Cleavage of methylated CCCGGG sequences containing either N4-methylcytosine or 5-methylcytosine with MspI, HpaII, SmaI, XmaI and Cfr9I restriction endonucleases.

The cleavage specificity of R.Cfr9I was determined to be C decreases CCGGG whereas the methylation specificity of M.Cfr9I was C4mCCGGG. The action of MspI, HpaII, SmaI, XmaI and Cfr9I restriction endonucleases on an unmethylated parent d(GGACCCGGGTCC) dodecanucleotide duplex and a set of oligonucleotide duplexes, containing all possible substitutions of either 4mC or 5mC for C in the CCCGGG sequence, was investigated. It was found that 4mC methylation, in contrast to 5mC, renders the CCCGGG site resistant to practically all the investigated endonucleases. The cleavage of methylated substrates with restriction endonucleases is discussed.     

Distribution of glomerular peripolar cells in different mammalian species

Summary Peripolar cells are granulated glomerular epithelial cells that form a cuff around the vascular pole of the glomerulus. Quantitation of these cells in 17 species of mammals (including man, several laboratory animals and a variety of other species) indicated that they were detectable by light microscopy in all but one of the mammals that were examined (the Australian hopping mouse). In adult mammals with detectable peripolar cells, the peripolar cell index (the percentage of randomly sectioned glomeruli that displayed peripolar cells in histological sections of kidney) ranged from 0.15 (for echidna) to 11.86 (for sheep). Newborn lambs and rats showed strikingly high values (23.30 and 10.76, respectively) compared with their adult counterparts. Using electron microscopy, peripolar cells were observed in all species that were examined, including the Australian hopping mouse. Morphologically, peripolar cells were similar in all species although their size and granule population varied. They showed a predominantly outer cortical glomerular distribution and a close anatomical relationship with the renin-containing myoepithelioid cells. These findings indicate that peripolar cells are present in a wide variety of species and support the view that such cells may play a significant role in the regulation of normal renal function.     

B-A and B-Z transitions in deoxyoligoduplexes containing 4- and 5- methylcytosine

Self-complementary oligodeoxynucleotides: GGACCCGGGTCC, GGA4mCCCGGGTCC, GGA5mCCCGGGTCC, CGCGCGCG, CG4mCGCGCG, CG5mCGCGCG were synthetized to study the contribution of methyl groups into the energetics of the three known cooperative transitions in DNA: helix-coil, B-A and B-Z With the use of circular dichroism and absorbtion methods the profiles of the above transitions were obtained by variation of temperature (helix-coil), trifluoroethanol fraction (B-A), NaCl and trifluorethanol contents (B-Z). On the basis of the transition widths and shifts of the transition points due to the methylations the energetics of the methyl groups was estimated. 5mC stabilizes the B form relatively the A form by 0.33 kcal/mol; while 4mC by 0.5 kcal/mol. In the B-Z transition 5 mC stabilizes the Z form by 0.28 kcal/mol relatively the B form; 4mC stabilizes also the Z form although by 0.14 kcal/mol only. Thus, these naturally occurring modifications could modulate substantially the ability of a DNA piece to shift into the A or Z form.     

At the border of ecological change: status and nest sites of the Lithuanian Black Stork Ciconia nigra population 2000–2006 versus 1976–1992

Recent trends in the European Black Stork Ciconia nigra population are geographically distinct: range expansion and adaptation to human activity dominate in western and central Europe, while declines—probably induced by landscape change—are reported in the east. We studied the large Lithuanian Black Stork population in the transition zone to explore whether, and how, the detrimental influences of recent Baltic landscape changes are balanced by the West European tendency of behavioural adaptation to human activity. Based on monitoring in sample plots, the current population was estimated at 650–950 pairs, indicating a significant decrease (possibly over 20%) during the last two decades. In comparison to the Latvian and Estonian populations, however, this decline is smaller, and the reproductive success remains at a high level [66% breeding success and 2.99 ± 0.97 (SD) fledglings per successful attempt, 2000–2006]; this north–south gradient suggests a climate-mediated impact of habitat degradation in the Baltic countries. The storks are also nesting closer to forest edges and in younger stands than 15–30 years ago, which has probably reduced the nest-tree limitation, as indicated by an increased use of large oaks. Thus, habitat degradation and adaptation seem to be taking place simultaneously in the Lithuanian Black Stork population, as was expected from its geographical location. In general, our study supports the view that, whenever possible, species conservation strategies and the use of indicator species should be geographically explicit.     

A review of non-DLVO interactions in environmental colloidal systems

The interaction and behavior of surfaces orcolloids is of quantitative significance inunderstanding the transport and fate ofcompounds and microorganisms in environmentalsystems. Historically, the DLVO model ofcolloid stability has described theseinteractions. This model finds its basis in aforce (energy) balance that comprisesattractive van der Waals and repulsiveelectrostatic interactions. Recently, the DLVOmodel has been found unable to fully describebiotic and abiotic colloidal behavior inaqueous media. The suspending phase (commonlywater) is often treated as a force (energy)transmitting or propagating medium. It isreasonable to believe that the structure ofwater may participate in a more significantfashion. Moreover, other moieties (sorbed anddissolved) may also have non-DLVO effects. Significant work has been focused on extendingthe precepts of the traditional DLVO model toaccommodate these non-DLVO forces (energies). This paper reviews many of the interactionsthat play a role in environmental systems andare not commonly subsumed by the traditionalDLVO model: e.g., hydrogen bonding and thehydrophobic effect, hydration pressure,non-charge transfer Lewis acid baseinteractions, and steric interactions.     

Towards redox active liquid crystalline phases of lipids: a monoolein/water system with entrapped derivatives of ferrocene

The phase and electrochemical behavior of the aqueous mixtures of monoolein (MO) and synthetic ferrocene (Fc) derivatives containing long alkyl chains-(Z)-octadec-9-enoylferrocene (1), (Z)-octadecen-9-ylferrocene (2), and ferrocenylmethyl (Z)-octadec-9-enoate (3)-were studied. At low hydration, the reversed micelles (L(2) phase) and cubic Q(230) phase of MO can accommodate relatively high amounts (>6 wt.%) of the Fc-derivative 2, whereas at high hydration, the pseudoternary cubic phase Q(224) is destabilized even at about 2 wt.% of this Fc. Increasing the Fc-derivative content induces L(alpha)-->L(2) and L(alpha)-->reversed bicontinuous cubic phase (Q(II))-->H(II) transitions depending upon hydration. A rough study of the MO system containing compounds 1 and 3 indicates very similar phase behavior to that of the MO/2/H(2)O system. Compound 2 apparently has no effect on the lipid monolayer thickness in the pseudoternary L(alpha), H(II) and Q(II) liquid crystalline phases of MO. Within a 3D-structure of the Q(224) phase, derivatives 1-3 exhibit electrochemical activity on the gold electrode. The one-electron redox conversion processes are electrochemically quasi-reversible and controlled by diffusion. The values of apparent diffusion coefficient (D(app)) and heterogeneous electron-transfer rate constant (k(s)) of Fcs are significantly lower in the cubic phase matrix when compared to the acetonitrile solution. By contrast, the MO H(II) phase with entrapped Fc-derivatives does not exhibit electrochemical activity on the electrode surface. It is suggested that the diffusional anisotropy and/or localized aggregation of compounds 1-3 within a 2D-structure of the H(II) phase account(s) for the latter observation.     

M.Smal is an N4-methylcytosine specific DNA-methylase.

An enzymatic activity rendering DNA immune to the action of the Smal restriction endonuclease in the presence of S-adenosyl-L-methionine has been detected in Serratia marcescens Sb. This methylase, M.Smal, modifies the second cytosine residue of the substrate sequence CCCGGG yielding N4-methylcytosine.     

Exploration of genetic factors resulting in abnormal disease in cattle experimentally challenged with bovine spongiform encephalopathy

ABSTRACT

Since the discovery of bovine spongiform encephalopathy (BSE), researchers have orally challenged cattle with infected brain material to study various aspects of disease pathogenesis. Unlike most other pathogens, oral BSE challenge does not always result in the expected clinical presentation and pathology. In a recent study, steers were challenged orally with BSE and all developed clinical signs and were sacrificed and tested. However, despite a similar incubation and clinical presentation, one of the steers did not have detectable PrP^Sc in its brain. Samples from this animal were analysed for genetic differences as well as for the presence of in vitro PrP^Sc seeding activity or infectivity to determine the BSE status of this animal and the potential reasons that it was different. Seeding activity was detected in the brainstem of the abnormal steer but it was approximately one million times less than that found in the normal BSE positive steers. Intra-cranial challenge of bovinized transgenic mice resulted in no transmission of disease. The abnormal steer had different genetic sequences in non-coding regions of the PRNP gene but detection of similar genotypes in Canadian BSE field cases, that showed the expected brain pathology, suggested these differences may not be the primary cause of the abnormal result. Breed composition analysis showed a higher Hereford content in the abnormal steer as well as in two Canadian atypical BSE field cases and several additional abnormal experimental animals. This study could point towards a possible impact of breed composition on BSE pathogenesis.     

Mycoplasma restriction-modification system MunI and its possible role in pathogenesis processes]

The restriction-modification system, named RMMunI, has been purified and characterised from Friend murine erythroleukemia cells. The site-specific endonuclease recognizes and cleaves the 5'C1AATTG nucleotide sequence. RMunI is an isoschizomer of RMfeI from Mycoplasma fermentans. Site-specific methylase modifies the second adenine residue in the same sequence (5'Cam6ATTG). It was established that the discovered enzymatic system is from mycoplasma which contaminates cell lines. Mycoplasma's DNA hybridizes with species-specific DNA probed for Mycoplasma fermentans and Mycoplasma arginini. The possible role of mycoplasmic restriction-modification enzymes in the process of acquired immune deficiency syndrome are discussed.