A DNA fragment fromStreptomyces fradiae increases the production of a metalloprotease inStreptomyces lividans

Streptomyces fradiae produces several extracellular proteases and many of these are inducible. An 8.8 kb DNA fragment of Streptomyces fradiae cloned on pIJ699 caused increased protease activity in Streptomyces lividans.Clones carrying this recombinant plasmid showed a significant delay in sporulation. A protein of 18 kDa was purified from the extracellular proteins secreted by the host carrying the recombinant plasmid. Further characterization showed that this protease is a metalloprotease.     

Micropropagation of selected somaclones ofBegonia andSaintpaulia

Begonia x elatior plantlets which regenerated from leaf disk callus showed variations in plant morphology, number of flowers per plant, and flower size. Variations in flowering period, number of flowers per plant, and flower morphology were observed in Saintpaulia ionantha L. plants directly regenerated from leaf disk explants. The cytokinins, benzylaminopurine and zeatin, tested in the culture medium did not affect the basic plant characteristics including flower colour which remained stable in both species. Micropropagation of selected somaclones having the desirable trait of high number of flowers per plant was stable in the MV2 and MV3 generations.     

Adherence ofMycobacterium leprae to the nasal mucosa is influenced by surface integrity and viability

The intranasal route is one of the main routes of Mycobacterium leprae infection and there is paucity of information regarding the mode of spread of the pattern. The adherence of M.leprae to the nasal mucosa, its trapping within the sinuses of the head, and its fate after entry into the host was studied using mouse model. A comparison of the adherence profile of M. leprae and Mycobacterium tuberculosis showed that while larger numbers of M. tuberculosis were demonstrated within lungs, greater numbers of M.leprae were present within the sinuses of the head. Adherence of M.leprae to the nasal mucosa was dependent on surface integrity since opsonization and heat killing resulted in decreased numbers of M.leprae in the nasal sinuses and a greater amount entering the lungs. The adherence appeared to the independent of the viability of the bacilli, as similar numbers of formalin-fixed, rifampicin-treated and viable M.leprae entered the lungs in the initial stages. However the numbers of rifampicin-treated M. leprae in the nasal sinuses were 12-fold lower than the numbers of viable M.leprae. These results indicated that both viability and surface integrity were important in the entry of M.leprae and it’s consequent dissemination.     

Cloning of acryIIIA endotoxin gene ofBacillus thuringiensis var.tenebrionis and its transient expression inindica rice

Damage caused to rice production by coleopteran insects like rice weevil (Sitophilus oryzae), a stored grain insect pest and rice hispa (Dicladispa armigera), a pest of the growing plant is quite high. In order to combat the damage, generation of insect resistant transgenic rice plant was considered desirable. CryIIIA endotoxin ofBacillus thuringiensis var.tenebrionis, a 65 kDa protein toxic to coleopteran insects, figured as the candidate gene product. Thus, the cryIIIA gene was isolated from a local isolate ofBacillus thuringiensis var.tenebrionis. The gene was tailored at the N-terminal end to its minimal size by using a synthetic ATG codon which replaced the first codon next to ATG of threonine to proline. This modification did not affect the functional property of the gene product. A chimeric construct of the modifiedcryIIIA gene was developed containing CaMV35S promoter andnos terminator for plant expression. The expressibility of thecryIIIA gene inindica rice was judged through test for transient expression in indica rice protoplasts.     

Follicular growth and kinetics in the Indian gerbil (Tatera indica) ovary during oestrous cycle

The growth of the follicle and oocyte in the Indian gerbil (Tatera indica) was a continuous process. The relationship between follicle and oocyte or its nucleus was log linear, represented by the equation log Y =a +b logX.A linear relationship (Y =a +bX)existed between the oocyte and its nucleus. The number of stages I and II follicles varied significantly during the oestrous cycle. Maximum percentage of stage I follicles was observed during oestrus and metoestrus, while stage II follicles were abundant during dioestrus, metoestrus and pro-oestrus. These follicles were significantly more in number than other types of the follicles. The occurrence of comparatively larger follicles during pro-oestrus and the presence of newly formed corpora lutea at oestrus, indicated ovulation in the early oestrus.     

Mammotrope heterogeneity in the pituitary gland of European ferret,Mustela putorius furo: An immunoelectron-microscopic study

Employing the superimposition technique of electron-microscopic immunocytochemistry ultrastructural heterogeneity of the mammotropes in the pituitary gland of the European ferret,Mustela putorius furo,was studied. On the basis of the size of their secretory granules, the mammotropes were classified into three subtypes, type-I, type-II and type-Ill, which may correspond to different developmental or physiological states of a single cell type. Simultaneous study of mammotropes and somatotropes in several pairs of serial semithin sections demonstrated the occasional occurrence of bihormonal somatomammotropes /mammosomatotropes which may represent a transitional stage of the progenitor stem-somatotrope during its differentiation into mammotrope; alternatively it may be a functional intermediate during the cross-transformation of somatotrope into mammotrope or vice versa.     

Efforts in diagnosing early leprosy using serological techniques

Skin scrapings obtained from the lesions of leprosy patients of all types showed 96 % positivity to the serum antibody competition test using monoclonal antibody (ML04)to 35 kDa antigen of Mycobacterium leprae. Further, in vitro culture of full thickness skin biopsies from lepromatous patients were noted to release IgG antibodies toM. leprae with a peak antibody response at 48 h. The significance of this local antibody response toM. leprae in skin has been discussed for its possible use in diagnosing early leprosy.     

Photoinhibition of photosynthesis without net loss of photosystem II components inPopulus deltoides

The photoinhibition of photosynthesis was investigated on intact attached leaves and isolated thylakoid membranes of Populus deltoides.Our studies demonstrate that in intact leaves photoinhibition takes place under high irradiance which is more pronounced at higher temperatures. No net loss of Dl and other proteins associated with photosystem II (PSII) were observed even after 64 % photoinhibition suggesting that the degradation of polypeptides associated with PSII is not the only key step responsible for photoinhibition as observed by other workers. Electron transport studies in isolated thylakoid membranes suggested water oxidation complex as one of the damaged site during high light exposure. The possible mechanisms of photoinhibition without net loss of D1 protein are discussed.     

Biochemical characterization of Silene alba α4-fucosyltransferase and Lewis a products

1,4-Fucosylation has been recently detected in Arabidopsis thaliana [Léonard et al. (2002), Glycobiology 12: 299–306], and corresponding enzymes have also been characterized in Beta vulgaris [Bakker et al. (2001), FEBS Lett, 507: 307–312], and Lycopersicum aesculentum [Wilson (2001), Glycoconjugate J., 18: 439–447]. Here we demonstrated fucosyltransferase activity (FucT) in Silene alba cells and tissues. The Fuc linkage to GlcNAc residues of the lactosamine moiety of the Type I acceptor was confirmed by mass spectrometry experiments. Le^a-glycoconjugates are found in the Golgi apparatus and plasma membrane of plant cells. In planta, the highest levels of activity were detected in seedlings, young roots and male flowers. The enzyme was stable up to 45C and the optimum pH of reaction was 8.0. The enzyme required Mg²⁺ or Mn²⁺ for activity and was inhibited by Zn²⁺ and ethylenediaminetetraacetic acid. Chemical modification of the enzyme with group-selective reagents revealed that selective modifications of arginine and lysine residues had no effect on enzyme activity. However the enzyme contains histidine and tryptophan residues that are essential for its activity. In contrast to human FUT3, the S. alba 4-FucT was insensitive to N-ethylmaleimide (NEM) treatment. Measurement of enzyme activity in S. alba cell fractions indicated that the enzyme is bound to microsomal membranes, furthermore a soluble isoform of the protein may be present. Published in 2005.     

Plant regeneration from protoplasts ofCapsicum annuum L. cv. California Wonder

Plant regeneration from mesophyll protoplasts of pepper,Capsicum annuum L. cv. California Wonder has been demonstrated via shoot organogenesis. Protoplasts isolated from fully expanded leaves of 3-week-old axenic shoots when cultured in TM medium supplemented with 1 mg l ⁻¹ NAA, 1 mg l ⁻¹ 2,4-D, 0 5 mg l ⁻¹ BAP (CM 1) resulted in divisions with a frequency ranging from 20–25 %. Antioxidant ascorbic acid and polyvinylpyrrolidone (PVP) in the medium and incubation in the dark helped overcome browning of protoplasts. Microcalli and macrocalli were formed in TM medium containing 2 mg l ⁻¹ NAA and 0.5 mg l ⁻¹ BAP (CM II) and MS gelled medium containing 2 mg 1 ⁻¹ NAA and 0 5 mg 1 ⁻¹ BAP (CM III), respectively. Regeneration of plantlets was possible via caulogenesis. Microshoots, 2–5 percallus appeared on MS gelled medium enriched with 0.5 mg l ⁻¹ IAA, 2mg l ⁻¹ GA and l0mg l ⁻¹ BAP (CM IVc). Rooting of microshoots was obtained on half strength gelled medium containing 1 mg l ⁻¹ NAA and 0.5mg l ⁻¹ BAP. Protoplasts isolated from cotyledons failed to divide and degenerated eventually.     

Genome analysis of amaranths: Determination of inter- and intra-species variations

Amaranths are an important group of plants and include grain, vegetable and ornamental types. Despite the economic importance of the amaranths, there is very little information available about the extent and nature of genetic diversity present in the genus Amaranthus at molecular level. We now report the randomly amplified polymorphic DNA (RAPD) profiles of different species of Amaranthus as well as different accessions of the species. These RAPD analyses have been carried out using 65 arbitrary sequence decamer primers. From the RAPD data, an UPGMA dendrogram illustrating the inter-as well as intra-species relationships has been computed. The putative hybrid origin of A.dubious from A. hybridus and A. spinosus is also ruled out by the RAPD data. The trends of species relationships amongst the amaranths determined by RAPDs is consistent with their cytogenetic and evolutionary relationships that have already been determined. NBRI Communication No:464 (N.S.).     

Purification and characterization of two major lectins fromVigna mungo (blackgram)

Blackgram (Vigna mungo L. Hepper)seeds contain two galactose-specific lectins, BGL-I and BGL-II. BGL-I was partially purified into two monomeric lectins which were designated as BGL-I-1 (94 kDa) and BGL-I-2 (89 kDa). BGL-II is a monomeric lectin of 83 kDA. The purified lectins were associated with galactosidase activities. BGL-I-1 and BGL-II were copurified with α-galactosidase activity while BGL-I-2 was largely associated with β-galactosidase activity. These lectins agglutinate trypsin treated rabbit erythrocytes, but not the human erythrocytes of A, B or O groups. They were stable between pH 3·5 and 7·5 for their agglutination. The lectins did not show any metalion requirement. They were inactivated at 50°C. The lectin activity was inhibited by D-galactose (0·1 mM). The Scatchard plots of galactose binding to these lectins are nonlinear and biphasic curves indicative of multiple binding sites. The data show that the monomeric lectins have both lectin and galactosidase activities suggestive of a bifunctional protein.     

The BglG group of antiterminators: a growing family of bacterial regulators

The product of the bglG gene of Escherichia coli was among the first bacterial antiterminators to be identified and characterized. Since the elucidation ten years ago of its role in the regulation of the bgl operon of E. coli,a large number of homologies have been discovered in both Gram-positive and Gram-negative bacteria. Often the homologues of BglG in other organisms are also involved in regulating β-glucoside utilization. Surprisingly, in many cases, they mediate antitermination to regulate a variety of other catabolic functions. Because of the high degree of conservation of the cis-acting regulatory elements, antiterminators from one organism can function in another. Generally the antiterminator protein itself is negatively regulated by phosphorylation by a component of the phosphotransferase system. This family of proteins thus represents a highly evolved regulatory system that is conserved across evolutionarily distant genuses.     

Within- and among-population variation in oviposition preference for urea-supplemented food inDrosophila melanogaster

Oviposition preference for ureasupplemented food was assayed by simultaneous choice trials on five pairs of closely related laboratory populations of Drosophila melanogaster.Each pair of populations had been derived from a separate ancestral population about 85 generations prior to this study. One population in each pair had been subjected to selection for larval tolerance to the toxic effects of urea; the other population served as a control. Considerable variation in oviposition preference was seen both within and among populations, with four of the ten populations showing a significant mean preference for ureasupplemented food. The degree of specificity shown by individual females was surprisingly high, leading to a bimodal distribution of oviposition preference in some populations. Overall, selection for larval tolerance to urea did not significantly affect oviposition preference. However, the data indicated that pairwise comparisons between randomly selected populations from the two larval selection regimes would lead to a range of possible outcomes, suggesting, in several cases, that selection for larval urea tolerance had led to significant differentiation of adult oviposition preference for urea in one or the other direction. The results, therefore, highlight the importance of population level replication and caution against the practice, common in ecological studies, of assaying oviposition preference in two populations that utilize different hosts in nature, and then drawing broad evolutionary inferences from the results.     

Evolutionary conservation of angiosperm flower development at the molecular and genetic levels

Flowers consist primarily of four basic organ types whose relative positions are universally conserved within the angiosperms. A model has been proposed to explain how a small number of regulatory genes, acting alone and in combination, specify floral organ identity. This model, known widely as the ABC model of flower development, is based on molecular generic experiments in two model organisms,Arabidopsis thaliana and Antirrhinum majus.Both of these species are considered to be eudicots, a clade within the angiosperms with a relatively conserved floral architecture. In this review, the application of the ABC model derived from studies of these typical eudicot species is considered with respect to angiosperms whose floral structure deviates from that of the eudicots. It is concluded that the model is universally applicable to the angiosperms as a whole, and the enormous diversity seen among angiosperms flowers is due to genetic pathways that are downstream, or independent, of the genetic programme that specifies floral organ identity.     

Substances interfering with phosphatidyl inositol signalling pathway affect ultradian rhythm ofDesmodium motorium

The ultradian rhythm of the lateral leaflets ofDesmodium motorium}(Houtt.) Merril. was recorded with a picture analysis method using a video camera and a computer. The periods are in the minute range and depend strongly on temperature. The phosphatidyl inositol signal chain might be involved in the ultradian rhythm of the lateral leaflet movement of Desmodium motorium:Myoinositol shortens the period length and reduces the known period lengthening effect of lithium ions. Neomycin, which inhibits the hydrolysis of phosphatidylinositol-4,5 -biphosphate to inositol-4-phosphate and diacylglycerin, lengthens the period of the rhythm at low concentrations (0.2 mM). Higher concentrations shorten the period, perhaps by activating G protein. Mastoparan, which activates G protein, shortens period likewise. The G protein agonists fluorid ion and ethanol are toxic for the lateral leaflets and could therefore not be used to test the involvement of G protein. The intracellular Ca ²⁺ antagonist 3,4,5-trinietlioxybeiizoic acid 8-(diethylamino)octylester lengthens the period of the rhythm. This indicates, that release of Cas ²⁺ from intracellular stores is important for the lateral leaflet movement rhythm.     

Determination and diagnostic value of CA9 mRNA in peripheral blood of patients with oral leukoplakia

Background: Oral leukoplakia is one of the most common oral premalignant disorder. The classical evaluation through tissue biopsy is not always valid to evaluate the risk of malignization.

Material and methods: RT-qPCR was performed on 47 blood samples (21 patients with leukoplakia, 2 with oral squamous cell carcinoma (OSCC), and 24 healthy patients) and on 11 tissue samples (3 leukoplakia, 4 OSCC, and 4 samples of healthy tissue).

Results: There are significant differences in expression between the different groups (F?=?4.057, p?=?.006). The Duncan post hoc test shows that the only group that differentiates is the tumour tissue. Using Wilcoxon test, different covariables of patients with leukoplakia were analysed with respect to the group of healthy patients and no significant differences were observed.

Conclusions: The diagnostic route through liquid biopsy has not been conclusive in this study, but there are significant differences in the levels analysed in the different tissue samples.     

hsp 83 mutation is a dominant enhancer of lethality associated with absence of the non-protein codinghsrω locus inDrosophila melanogaster

Thehrsω or the 93D heat shock locus ofDrosophila melanogaster, which does not code for any protein, has an important role in development since nullosomy of this locus in transheterozygotes for two overlapping deficiencies, viz.,Df(3R) e ^Gp4 (eGp4) andDf(3R)GC14 (GC14), is known to cause a high (∼ 80%) mortality with the small number of escapee nullosomic flies being sterile, weak and surviving for only a few days. We now show that a majority of thehsrω-nulosomics die as embryo and that the 20% escapee embryos develop slower compared to their sibs carrying either one or two copies of thehsrω locus but after hatching survive to pupal/imago stage. Most interestingly, we further show that when onehsp83 mutant allele (hsp83 ^e4A) is introduced ineGp4/GC14 trans-heterozygotes, practically none of thehsrω-nullosomic embryos develop beyond the 1st instar larval stage. The specificity of this interaction betweenhsp83 andhsrω genes was further confirmed by examining the effect of thehsp83 mutant allele on other mutations in the 93D cytogenetic region. Therefore, we conclude that thehsp83 mutation acts as a dominant enhancer of the lethality associated with nullosomy for thehsrω gene. The observed genetic interaction between these two members of the heat shock gene family during normal embryonic development ofDrosophila reveals novel aspects of their biological functions.     

Immunoprophylactic studies on cell wall associated proteins ofMycobacterium tuberculosis H37Ra

The cell wall protein peptidoglycan complex (CW-PPC) of Mycobacterium tuberculosis H₃₇Ra was isolated through sequential extraction of lipids, carbohydrates and soluble proteins. CW-PPC emulsified in FIA was found to induce significant protection in mice against challenge with LD₅₀ dose ofM. tuberculosis H₃₇Rv. To identify the immunoprotective components of CW-PPC, the proteins in avid association with peptidogican were dissociated by chemical treatment with trifluoromethanesulthonic acid (CF₃CO₃H): anisole (2:1). Immunoreactivity of total (CW-Pr) as well as its component proteins i.e., 71, 60 and 45 kDa proteins of cell wall was studied in animals immunized with CW-Pr-FIA. The 71 kDa protein was found to be most immunoreactive giving higher T-cell sensitization and humoral responses. Further, immunization of mice with 71 kDa-FIA demonstrated enhanced T- and B- cell responses. Mice immunized with 71 kDa-FIA gave significantly higher protection (P ≤ 0.05) against intravenous challenge with LD₅₀ dose ofM. tuberculosis H₃₇Rv, than BCG immunized animals. The results indicate the potential of 71 kDa cell wall protein as a suitable candidate for Cthe subunit vaccine.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.