Polyamines, both common and uncommon, under heat stress in rice (Oryza sativa) callus

Enhanced production and accumulation of free and conjugated polyamines as well as increased activities of their biosynthetic enzymes in plants have been associated with heat stress. Perchloric acid-soluble free, as well as conjugated polyamines, and their metabolic enzymes were studied under 45°C heat stress in callus raised from heat-tolerant and -sensitive rice cultivars. The levels of free and conjugated polyamines, as well as arginine decarboxylase (EC 4.1.1.19) and polyamine oxidase (EC 1.4.34) activities were higher in tolerant than in sensitive callus under non-stressed conditions. Heat stress caused greater accumulation of free and conjugated polyamines in callus of the heat-tolerant cultivar N22 than in that of the heat-sensitive cultivar IR8. In particular, the uncommon polyamines norspermidine and norspermine were detected in cv. N22, which increased appreciably during stress, but they were not detected in callus of cv. IR8. Arginine decarboxylase and polyamine oxidase activities increased to a larger extent in N22 than in IR8 callus during stress, activities that were well correlated with the increased levels of common and uncommon polyamines. Increased levels of transglutaminase activity indicated the high titre of conjugated polyamines.     

DNA fingerprinting of human cell lines using PCR amplification of fragment length polymorphisms

Summary Methods for monitoring cell line identification and authentication include species-specific immunofluorescence, isoenzyme phenotyping, chromosome analysis, and DNA fingerprinting. Most previous studies of DNA fingerprinting of cell lines have used restriction fragment length polymorphism analysis. In this study, we examined the utility of an alternative and simpler method of cell line DNA fingerprinting—polymerase chain reaction (PCR) amplification of fragment length polymorphisms. Fourteen human cell lines previously found by other methods to be either related or disparate were subjected to DNA fingerprinting by PCR amplification of selected fragment length polymorphism loci. Cell identification patterns by this method were concordant with those obtained by isoenzyme phenotyping and restriction fragment length polymorphism-DNA fingerprinting, and were reproducible within and between assays on different DNA extracts of the same cell line. High precision was achieved with electrophoretic separation of amplified DNA products on high resolution agarose or polyacrylamide gels, and with fragment length polymorphism (FLP) loci-specific “allelic ladders” to identify individual FLP alleles. Determination of the composite fingerprint of a cell line at six appropriately chosen fragment length polymorphism loci should achieve a minimum discrimination power of 0.999. The ability of PCR-based fragment length polymorphism DNA fingerprinting to precisely and accurately identify the alleles of different human cell lines at multiple polymorphic fragment length polymorphism loci demonstrates the feasibility of developing a cell line DNA fingerprint reference database as a powerful additional tool for future cell line identification and authentication.     

The formation of germtubes by candida albicans,when grown with Staphylococcus pyogene,Escherichia coli,Klebsiella pneumoniae,Lactobacilius acidophilus and Proteus vulgaris

The formation of germtubes by twelve clinical isolates of C. albicans was studied in human serum containing per millilitre 10³ to 10⁹ organisms as: Staphylococcus pyegene, Escherichia coli, Klebsiella pneumoniae, Lactobacillus acidophilus and Proteus vulgaris. All the five bacteria inhibited formation of germtubes by C. albicans at all concentrations and the percent germtube formed diminished with increasing concentration of the bacteria. Lactobacillus acidophilus inhibited the formation of germtubes maximally followed by Staphylococcus pyogene, Escherichia coli and Klebsiella pneumoniae. Proteus vulgaris in the concentrations of 10³ to 10⁷ bacteria per millilitre produced only insignificant inhibition of formation of germtubes by C. albicans. Since germtubes of C. albicans are invasive, it is suggested that inhibition of blastospo-regermtube transformation may be significantly responsible for prevention of infection by C. albicans by coexisting bacterial flora.     

Breakdown ofd-glucose by mixed cultures ofEscherichia coli,Desulfovibrio vulgaris,andChromatium vinosum

Experiments with mixed cultures ofEscherichia coli, Desulfovibrio vulgaris, andChromatium vinosum were conducted using a synthetic medium with glucose as the substrate. The bacterial number and the changes in the chemical factors were determined during the development of the mixed culture. (i) Fermentation of glucose byE. coli produces organic acids (formic, acetic, lactic, and succinic) and alcohol. The growth-yield constant K (cell dry weight per weight solid substrate) does not exceed 0.05. (ii) In the mixed culture ofE. coli andD. vulgaris, the reduction of sulfate is accompanied by a total consumption of formate, lactate, and alcohol and an increase in the sulfide and acetate content. (iii) When the three physiologically different species are allowed to grow simultaneously, there is no accumulation of catabolites in the medium and the growth yield constant increases to 0.46. Maximum phototrophic production requires the presence of bothE. coli andD. vulgaris. A low substrate concentration and the simultaneous growth of the three organisms are other factors that contribute towards a high output. The biochemical parameters of the medium are influenced to a large extent by the glucose level. The results suggest that the behavior of the strains is different in pure and mixed cultures.     

Interaction of cobalt(II) and copper(II) hydroxamates with polyriboadenylic acid: An insight into RNA based drug designing

The polyadenylic acid [poly(A)] tail of mRNA plays a noteworthy role in the initiation of the translation, maturation, and stability of mRNA. It also significantly contributes to the production of alternate proteins in eukaryotic cells. Hence, it has recently been recognized as a prospective drug target. Binding affinity of bis(N-p-tolylbenzohydroxamato)Cobalt(II), [N-p-TBHA-Co(II)] (1) and bis(N-p-naphthylbenzohydroxamato)Copper(II), [N-p-NBHA-Cu(II)] (2) complexes with poly(A) have been investigated by biophysical techniques namely, absorption spectroscopy, fluorescence spectroscopy, diffuse reflectance infrared Fourier transform spectroscopy, circular dichroism spectroscopy, viscometric measurements and through molecular docking studies. The intrinsic binding constants (K_b) of complexes were determined following the order of N-p-TBHA-Co(II)] > N-p-NBHA-Cu(II), along with hyperchromism and a bathochromic shift for both complexes. The fluorescence quenching method revealed an interaction between poly(A)-N-p-TBHA-Co(II)/poly(A)-N-p-NBHA-Cu(II). The mode of binding was also determined via the fluorescence ferrocyanide quenching method. The increase in the viscosity of poly(A) that occurred from increasing the concentration of the N-p-TBHA-Co(II)/N-p-NBHA-Cu(II) complex was scrutinized. The characteristics of the interaction site of poly(A) with N-p-TBHA-Co(II)/N-p-NBHA-Cu(II) were adenine and phosphate groups, as revealed by DRS-FTIR spectroscopy. Based on these observations, a partial intercalative mode of the binding of poly(A) has been proposed for both complexes. Circular dichroism confirmed the interaction of both the complexes with poly(A). The molecular docking results illustrated that complexes strongly interact with poly(A) via the relative binding energies of the docked structure as ?259.39eV and ?226.30eV for N-p-TBHA-Co(II) and N-p-NBHA-Cu(II) respectively. Moreover, the binding affinity of N-p-TBHA-Co(II) is higher in all aspects than N-p-NBHA-Cu(II) for poly(A).     

Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication

Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3D(pol), in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3D(pol)-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3D(pol) was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3D(pol) could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3D(pol) to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu(6) to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3D(pol) in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.