Solid phase immunoenzyme analysis of the immunospecificity of DNA from calf spleen alkylated by thiophosphamide

DNA binding activity of rabbit antiserum against calf spleen DNA's modified by thiophosphamide (DNA-T) was studied by means of solid enzyme immunoassays (ELISA). The studies demonstrated the preferential binding of the immobilized DNA-T compared to immobilized single-stranded DNA (ss-DNA) and only small preference compared to native DNA. Two antisera against DNA-T were purified by affinity chromatography on a ss-DNA-CNBr agarose from antibodies to calf spleen ss-DNA. They interacted only with the immobilized DNA-T, but not with ss-DNA or native DNA. These results demonstrated that DNA modification by thiophosphamide, decreases the immunogenicity of usual nitrogen-containing DNA bases, but detected new immunogenic specificity for adducts. Detection of new immunogenic specificity in DNA's alkylated by thiophosphamide, resulted in the development of a sensitive enzyme immunoassay for the detection of these adducts in nucleic acids, in monitoring their formation, persistence and repair damages in DNA.     

Electrophysiological analysis of corticofugal influences on preoptic neurons

Evoked potential (EPs) and responses of the medial (MPO) and lateral (LPO) preoptic region (RPO) and adjacent structures of the hypothalamus to stimulation of the prefrontal (area 8) and cingulate (area 24) cortex, piriform lobe (periamygdaloid cortex), and hippocampus (area CA3) were investigated in acute experiments on cats under ketamine anesthesia. The most pronounced EPs were observed in the RPO after stimulating the piriform and cingulate cortex. A close relation was found between neuronal responses and EP components. The majority of neurons responding to stimulation of various cortical structures were localized in the LPO, where primarily excitatory responses dominate. The MPO contained somewhat fewer neurons responding to cortical stimulation, and the dominant response here was primarily inhibitory. The ratio of inhibitory and excitatory responses in the LPO was 0.6:1 and in the MPO 5.8:1. Primarily in-inhibitory responses dominated also in the LPO zone adjacent to the bed nucleus stria terminalis (BST) and primarily excitatory in the region surrounding the supraoptic nucleus (SO) (respective ratios 4.9:1 and 0.7:1). The RPO is a broad convergence zone, where 3/4 of the neurons responded to stimuli of two and more cortical regions.A. M. Gorky Medical Institute, Ukrainian Minstry of Health, Donetsk. Translated from Neirofiziologiya, Vol. 23, No. 6, pp. 709–719, November–December, 1991.     

Firing activity of preoptic and supraoptic neurons upon mild alterations in osmo- and glucohomeostasis

Responses of neurons of the lateral (LPO) and medial (MPO) subdivisions of the preoptic region (RPO) and of the supraoptic nucleus (SO) of the hypothalamus at infusions of up to 400 µl of a hypotonic (0.2%) or a hypertonic (0.3%) NaCl solution or an isotonic (5.5%) glucose solution into the homolateral internal carotid artery were studied in acute experiments on ketamine-anesthetized cats. Changes in the firing frequency were exhibited by 69% of the examinedRPO andSO neurons, the response being of four different types: a monophasic increase (1st-type) or a monophasic decrease (2nd-type) in the activity; biphasic responses where an initial frequency rise was followed by inhibition (3rd-type) and vice versa (4th-type). Of all the neuronal responses to all stimulations, 50% (121/245) were of the 1st type; 11% (26/245), of the 2nd type; 5% (14/245), of the 3rd type; and 3% (7/245), of the 4th type. No neurons with 1st-type responses to infusion of the hypotonic NaCl solution were found in the medial sections of theMPO, and of the hypertonic solution, in the lateral sections of theLPO. Neurons with 2nd-type responses to infusion of hypo- and hypertonic NaCl solutions were almost solely detected in theSO, whereas units with such responses to glucose infusions were observed only in theRPO, being fully absent in theSO, where this stimulation gave rise only to 1st-type responses. Neurons with 3rd- and 4th-type responses to hypo- and hyperosmotic stimulations were detected predominantly in theMPO andSO, and to glucose stimulation, in theMPO andLPO.Neirofiziologiya/Neurophysiology, Vol. 25, No. 4, pp. 281–291, July–August, 1993.     

Catalytic antibodies in the bone marrow and other organs of Th mice during spontaneous development of experimental autoimmune encephalomyelitis associated with cell differentiation

Exact mechanisms of autoimmune disease development are still yet unknown. However, it is known that the development of autoimmune diseases is associated with defects in the immune system, namely, the violation of the bone marrow hematopoietic stem cells (HSCs) differentiation profiles. Different characteristics of autoimmune reaction development in experimental autoimmune encephalomyelitis (EAE) prone Th mice characterizing T-lymphocytes response were analyzed using standard approaches. Profiles of several HSCs differentiation of bone marrow (BFU-E, CFU-E, CFU-GM, CFU-GEMM, T- and B-lymphocytes) of Th male and female mice during spontaneous development of EAE were noticeably different. Patterns of total lymphocytes, B- and T-cells proliferation in several different organs (bone marrow, blood, spleen, thymus, and lymph nodes) were also remarkably different. In addition, there were in time noticeable differences in their changes for some organs of male and female mice. Characters of changes in the profiles of CD4 and CD8 cells proliferation in some organs not always coincide with those for total T lymphocytes. The changes in the differentiation profiles of HSCs and the level of lymphocytes proliferation in the bone marrow and other organs were associated with the increase in the concentration of antibodies against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, and catalytic antibodies hydrolyzing these substrates. Despite some differences in changes in the analyzed parameters, in general, the spontaneous development of EAE in male and female mice occurs to some extent in a comparable way.

     

Photoinactivation of microorganisms in the presence of photosensitizers

Two photosensitizing preparation for the photodynamic therapy of oncological diseases have been obtained. As revealed in this study, under experimental conditions their action suppresses the growth of Enterococcus faecalis and Staphylococcus aureus, which may be of great importance for the treatment of infections. And in contrast to antibiotic therapy, their use may not affect the development of normal intestinal microflora. The study has shown that their derivatives with the residues of carbonic (or muramic) acids more intensively penetrate microorganisms through their cell walls. This makes in possible to expand the spectrum of action of the preparations. The proposed method permits the rapid analysis of the photochemical activity of preparation for photodynamic therapy in the process of their development.     

4-(4-R-phenylamino)-5-methoxy-1,2-benzoquinones are new selective inhibitors of carbon tetrachloride-initiated free radical reactions in liver.

The action of several 1,2-benzoquinone derivatives on free radical processes initiated by carbon tetrachloride was studied. Among them a substance that effectively inhibited lipid peroxidation without substantial influence on covalent binding of radical products of metabolic cleavage of carbon tetrachloride as well a substance that equally inhibited both processes were found. It was shown that 1,2-benzoquinones can be useful tool for investigation of free radical mechanisms of carbon tetrachloride-initiated liver cell damage in vivo.     

Small scale genetic alterations contribute to increased mutability at the X-linked Hprt locus in vivo in Blm hypomorphic mice

BLM, the gene mutated in Bloom syndrome (BS), encodes an ATP-dependent RecQ DNA helicase that is involved in the resolution of Holliday junctions, in the suppression of crossovers and in the management of damaged replication forks. Cells from BS patients have a characteristically high level of sister chromatid exchanges (SCEs), and increased chromosomal aberrations. Fibroblasts and lymphocytes of BS patients also exhibit increased mutation frequency at the X-linked reporter gene HPRT, suggesting that BLM also plays a role in preventing small scale genomic rearrangements. However, the nature of such small scale alterations has not been well characterized. Here we report the characterization of Hprt mutations in vivo in Blm hypomorphic mice, Blm^tm1Ches/Blm^tm3Brd. We found that the frequency of Hprt mutants was increased about 6-fold in the Blm^tm1Ches/Blm^tm3Brd mice when compared to Blm^tm3Brd heterozygous mice or wildtype mice. Molecular characterization of Hprt gene in the mutant clones indicates that many of the mutations were caused by deletions that range from several base pairs to several thousand base pairs. While deletions in BLM-proficient somatic cells are often shown to be mediated by direct repeats, all three deletion junctions in Hprt of Blm^tm1Ches/Blm^tm3Brd mice were flanked by inverted repeats, suggesting that secondary structures formed during DNA replication, when resolved improperly, may lead to deletions. In addition, single base pair substitution and insertion/deletion were also detected in the mutant clones. Taken together, our results indicated that BLM function is important in preventing small scale genetic alterations. Thus, both large scale and small scale genetic alterations are elevated when BLM is reduced, which may contribute to loss of function of tumor suppressor genes and subsequent tumorigenesis.     

AccuTyping: new algorithms for automated analysis of data from high-throughput genotyping with oligonucleotide microarrays

Microarray-based analysis of single nucleotide polymorphisms (SNPs) has many applications in large-scale genetic studies. To minimize the influence of experimental variation, microarray data usually need to be processed in different aspects including background subtraction, normalization and low-signal filtering before genotype determination. Although many algorithms are sophisticated for these purposes, biases are still present. In the present paper, new algorithms for SNP microarray data analysis and the software, AccuTyping, developed based on these algorithms are described. The algorithms take advantage of a large number of SNPs included in each assay, and the fact that the top and bottom 20% of SNPs can be safely treated as homozygous after sorting based on their ratios between the signal intensities. These SNPs are then used as controls for color channel normalization and background subtraction. Genotype calls are made based on the logarithms of signal intensity ratios using two cutoff values, which were determined after training the program with a dataset of approximately 160,000 genotypes and validated by non-microarray methods. AccuTyping was used to determine >300,000 genotypes of DNA and sperm samples. The accuracy was shown to be >99%. AccuTyping can be downloaded from http://www2.umdnj.edu/lilabweb/publications/AccuTyping.html.     

Low-molecular peptides from tissues of hibernating and cold-adapted animals influence cell proliferation in the Ehrlich ascitic carcinoma

Polypeptides with cytostatic activity are known to be present in animal tissues during winter dormancy. A 1-10 kDa polypeptide fraction with cytostatic activity was obtained from brain tissue of hibernating ground squirrels and cold-adapted Yakut horses. The pattern of cytostatic activity of this fraction towards tumor cells is of great interest. We present results testifying to cytostatic activity of this fraction towards the Ehrlich ascitic carcinoma cells. The cytostatic effect is realized in tumor cells at the genetic level.     

TGFBI gene mutations in the Ukrainian patients with inherited corneal stromal dystrophies

Mutations Arg124Cys, Thr538Arg, Arg555Thr, Arg555Gln, Leu558Pro, and His626Arg in TGFBI gene were analyzed by polymerase chain reaction and restriction in 84 patients with various forms of corneal stromal dystrophies from 49 unrelated families and 29 clinically healthy relatives of these patients. A new mutation in TGFBI gene, Leu558Pro, was identified in the patients with atypical lattice dystrophy. The haplotypes of four microsatellite markers surrounding TGFBI gene region were analyzed in 22 families. The data on association of genotype and phenotype suggest that the analysis of TGFBI gene mutations is important for differential diagnostics of corneal dystrophies.