-kDa subunits. The activity is maximum at pH 6.5 and the Km values for dNTPs vary between 3-9 microM. The enzyme possesses an intrinsically associated exonuclease activity which functions in the mismatch repair during DNA synthesis. Both polymerase and 3'-->5' exonuclease activities are associated with the 215-kDa subunit. By itself, DNA polymerase epsilon is processive and the catalytic activity is not enhanced by externally added bPCNA (Bombyx-proliferating cell nuclear antigen, an auxiliary protein for DNA polymerase delta). The enzyme resembles polymerase delta in having the exonuclease activity and in its response to aphidicolin or substrate analogs, but could be distinguished from the latter by its lack of response to the bPCNA and sensitivity to dimethyl sulfoxide. The two enzymes show partial immunological cross-reactivity with each other but no immunological relatedness to polymerase alpha. The absence of the repair enzyme DNA polymerase beta and the presence of substantial levels of polymerase epsilon in the silk glands suggest a possible role for the latter in DNA repair in that tissue.

Nucleotide sequence of the first cosmid from the Mycobacterium leprae genome project: structure and function of the Rif-Str regions

The nucleotide sequence of cosmid B1790, carrying the Rif-Str regions of the Mycobacterium leprae chromosome, has been determined. Twelve open reading frames were identified in the 36716bp sequence, representing 40% of the coding capacity. Five ribosomal proteins, two elongation factors and the β and β'subunits of RNA polymerase have been characterized and two novel genes were found. One of these encodes a member of the so-called ABC family of ATP-binding proteins while the other appears to encode an enzyme involved in repairing genomic lesions caused by free radicals. This finding may well be significant as M. leprae, an intracellular pathogen, lives within macrophages.

Nutritional evaluation of silage made from the toxic weed parthenium hysterophorus in animals

After ensilation, the toxic Compositae weed Parthenium hysterophorus was devoid of the toxic principle parthenin. Laboratory-scale ensilation indicated that no parthenin was detectable after 5 wk of anaerobic fermentation. For animal feeding studies, silage was made on a large scale from Parthenium mixed with maize or from Parthenium alone. Crossbred bull and buffalo bull calves were fed diets containing the silages, or control diet without silage, for 12 wk. The animals consumed both silages with relish, and body weight gains of silage-fed calves did not differ from those of the controls. The digestibilities of dry matter, fibre and nitrogen-free extract were greater with the control diet, but the biological value of proteins tended to be greater with the silage-containing diets. Haematological studies indicated no significant differences between experimental and control groups in selected parameters, except for a reduction in blood urea nitrogen in the animals fed silage. The possible causes for these biochemical alterations are discussed. Since the nutritive value of Parthenium silage compares favourably with the standard diet, and Parthenium seeds collected from the silage did not germinate, we suggest that ensilation can be used as an additional method in the containment and eradication of these plants, which grow wild in India.

Purification and characterization of an extracellular β-glucosidase from the thermophilic fungus Sporotrichum thermophile and its influence on cellulase activity

Multiple forms of beta-glucosidase (EC 3.2.1.21) of Sporotrichum thermophile were produced when the fungus was grown in a cellulose medium. One beta-glucosidase was purified 16-fold from 6-d-old culture filtrates by ion-exchange and gel-filtration chromatography. The purified enzyme was free of cellulase activity. It hydrolysed aryl beta-D-glucosides and beta-D-linked diglucosides. It was optimally active at pH 5.4, at 65-degrees-C. The apparent K(m) values for p-nitrophenyl beta-D-glucoside (PNPG) and cellobiose were 0.29 and 0.83 mm, respectively. Glucose, fucose, nojirimycin and gluconolactone inhibited beta-glucosidase competitively. At high (> 1 mm) substrate concentration, beta-glucosidase catalysed a parallel transglycosylation reaction. The transglycosylation product formed from cellobiose appeared to be a beta-linked tetramer of glucose. Admixtures of beta-glucosidase and cellulase components showed that the concept of cellobiose inhibition of cellulases was not valid for all components of the cellulase system of S. thermophile. Beta-Glucosidase supplementation also stimulated cellulose hydrolysis by cellulases when there was no accumulation of cellobiose in reaction mixture.

Reduction behaviour of Fe3+/Al2O3 obtained from the mixed oxalate precursor and the formation of the Fe0-Al2O3 metal-ceramic composite

Reduction behaviour of Fe3+/Al2O3 obtained by the decomposition of the oxalate precursor has been investigated by employing X-ray diffraction (XRD), Mössbauer spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. Calcination of Fe3+/Al2O3 at or below 1070 K yields mainly a poorly ordered, fine particulate form of ?-Al2�xFexO3. Calcination at or above 1220 K yields ?-Al2�xFexO3. Reduction of Fe3+/Al2O3 samples calcined at or below 1070 K gives the FeAl2O4 spinel on reduction at 870 K; samples calcined at or above 1220 K give Al2-xFexO3 with a very small proportion of metallic iron. Fe3+/Al2O3 samples calcined at 1220 K or above yield metallic iron and a very small proportion of the spinel on reduction below 1270 K. In the samples reduced at or above 1270 K, the main product is metallic iron in both ferromagnetic and superparamagnetic forms. The oxalate precursor route yields more metallic iron than the sol�gel route.

The 'neighbour effect' and its role in shaping the positional pattern of seed development in fruits: An illustration from the pods of Erythrina suberosa

Plants exhibit certain intra-fruit positional patterns in the development of seeds. These patterns have been generally interpreted to be a consequence of resource and fertilization gradients. However, such positional patterns might also be shaped by the 'neighbour effect', wherein formation and development of a seed at any position might positively or negatively influence those of other seeds in the neighbourhood. In this article, we examine the role of such neighbour effect in shaping the positional pattern of seeds in the pods of Erythrina suberosa. The results suggest the existence of a positive neighbour effect leading to a higher frequency of seeds in contiguous positions.

A survey of indian logic from the point of view of computer science

Indian logic has a long history. It somewhat covers the domains of two of the six schools (darsanas) of Indian philosophy, namely, Nyaya and Vaisesika. The generally accepted definition of Indian logic over the ages is the science which ascertains valid knowledge either by means of six senses or by means of the five members of the syllogism. In other words, perception and inference constitute the subject matter of logic. The science of logic evolved in India through three ages: the ancient, the medieval and the modern, spanning almost thirty centuries. Advances in Computer Science, in particular, in Artificial Intelligence have got researchers in these areas interested in the basic problems of language, logic and cognition in the past three decades. In the 1980s, Artificial Intelligence has evolved into knowledge-based and intelligent system design, and the knowledge base and inference engine have become standard subsystems of an intelligent system. One of the important issues in the design of such systems is knowledge acquisition from humans who are experts in a branch of learning (such as medicine or law) and transferring that knowledge to a computing system. The second important issue in such systems is the validation of the knowledge base of the system i.e. ensuring that the knowledge is complete and consistent. It is in this context that comparative study of Indian logic with recent theories of logic, language and knowledge engineering will help the computer scientist understand the deeper implications of the terms and concepts he is currently using and attempting to develop.

Characterization of a variant glucose-6-phosphate dehydrogenase from the south Indian population.

Glucose-6-phosphate dehydrogenase (G6PD) is coded by a gene on the X-chromosome. Earlier studies have shown that the South Indian population has a high incidence of this enzyme deficiency. The electrophoretic mobility, pH optimum and the K-m values for G6PD from normal and variant individuals were identical. However, the specific activity of the variant enzyme was 8 times less compared to the value of the normal enzyme. Western blot analysis of partially purified G6PD from normal and variant individuals performed using equal amounts of total protein showed that the variant protein was 3 times less in concentration. Similar analysis performed using protein corresponding to equal enzyme activity units in the normal and variant samples showed that the variant enzyme was 2.25 times less efficient compared to the normal enzyme. RNA dot blot analysis using full length G6PD cDNA probe (PGDT5B, a kind gift from Prof. L Luzzatto) revealed that lymphocytes from normal and variant individuals had equal amounts of G6PD specific mRNA.