Effects of benzo[a]pyrene-deoxyguanosine lesions on DNA methylation catalyzed by EcoRII DNA methyltransferase and on DNA cleavage effected by EcoRII restriction endonuclease

DNA methylation is an important cellular mechanism for controlling gene expression. Whereas the mutagenic properties of many DNA adducts, e.g., those arising from polycyclic aromatic hydrocarbons, have been widely studied, little is known about their influence on DNA methylation. We have constructed site-specifically modified 18-mer oligodeoxynucleotide duplexes containing a pair of stereoisomeric adducts derived from a benzo[a]pyrene-derived diol epoxide [(+)- and (-)-r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, or B[a]PDE] bound to the exocyclic amino group of guanine. The adducts, either (+)- or (-)-trans-anti-B[a]P-N(2)-dG (G*), positioned either at the 5'-side or the 3'-side deoxyguanosine residue in the recognition sequence of EcoRII restriction-modification enzymes (5'-...CCA/TGG...) were incorporated into 18-mer oligodeoxynucleotide duplexes. The effects of these lesions on complex formation and the catalytic activity of the EcoRII DNA methyltransferase (M.EcoRII) and EcoRII restriction endonuclease (R.EcoRII) were investigated. The M.EcoRII catalyzes the transfer of a methyl group to the C5 position of the 3'-side cytosine of each strand of the recognition sequence, whereas R.EcoRII catalyzes cleavage of both strands. The binding of R.EcoRII to the oligodeoxynucleotide duplexes and the catalytic cleavage were completely abolished when G was positioned at the 3'-side dG position (5'-...CCTGG*...). When G* was at the 5'-side dG position, binding was moderately diminished, but cleavage was completely blocked. In the case of M.EcoRII, binding is diminished by factors of 5-30 but the catalytic activity was either abolished or reduced 4-80-fold when the adducts were located at either position. Somewhat smaller effects were observed with hemimethylated oligodeoxynucleotide duplexes. These findings suggest that epigenetic effects, in addition to genotoxic effects, need to be considered in chemical carcinogenesis initiated by B[a]PDE, since the inhibition of methylation may allow the expression of genes that promote tumor development.     

Dependence of the effect of RNAase from Bacillus intermedius on the growth of baker's yeast on the concentration of the exogenous enzyme

Bacillus intermedius RNase added at a low concentration (0.001 microgram/ml) stimulated yeast growth, while a high RNase concentration (1500 micrograms/ml) was inhibitory to yeast growth. The inhibitory effect of RNase was transient and correlated with the increase in the trehalose pool of yeast cells. The number of unbudded cells in the yeast population tended to decrease under the action of low concentrations of bacillar RNase and to increase under the action of high concentrations of this enzyme.     

Effect of membrane-active microbial autoregulators on the growth of cultured ras-transformed fibroblasts

Differential effects on proliferation of individual vs. combined administration of high- and low-molecular-weight microbial autoregulators (extracellular RNase from Bacillus subtilis and anabiosis-inducing factor d1) are reported for the first time for cultured cells of higher eukaryotes. Proliferation of ras-transformed mouse fibroblasts was affected by both autoregulators dose-dependently. The cytotoxic activity of individual regulators was directly related to their concentration. Unlike RNase, factor d1 (which functions as a chemical chaperone) exerted reversible effects. Studies of the effects of combined administration of the autoregulators demonstrated that pretreatment of the cells with low-dose d1 decreased the toxicity of RNase. Higher doses of d1 were required to attenuate the effects of toxic agents with more pronounced membrane tropism. The results obtained suggest that a universal system regulating the physiological activity of cells is operative in taxonomically remote organisms. The operation of the system is based on sequential changes in the structural organization and function of subcellular structures, induced by low- and high-molecular-weight autoregulators.     

Effect of cross-fostering on various physiological and behavioral features in Wistar and genetically cataleptic rats

In the GC rats reared by their natural mothers the plasma corticosterone concentration and open field locomotion were lower than in Wistar rats whereas the startle-reflex amplitude was higher. Cross-fostering did not affect the plasma corticosterone concentration or the startle-reflex amplitude. A negative correlation between these parameters was found. An open field test yielded lower results in Wistar rats reared by foster mothers belonging to the GC strain whereas the reverse cross-fostering did not affect the locomotion in GC rats. The data suggest that fostering affects the open field behaviour whereas cross-fostering effects depend on the genotype of fostered litter.     

Role of an active site adenine in hairpin ribozyme catalysis

The hairpin ribozyme is a small catalytic RNA that accelerates reversible cleavage of a phosphodiester bond. Structural and mechanistic studies suggest that divalent metals stabilize the functional structure but do not participate directly in catalysis. Instead, two active site nucleobases, G8 and A38, appear to participate in catalytic chemistry. The features of A38 that are important for active site structure and chemistry were investigated by comparing cleavage and ligation reactions of ribozyme variants with A38 modifications. An abasic substitution of A38 reduced cleavage and ligation activity by 14,000-fold and 370,000-fold, respectively, highlighting the critical role of this nucleobase in ribozyme function. Cleavage and ligation activity of unmodified ribozymes increased with increasing pH, evidence that deprotonation of some functional group with an apparent pK(a) value near 6 is important for activity. The pH-dependent transition in activity shifted by several pH units in the basic direction when A38 was substituted with an abasic residue, or with nucleobase analogs with very high or low pK(a) values that are expected to retain the same protonation state throughout the experimental pH range. Certain exogenous nucleobases that share the amidine group of adenine restored activity to abasic ribozyme variants that lack A38. The pH dependence of chemical rescue reactions also changed according to the intrinsic basicity of the rescuing nucleobase, providing further evidence that the protonation state of the N1 position of purine analogs is important for rescue activity. These results are consistent with models of the hairpin ribozyme catalytic mechanism in which interactions with A38 provide electrostatic stabilization to the transition state.     

Dual manifestation of catatonic reaction in rats

Results of genetic, neurophysiological, neurochemical and pharmacological suggest that the cataleptic freezing and "nervousness" observed in the cataleptic rat strain GC have a common mechanism. There seems to be a physiological factor causing catalepsy, upon reaching a certain level of intensity, to be transformed into "nervousness", which is observed both at different period and/or moment of individual life and in the processes of breeding the strain for predisposition to catalepsy.     

Protein remodeling of the heart ventricles in hereditary hypertriglyceridemic rat: effect of ace-inhibition

The aim of this study was to determine whether protein remodeling of the heart ventricles and remodeling of the aorta were present in hereditary hypertriglyceridemic (hHTG) rats and whether treatment with the angiotensin-converting enzyme inhibitor, captopril could prevent these alterations. Three groups of rats were investigated in a four week experiment control Wistar /C/rats, hHTg rats, hHTg rats given captopril (100 mg/kg/day) (hHTg + CAP). In the hHTg group, the increased systolic blood pressure (SBP) was associated with hypertrophy of the LV and RV. Protein profile analysis revealed an enhancement of metabolic protein concentration in both ventricles. The concentration of total collagenous proteins was not changed in either ventricles. However, alterations in composition of cardiac collagen were detected, characterized by higher concentration of hydroxyproline in pepsin-insoluble fraction and lower concentration of hydroxyproline in pepsin soluble faction in the LV. Hypertrophy of aorta, associated with the reduction of nitric oxide dependent relaxation, was also present in hHTG rats. Captopril normalized SBP, reduced left ventricular hypertrophy (LVH), diminished metabolic protein concentration in both ventricles, and improved NO-dependent relaxation of the aorta. Furthermore, captopril partially reversed alterations in hydroxyproline concentration in soluble and insoluble collagenous fractions of the LV. We conclude that hypertrophy of both ventricles and the aorta are present in hHTG rats, along with protein remodeling of both ventricles. Captopril partially prevented left ventricular hypertrophy development and protein remodeling of the myocardium.     

Role of mitochondria in the pheromone- and amiodarone-induced programmed death of yeast

Although programmed cell death (PCD) is extensively studied in multicellular organisms, in recent years it has been shown that a unicellular organism, yeast Saccharomyces cerevisiae, also possesses death program(s). In particular, we have found that a high doses of yeast pheromone is a natural stimulus inducing PCD. Here, we show that the death cascades triggered by pheromone and by a drug amiodarone are very similar. We focused on the role of mitochondria during the pheromone/amiodarone-induced PCD. For the first time, a functional chain of the mitochondria-related events required for a particular case of yeast PCD has been revealed: an enhancement of mitochondrial respiration and of its energy coupling, a strong increase of mitochondrial membrane potential, both events triggered by the rise of cytoplasmic [Ca2+], a burst in generation of reactive oxygen species in center o of the respiratory chain complex III, mitochondrial thread-grain transition, and cytochrome c release from mitochondria. A novel mitochondrial protein required for thread-grain transition is identified.     

CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex

CLIP-associating protein (CLASP) 1 and CLASP2 are mammalian microtubule (MT) plus-end binding proteins, which associate with CLIP-170 and CLIP-115. Using RNA interference in HeLa cells, we show that the two CLASPs play redundant roles in regulating the density, length distribution and stability of interphase MTs. In HeLa cells, both CLASPs concentrate on the distal MT ends in a narrow region at the cell margin. CLASPs stabilize MTs by promoting pauses and restricting MT growth and shortening episodes to this peripheral cell region. We demonstrate that the middle part of CLASPs binds directly to EB1 and to MTs. Furthermore, we show that the association of CLASP2 with the cell cortex is MT independent and relies on its COOH-terminal domain. Both EB1- and cortex-binding domains of CLASP are required to promote MT stability. We propose that CLASPs can mediate interactions between MT plus ends and the cell cortex and act as local rescue factors, possibly through forming a complex with EB1 at MT tips.     

The carboxy-terminal domain of the DExDH protein YxiN is sufficient to confer specificity for 23S rRNA

DE x DH proteins are believed to modulate the structures of RNAs and ribonucleoprotein complexes by disrupting RNA helices and RNA-protein interactions. All DE x DH proteins contain a two-domain catalytic core that enables their RNA-dependent ATPase and RNA helicase activities. The catalytic core may be flanked by ancillary domains that are proposed to confer substrate specificity and facilitate the unique functions of individual proteins. The Escherichia coli DE x DH protein DbpA and its Bacillus subtilis ortholog YxiN have similar 75aa carboxy-terminal domains, and both proteins are specifically targeted to 23S rRNA. Here we demonstrate that the carboxy-terminal domain of YxiN is sufficient to confer RNA specificity by characterizing a chimera in which this domain is appended to the core domains of E.coli SrmB, a DE x DH protein with no apparent substrate specificity. Both the RNA-dependent ATPase and RNA helicase activities of the chimera are specifically activated by 23S rRNA and abolished by sequence changes within hairpin 92, a critical recognition element for Y x iN. These data support a model in which the carboxy-terminal domain binds hairpin 92 to target the protein to 23S rRNA.