Karyotype of Mesembryanthemum crystallinum (Aizoaceae) studied by chromosome banding,FISH with rDNA probes and immunofluorescence detection of DNA methylation

The karyotype of the common ice plant Mesembryanthemum crystallinum L. (Aizoaceae) was studied using Chromomycin A₃ (CMA)/4′,6-diamidino-2-phenylindole (DAPI) staining, fluorescence in situ hybridization with 5S and 18S–5.8S–25S rDNA probes, DAPI/C-banding and immunodetection of 5-methylcytosine. A single bright CMA-band was revealed on the satellite chromosome, whose location was coincided with a position of a site of 18S–5.8S–25S rRNA genes. A site of 5S rRNA genes was observed on one of the other chromosomes. Relatively large DAPI/C-bands were mainly localized in the pericentromeric regions of the chromosomes. DAPI/C-banding patterns allowed us to identify all the chromosomes in the karyotype of M. crystallinum. The methylation of euchromatic chromosome regions was weaker as compared with heterochromatic DAPI/C-bands, which were hypermethylated. The obtained results may provide opportunities for investigating, at the chromosomal level, the genomic changes occurring in M. crystallinum either under salinization or under the action of other stress factors.     

The influence of conditions of <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> K-4 strain cultivation on surface-active substances synthesis

It has been observed that the Acinetobacter calcoaceticus K-4 strain produces surface-active substances (SAS) while growing either on hydrophilic (ethanol) or on hydrophobic substrates (hexadecane). Maximal SAS synthesis (with a conditional SAS concentration of 3.6; emulsifying activity of culture liquid dissolved in 50 times equal to 96%) was detected with growth on an ethanol-containing medium with the addition of urea, yeast autolysate and microelements, C/N ratio 60:1 and 10% inoculate, cultivated on ethanol-containing medium by the end of the exponential phase of growth. With respect to its chemical nature, extracellular SAS synthesized by A. calcoaceticus K-4 growing on ethanol-containing medium under optimal cultivating conditions form a glycolipid-aminolipid complex.     

Methods for the design and analysis of oligodeoxynucleotide-based DNA (cytosine-5) methyltransferase inhibitors

Several second-generation inhibitors of DNA (cytosine-5) methyltransferases based on studies of modified synthetic oligodeoxynucleoides have been described. As an aid to studies of these inhibitors, we present an electronic structure-based algorithm that can be used as a method for predicting the nature of the expected inhibition by any noncytosine nucleotide target. Targeting by the major human enzyme (hDnmt1) is governed by the presence of a three-nucleotide motif. In hemimethylated DNA, this motif consists of a 5-methylcytosine targeting signal that causes the enzyme to probe the opposite strand for a normally paired guanosine or inosine residue and attempt to methylate the residue 5' to that site. As a demonstration of the method, we apply these rules to the design and characterization of a novel oligodeoxynucleotide inhibitor of hDnmt1. This inhibitor takes advantage of the three-nucleotide recognition motif characteristic of hDnmt1 and shows that the enzyme is inhibited in vitro by non-CG methylation which targets the enzyme to normally basepaired but unproductive nucleotides such as dG, dA, and dT. Kinetic analysis at constant S-adenosyl-L-methionine concentration shows that representative inhibitory oligodeoxynucleotides are best viewed as weakly productive components of systems containing two DNA substrates. This model suggests that the most effective inhibitors are those with very low apparent Vmax and very low Km values. Oligodeoxynucleotides containing mispaired and unproductive targets such as dG, dA, dT, and dU are also inhibitory as secondary substrates for the human enzyme. Biologically, fail-safe mechanisms identified by the ab initio approach appear to be active in preventing potentially mutagenic deamination of dihydrocytosine and enzymatic methylation of dU.     

Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.     

Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously

A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simultaneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on Pfu polymerase mix, which has a proofreading activity. We successfully assembled (and confirmed by sequencing) seven different linear constructs ranging from 3 to 20 kb, including two 20 kb products (from fragments of 11, 1.7 and 7.5 kb), two 10.8 kb constructs, and two constructs of 6.1 and 6.2 kb, respectively. Accuracy of the PCR fusion is greater than or equal to one error per 6.6 kb, which is consistent with the expected error rate of the PCR mix. The method is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as somatic cell knockout in human cells or creation of whole genomes of viruses for vaccine research.     

Use of claydite-immobilized oil-oxidizing microbial cells for purification of water from oil

Oil-oxidizing bacteria were isolated from oil-polluted soil and water samples and identified as Acinetobacter calcoaceticus K-4, Nocardia vaceinii K-8, Rhodococcus erythropolis EK-1, and Mycobacterium sp. K-2. It was found that immobilization of the bacteria on an expanded clay aggregate accelerated their growth and consumption of hydrocarbon substrates. It was also found that water polluted with 100 mg/l oil could be purified with Rhodococcus erythropolis EK-1 and Nocardia vaceinii K-8 cells immobilized in this way. The dependence of the degree of water purification on its flow rate, aeration, and availability of nitrogen and phosphorus sources was determined. The efficiency of water purification from oil by immobilized Rhodococcus erythropolis EK-1 cells at high flow rates (of up to 0.68 l/h), low aeration (of 0.1 l/l per min) and an intermittent supply of 0.01% diammonium phosphate reached 99.5-99.8%.     

Blunt trauma to large vessels: a mathematical study

Background  

Blunt trauma causes short-term compression of some or all parts of the chest, abdomen or pelvis and changes hemodynamics of the blood. Short-term compression caused by trauma also results in a short-term decrease in the diameter of blood vessels. It has been shown that with a sudden change in the diameter of a tube or in the direction of the flow, the slower-moving fluid near the wall stops or reverses direction, which is known as boundary layer separation (BLS). We hypothesized that a sudden change in the diameter of elastic vessel that results from compression may lead not only to BLS but also to other hemodynamic changes that can damage endothelium.