Glucocorticoid-mediated degradation of DNA in thymocytes of rats with transplantable Zajdela ascites hepatoma

Tumour growth was shown to be associated with DNA breakdown in thymocytes of rats bearing Zajdela ascites hepatomas. The tumour action on the thymus is mediated through adrenal glands since bilateral adrenalectomy completely prevents DNA breakdown in thymocytes. Using Southern hybridization of DNA genome with probes for histone, ribosomal and heat shock gene (hsp 70), it was shown that the degradation products of specific DNA sequences are as heterogenous as those of total DNA, although marked differences in appearance of nucleosomal ladder were seen. These data were interpreted to indicate different patterns of DNA breakdown in dying thymocytes. DNA breakdown in thymocytes in vivo and in isolated rat liver nuclei in vitro seems to proceed by similar mechanisms.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Extramedullary hematopoiesis in a case of benign mixed mammary tumor in a female dog: cytological and histopathological assessment

Backgroud  

Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case.     

Mutation scanning of short amplicons: optimization of DNA melting conditions

High resolution melting analysis (HRMA) using special "saturating" fluorescent dyes is a new and very effective approach to genotyping and mutation scanning. HRMA, which is carried out usually just after PCR without any intermediate manipulations (the "closed tube" format), is simple and high-throughput method excluding sample cross-contaminations. The "closed tube" format makes, however, HRMA dependent on PCR mixes and, as such, limits its capability. The "open tube" format (post-PCR amplicon shortening and optimization of the ionic medium) proposed by us earlier, although somewhat more laborious, significantly increases sensitivity of the method and makes it possible to scan mutations in the short amplicons using conventional SYBR Green I dye and a standard (not adapted specifically for HRMA) real-time PCR instrument. Detection of mutant K-RAS in DNA of clinical specimens (tumor tissues, formalin-fixed paraffin-embedded samples) reveals equal, at least, sensitivity of this method as compared with the HRMA and much higher as compared with Sanger sequencing. The problem of false-negative results in mutation scanning of K-RAS, which is highly important in some forms of cancer, is discussed.     

Study of insulin receptors on liver and hepatoma cell plasma membranes]

Plasma membranes (PM) of helathy rats as well as those hepatomas and PM of tumor-bearing rats were found (radioreceptor assay) to have apparently two groups of receptors for insulin with a high and low affinity for the hormone respectively and different insulin-binding capacities. Kass values of the receptors with a high affinity for insulin are drastically decreased in the PM of ascites Zajdela hepatoma (AZH) and of solid hepatoma 27 (SH-27) as well as in the liver of SH-27-bearing animals, but not in the liver of the AZH-bearing rats. Kass values of the receptors with a low affinity for insulin in the PM of AZH and in those of the liver of AZH-bearing rats appear nearly normal. In contrary, above Kass the receptors in the PM of SH-27 and SH-27-bearing animals are significantly decreased. The insulin-binding capacity of the receptors with a high affinity for the hormone in the PM of SH-27, AZH and the liver of both tumor-bearing rats is shown to be significantly higher than that in the PM of normal animals. The same property of the receptors with a low is affinity in the PM of the hepatomas and theliver of their hosts is also increased, especially in the PM of SH-27 and of the liver of SH-27-carring rats.     

Scanning of mutations in short amplicons: Optimization of DNA melting method

The high resolution melting analysis (HRMA) is a new highly efficient method for genotyping and mutation scanning. HRMA is conducted immediately after PCR in closed-tube format, which enables the high throughput of the method. However, the closed-tube format makes HRMA dependent on the conditions of PCR and, thus, limits its capabilities. The open-tube format, which we have already developed (postamplification shortening of amplicons and optimized composition of ion medium), is applicable to the scanning of mutations of the K-RAS oncogene in tumor tissue and formalin-fixed paraffin-embedded samples. It is found that the open-tube format of DNA melting significantly increases the sensitivity of finding mutant alleles when using instruments both with and without an HRMA module. The higher sensitivity of the DNA melting compared to “Sanger” sequencing allows one to decrease the number of false-negative results of the mutation test, which is highly important for some forms of cancer.     

DNA melting analysis: application of the "open tube" format for detection of mutant KRAS

High-resolution melting (HRM) analysis is a very effective method for genotyping and mutation scanning that is usually performed just after PCR amplification (the “closed tube” format). Though simple and convenient, the closed tube format makes the HRM dependent on the PCR mix, not generally optimal for DNA melting analysis. Here, the “open tube” format, namely the post-PCR optimization procedure (amplicon shortening and solution chemistry modification), is proposed. As a result, mutation scanning of short amplicons becomes feasible on a standard real-time PCR instrument (not primarily designed for HRM) using SYBR Green I. This approach has allowed us to considerably enhance the sensitivity of detecting mutant KRAS using both low- and high-resolution systems (the Bio-Rad iQ5–SYBR Green I and Bio-Rad CFX96–EvaGreen, respectively). The open tube format, though more laborious than the closed tube one, can be used in situations when maximal sensitivity of the method is needed. It also permits standardization of DNA melting experiments and the introduction of instruments of a “lower level” into the range of those suitable for mutation scanning.     

Tube gel isotachophoresis: A method for quantitative isolation of nucleic acids from diluted solutions

The technique of isotachophoresis is intended for separation of molecules having different electrophoretic mobilities in a nonhomogeneous electric field. Since the mobility of nucleic acids in water solutions is uniform and does not depend on their size (because of a uniform distribution of negatively charged phosphate groups along the molecule), isotachophoresis will concentrate rather than separate them in the mobile borderline zone between the rapid (Cl⁻) and the slow (β-alanine⁻) anions. This idea served as the basis for elaboration of a novel method for isolation of nucleic acids from diluted solutions. Advantages of the method include quantitative yield (regardless of molecule size), high degree of concentration, and the ability to visually monitor the process. The method may find applications in nucleic acid isolation from highly degraded forensic and clinical samples, from bodily fluids in particular, and thereby promote development of this important direction of diagnostics.