Characterization of poly(ADP-ribosyl)ated domains of rat pachytene chromatin.

Poly(ADP-ribosyl)ation of nuclear proteins was several-fold higher in the pachytene spermatocytes than in the premeiotic germ cells of the rat. Among the histones of the pachytene nucleus, histone subtypes H2A, H1 and H3 were poly(ADP-ribosyl)ated. Based on the immunoaffinity fractionation procedure of Malik, Miwa, Sugimara & Smulson [(1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2554-2558] we have fractionated DNAase-II-solubilized chromatin into poly(ADP-ribosyl)ated chromatin (PAC) and non-poly(ADP-ribosyl)ated chromatin (non-PAC) domains on an anti-[poly(ADP-ribose)] IgG affinity matrix. Approx. 2.5% of the pachytene chromatin represented the PAC domains. A significant amount of [alpha-32P]dATP-labelled pachytene chromatin (labelled in vitro) was bound to the affinity matrix. The DNA of pachytene PAC domains had internal strand breaks, significant length of gaps and ligatable ends, namely 5'-phosphoryl and 3'-hydroxyl termini. On the other hand, the PAC domains from 18 h regenerating liver had very few gaps, if any. The presence of gaps in the pachytene PAC DNA was also evident from thermal denaturation studies. Although many of the polypeptides were common to the PAC domains of both pachytene and regenerating liver, the DNA sequences associated with these domains were quite different. A 20 kDa protein and the testis-specific histone H1t were selectively enriched in the pachytene PAC domains. The pachytene PAC domains also contained approx. 10% of the messenger coding sequences present in the DNAase-II-solubilized chromatin. The pachytene PAC domains, therefore, may represent highly enriched DNA-repair domains of the pachytene nucleus.     

Hydrogen peroxide permeation across liposomal membranes: a novel method to assess structural flaws in liposomes

Hydrogen peroxide permeation across large multilamellar vesicles of defined and complex lipid composition was shown to obey precise kinetic relationships for the activity of the occluded catalase. Careful assay conditions precluded simultaneous peroxidative damage to the lipids. The kinetic data was consistent with a barrier role for the bilayer for hydrogen peroxide permeation. More interestingly, hydrogen peroxide permeation across liposomes of complex lipid mixtures exhibited osmotic inhibition of permeation of hydrogen peroxide. On the other hand, purified egg lecithin vesicles did not exhibit any effect of external osmolality on hydrogen peroxide permeation in an experimentally defined non-lytic zone of external osmolarity. These results argue in favour of a heterogeneous, heteroporous structure of bilayers with complex lipid composition.     

Developmental and Dimensional Changes in the Cambial Fusiform Initials ofHoloptelea integrifolia (Roxb.) Planch.

Divisional activity, intrusive growth of the cell wall and loss of fusiform initials have been studied in Holoptelea integrifolia. The dimensional changes in relation to mean length, length frequency, mean width and length variation in relation to fibre length have also been analysed.     

Effects of nucleotide bromination on the stabilities of Z-RNA and Z-DNA: a molecular mechanics/thermodynamic perturbation study

The structures of ZI- and ZII-form RNA and DNA oligonucleotides were energy minimized in vacuum using the AMBER molecular mechanics force field. Alternating C-G sequences were studied containing either unmodified nucleotides, 8-bromoguanosine in place of all guanosine residues, 5-bromocytidine in place of all cytidine residues, or all modified residues. Some molecules were also energy minimized in the presence of H2O and cations. Free energy perturbation calculations were done in which G8 and C5 hydrogen atoms in one or two residues of Z-form RNAs and DNAs were replaced in a stepwise manner by bromines. Bromination had little effect on the structures of the energy-minimized molecules. Both the minimized molecular energies and the results of the perturbation calculations indicate that bromination of guanosine at C8 will stabilize the Z forms of RNA and DNA relative to the nonbrominated Z form, while bromination of cytidine at C5 stabilizes Z-DNA and destabilizes Z-RNA. These results are in agreement with experimental data. The destabilizing effect of br5C in Z-RNAs is apparently due to an unfavorable interaction between the negatively charged C5 bromine atom and the guanosine hydroxyl group. The vacuum-minimized energies of the ZII-form oligonucleotides are lower than those of the corresponding ZI-form molecules for both RNA and DNA. Previous x-ray diffraction, nmr, and molecular mechanics studies indicate that hydration effects may favor the ZI conformation over the ZII form in DNA. Molecular mechanics calculations show that the ZII-ZI energy differences for the RNAs are greater than three times those obtained for the DNAs. This is due to structurally reinforcing hydrogen-bonding interactions involving the hydroxyl groups in the ZII form, especially between the guanosine hydroxyl hydrogen atom and the 3'-adjacent phosphate oxygen. In addition, the cytidine hydroxyl oxygen forms a hydrogen bond with the 5'-adjacent guanosine amino group in the ZII-form molecule. Both of these interactions are less likely in the ZI-form molecule: the former due to the orientation of the GpC phosphate away from the guanosine ribose in the ZI form, and the latter apparently due to competitive hydrogen bonding of the cytidine 2'-hydroxyl hydrogen with the cytosine carbonyl oxygen in the ZI form. The hydrogen-bonding interaction between the cytidine hydroxyl oxygen and the 5'-adjacent guanosine amino group in Z-RNA twists the amino group out of the plane of the base. This may be responsible for differences in the CD and Raman spectra of Z-RNA and Z-DNA.     

An in vitro demonstration of peroxisome proliferation and increase in peroxisomal β-oxidation system mRNAs in cultured rat hepatocytes treated with ciprofibrate

Using the normal adult rat hepatocytes, plated on rat tail collagen-coated dishes and fed a chemically defined medium, we demonstrate here that ciprofibrate at 0.1 mM concentration, increases significantly the mRNA levels of fatty acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and thiolase (the three enzymes of the β-oxidation system), and causes peroxisome proliferation. Increase in mRNA levels of these genes was evident within 1 h and was maximal 24 h after the addition of ciprofibrate. In hepatocytes cultured in the absence of ciprofibrate, the basal levels of these enzymes were low and further declined with time. Concomitant treatment of hepatocytes with cycloheximide did not inhibit or superinduce the mRNA levels, indicating that this induction may represent a primary (direct) effect of this compound on the expression of these genes and does not apparently involve short-lived repressor protein(s).     

Isolation and identification of Pseudomonas spp. from Schirmacher Oasis, Antarctica.

Ten cultures of Pseudomonas spp. were established from soil samples collected in and around a lake in Antarctica. Based on their morphology, biochemical and physiological characteristics, and moles percent G + C of their DNA, they were identified as P. fluorescens, P. putida, and P. syringae. This is the first report on the identification of Pseudomonas spp. from continental Antarctica.     

c-AMP-induced c-fos expression in cells of melanocyte origin

The expression of the c-fos gene in murine cells of melanocyte origin in response to cAMP-elevating agents has been examined. Accumulation of c-fos mRNA at a high level as a consequence of these treatments precedes both proliferative and cytodifferentiative changes in non-tumorigenic or tumorigenic cell lines.     

Carcinoscorpius rotunda cauda amoebocyte lysate for detection of endotoxins--its preparation, stability, sensitivity and comparison with Limulus amoebocyte lysate

Carcinoscorpius amoebocyte lysate (CAL) was prepared from C. rotunda cauda by a modification of the method described by Mahalanabis et al. [Indian J Med Res, 70 (1979) 35]. Seasonal variation as well as batch variation was observed in the yield of haemolymph and the total lysate protein. In the presence of E. coli lipopolysaccharide (pure, free endotoxin) and E. coli and Salmonella cell suspensions (bound endotoxin), the CAL formed a gel after incubation at 37 degrees C. The gelling time varied from 10-90 min depending on the concentration of endotoxin used; higher concentrations formed gel more rapidly. The endotoxin detection capacity (sensitivity) of the lysate preparations was influenced by the season in which prepared, but not by the total protein content. Ten fold increase in the sensitivity was achieved by a purification step using chloroform. Although subsequent frozen storage with or without lyophilization did not alter the initial sensitivity, it was either decreased considerably or lost totally when the lysate was stored for 4 months at 4 degrees C or for 2 months at 30 degrees C. Under the same conditions, Limulus lysate was more stable. The lost sensitivity could not be regained by the incorporation of divalent cations (Ca2+ and Mg2+). The CAL preparations in general were able to detect as little as 10-100 pg of endotoxin or as few as 10(3) cells of E. coli or 10(4) cells of Salmonella and were comparable to LAL. CAL could be used successfully in lieu of Limulus amoebocyte lysate in the detection and assay of endotoxins.     

Site-specific integration in Streptomyces ambofaciens: localization of integration functions in S. ambofaciens plasmid pSAM2.

In Streptomyces ambofaciens ATCC 15154, an 11.1-kilobase element, pSAM2, exists as a single integrated copy in the chromosome. In S. ambofaciens 3212 (a derivative of ATCC 15154), pSAM2 exists as a free, circular plasmid as well as an integrated element. BclI fragments from the free form of pSAM2 were cloned into an Escherichia coli plasmid vector. By using gene transplacement methods, the chromosomally integrated form of pSAM2 was marked with a gene coding for apramycin resistance. This enabled us to isolate both a segregant that had lost the integrated pSAM2 element and a cosmid clone containing integrated pSAM2 along with the flanking chromosomal sequences. One of the BclI fragments derived from free pSAM2 was shown to contain all the plasmid-specified information required to direct site-specific recombination in a derivative of S. ambofaciens lacking the resident pSAM2 element as well as in a number of other Streptomyces strains. The attachment sites used by the plasmid and the chromosome in site-specific recombination and the junctions created after integration were cloned and sequenced. Certain structural features in common with other integrating elements in actinomycetes were noted.