Organization and bidirectional transcription of H2A, H2B and H4 histone genes in rice embryos

Our current understanding of the evolution of the histone gene family suffers from a lack of information on plant histone genes1. With a view to gathering some much needed information on these genes, we studied a rice genomic clone in pBR322 carrying H2A, H2B and H4 histone genes on a DNA fragment2 of 6.64 kilobases (kb). A restriction map of the insert was constructed and the organization of the three genes on this insert was determined. H2A and H2B histone genes were located at one end of the insert and H4 gene at the other with a 3.1 kb spacer in between. This cluster of three histone genes was found to be transcribed in a bidirectional fashion with H2A and H2B genes being encoded by one strand and the H4 gene by the other. These results indicate that plant histone gene organization differs from that of the sea urchin, but shows many similarities to the systems in other animals.

Tissue culture of forest trees: Clonal multiplication of Eucalyptus grandis L

Axillary shoot proliferation was obtained using explants of Eucalyptus grandis L. juvenile and mature stages on a defined medium. Murashige and Skoog medium (MS) supplemented with benzyladenine (BA), naphthalene acetic acid (NAA) and additional thiamine. Excised shoots were induced to root on a sequence of three media: (1) White's medium containing indoleacetic acid (IAA), NAA and indole butyric acid; (IBA), (2) half-strength MS medium with charcoal and (3) half-strength MS liquid medium. The two types of explants differed in rooting response, with juvenile-derived shoots giving 60% rooting and adult-derived ones only 35%. Thus, the factors limiting cloning of selected trees in vitro are determined to be those controlling rooting of shoots in E. grandis.

In vitro propagation of Eucalyptus grandis L. by tissue culture

Multiple shoots were induced from nodal segments of five year old trees of Eucalyptus grandis L. on solid medium containing Murashige and Skoog's (MS) Basal medium supplemented with additional thiamine, BAP and NAA. Rooting could be achieved from shoot culture on half strength MS salts or white's medium supplemented with low auxins like IAA, IBA and NAA.

Self-Organization of n-Alkane Chains in Water: Length Dependent Crossover from Helix and Toroid to Molten Globule

We demonstrate a chain length dependent crossover in the structural properties of linear hydrocarbon (n-alkane) chains using detailed atomistic simulations in explicit water. We identify a number of exotic structures of the polymer chain through energy minimization of representative snapshots collected from molecular dynamics trajectory. While the collapsed state is ring-like (circular) for small chains (CnH2n+2; n <= 20) and spherical for very long ones (n = 100), we find the emergence of ordered helical structures at intermediate lengths (n similar to 40). We find different types of disordered helices and toroid-like structures at n = 60. We also report a sharp transition in the stability of the collapsed state as a function of the chain length through relevant free energy calculations. While the collapsed state is only marginally metastable for C20H42, a clear bistable free energy surface emerges only when the chain is about 30 monomers long. For n = 30, the polymer exhibits an intermittent oscillation between the collapsed and the coil structures, characteristic of two stable states separated by a small barrier.

Culture Condition Dependent Changes Of 4-Thiouridine In The Transfer-Rna Of Azotobacter-Vinelandii

4..T~iouridine, a thionucleoside present in the transfer RNA of the free living, nitrogen-fixing ?actenu~ Azotobacter »inelandii shows a culture condition dependent change. When thebacterium IS grown Intheabsen~e ofanyfixed nit~ogen thetRNA contains 4-thiouridine to theextent of 45% of the total sulphur Incorporated. This gets reduced to 5%when the bacterium is grown in the presen~e of.e~ces~ ofamm~nium salt.Instead, a new thionucleoside which appears to be a derivative of 4-thloundlne IS found In the tRNA to the extent of 28%of the total sulphur incorporated.

Modulation of arginine decarboxylase activity in cucumber (Cucumis sativus) cotyledons in short-term organ culture

Among the various amines administered to excisedCucumis sativus cotyledons in short-term organ culture, agmatine (AGM) inhibited arginine decarboxylase (ADC) activity to around 50%, and putrescine was the most potent entity in this regard. Homoarginine (HARG) dramatically stimulated (3- to 4-fold) the enzyme activity. Both AGM inhibition and HARG stimulation of ADC were transient, the maximum response being elicited at 12 h of culture. Mixing experiments ruled out involvement of a macromolecular effector in the observed modulation of ADC. HARG-stimulated ADC activity was completely abolished by cycloheximide, whereas AGM-mediated inhibition was unaffected. Half-life of the enzyme did not alter on treatment with either HARG or AGM. The observed alterations in ADC activity are accompanied by change in Km of the enzyme. HARG-stimulated ADC activity is additive to that induced by benzyladenine (BA) whereas in presence of KCl, HARG failed to enhance ADC activity, thus demonstrating the overriding influence of K+ on amine metabolism.

Isolation and Culture of a Thermophilic Fungus, Melanocarpus albomyces, and Factors Influencing the Production and Activity of Xylanase

Summary: An uncommon thermophilic fungus, Melanocarpus albomyces, was isolated from soil and compost by incubating samples in a glucose/sorbose/asparagine liquid medium, followed by enrichment culture in medium containing sugarcane bagasse as carbon source. The culture filtrate protein of the fungus grown in the presence of bagasse or xylose hydrolysed xylan and some other polysaccharides but cellulose was not hydrolysed. High extracellular xylanase (EC 3.2.1.8) activity was produced by cultures grown on xylose or hemicellulosic materials. The enzyme was induced in glucose-grown washed mycelia in response to addition of xylose or xylan but not by alkyl or aryl β-D-xylosides. Cultures produced higher enzyme yields in shaken flasks than in a fermenter. Gel-filtration chromatography of culture filtrate protein showed the presence of two isoenzymes of xylanase, whose relative proportions varied with the carbon source used for growth. The extent of hydrolysis of heteroxylans or the hemicellulosic fraction of bagasse by culture filtrate protein preparations was greater when the cultures had been grown on bagasse rather than xylose as the inducing substrate. The activity of xylanase preparations was increased when an exogenous β-glucosidase was added.

In vitro culture of the allergenic weed Parthenium hysterophorus L

Excised stem, leaf segments and whole flower of the allergenic weed P. hysterophorus were cultured on Murashighe and Skoog's basal medium supplemented with hormones. Shoot buds readily formed in the stem callus cultured on MS Medium supplemented with IAA and BAP or Kinetin. The leaf callus formed roots alone in a wide variety of media. Suspension cultures were initiated from the leaf and stem callus. The leaf callus elicited a positive patch test response for delayed hypersensitivity in 4 patients suffering from Parthenium dermatitis, thus indicating its ability to synthesise the allergenic principle(s).

1-D hydrogen-bonded organization of hexanuclear {3d-4f-5d} complexes: evidence for slow relaxation of the magnetization for [{LMe2Ni(H2O)Ln(H2O)4.5}2{W(CN)8}2] with Ln = Tb and Dy

Heterometallic {3d-4f-5d} aggregates with formula [{LMe2Ni(H2O)Ln(H2O)4.5}2{W(CN)8}2]·15H2O, (LMe2 stands for N,N-2,2-dimethylpropylenedi(3-methoxysalicylideneiminato) Schiff-base ligand) with Ln = Gd, Tb, Dy, have been obtained by reacting bimetallic [LMe2Ni(H2O)2Ln(NO3)3] and Cs3{W(CN)8} in H2O. The hexanuclear complexes are organized in 1-D arrays by means of hydrogen bonds established between the solvent molecules coordinated to Ln and the CN ligands of an octacyanometallate moiety. The X-ray structure was solved for the Tb derivative. Magnetic behavior indicates ferromagnetic {W–Ni} and {Ni–Ln} interactions (JNiW = 18.5 cm-1, JNiGd = 1.85 cm-1) as well as ferromagnetic intermolecular interactions mediated by the H-bonds. Dynamic magnetic susceptibility studies reveal slow magnetic relaxation processes for the Tb and Dy derivatives, suggesting SMM type behavior for these compounds.

Spatiotemporal Organization of AT- and GC-rich DNA and Their Association With Transition Proteins TP1 and TP2 in Rat Condensing Spermatids

Transition protein 1 (TP1) and TP2 replace histones during midspermiogenesis (stages 12-15) and are finally replaced by protamines. TPs play a predominant role in DNA condensation and chromatin remodeling during mammalian spermiogenesis. TP2 is a zinc metalloprotein with two novel zinc finger modules that condenses DNA in vitro in a GC-preference manner. TP2 also localizes to the nucleolus in transfected HeLa and Cos-7 cells, suggesting a GC-rich preference, even in vivo. We have now studied the localization pattern of TP2 in the rat spermatid nucleus. Colocalization studies using GC-selective DNA-binding dyes chromomycin A3 and 7-amino actinomycin D and an AT-selective dye, 4',6-diamidino-2-phenylindole, indicate that TP2 is preferentially localized to GC-rich sequences. Interestingly, as spermatids mature, TP2 and GC-rich DNA moves toward the nuclear periphery, and in the late stages of spermatid maturation, TP2 is predominantly localized at the nuclear periphery. Another interesting observation is the mutually exclusive localization of GC- and AT-rich DNA in the elongating and elongated spermatids. A combined immunofluorescence experiment with anti-TP2 and anti-TP1 antibodies revealed several foci of overlapping localization, indicating that TP1 and TP2 may have concerted functional roles during chromatin remodeling in mammalian spermiogenesis.

A new genotype of Neurospora crassa that selectively produces abundant microconidia in submerged shake culture

A new microcycle microconidiating strain (mcm) ofNeurospora crassa was derived by successive backcrosses of a soil isolate to a laboratory wild-type strain. Surface grown cultures show normal wild-type vegetative morphology. However, under continuous agitation in liquid cultures at 22°C, macroconidial germlings bypass the usual mycelial phase and produce abundant numbers of uninucleate microconidia within 24 h. Temperature and culture media affect the production of microconidia.

Hormonal modulation of riboflavin carrier protein secretion by immature rat Sertoli cells in culture

We report here that a protein species with biochemical and immunological similarity with chicken egg riboflavin carrier protein (RCP) is synthesized and secreted by immature rat Sertoli cells in culture. When quantitated by a specific heterologous radioimmunoassay, optimal concentrations of FSH (25 ng/ml) brought about 3-fold stimulation of RCP secretion. FSH, in the presence of testosterone (10−6 M) brought about 6-fold stimulation of secretion of RCP over the control cultures which were maintained in the absence of these two factors. The aromatase inhibitor (1,4,6-androstatrien-3,17-dione) curtailed 85% of the enhanced secretion of RCP, suggesting that the hormonal stimulation is mediated through in situ synthesized estrogen and this could be confirmed with exogenous estradiol-17 β which brought about 3 — fold enhancement of secretion of RCP at a concentration of 10−6 M. When tamoxifen (10 μM) was added along with FSH and testosterone, there was 75% decrease in the enhanced secretion of RCP. Addition of this anti-estrogen together with exogenous estradiol resulted in 55% decrease in elevated levels of RCP. Cholera toxin (1 μg/ml) and 8-bromo-cyclic AMP (0.5 mM) mimicked the action of FSH on the secretion of RCP thus suggesting that FSH stimulation of RCP production may be mediated through cyclic AMP. These findings suggest that estrogen mediates RCP induction in hormonally stimulated sertoli cells presumably to function as the carrier of riboflavin to the developing germ cells through blood-testis barrier in rodents.

Phenotypic and Functional Characterization of Human Mammary Stem/Progenitor Cells in Long Term Culture

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages. Methodology: Single cell suspensions derived from human breast `organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres. Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture. Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance.