Obstacles and limitations of transfer factor biological activity assay design.

Transfer factor (TF) is a heterogeneous mix of low-molecular weight molecules obtained from dialyzed leukocyte extract that is capable of transferring cell-mediated immunity. As an immunostimulatory drug TF is used to improve treatment of infectious diseases, allergies, cancer and immune deficiencies. The main benefit of TF preparations as immunotherapeutic agents is the induction of a rapid immune response and the potential of TF as an adjuvant in combination with other drugs might lead to development of novel approaches to combat various diseases in the future. The process of TF preparation is rather simple. However, with respect to fact that TF is composed by several multifunction molecules, it is likely that during the activity measurement based only on one single parameter, other TF biological activities might be overlooked. In addition, separated TF components might display synergetic activity effect. According to recent European Pharmacopoeia there is no general protocol for immuno-stimulatory drugs (including TF) activity measurement available. Nevertheless, for the process of TF preparation, control of input material and for final pharmaceutical product batches it is inevitable to guaranty proper quality control including appropriate in vivo or in vitro test(s) for TF biological activity assay. The animal-origin materials and in vivo assays convey a considerable logistic, ethic and economic problem, meanwhile the available in vitro assays have been reported with limited reproducibility and sometimes contradictory results. The currently used method for testing biological activity of TF is the in vitro MTT cells proliferation assay that is recognized by control authorities in Slovak Republic. Keywords: immune system; transfer factor; dialysable leukocyte extract; diseases; MTT cells proliferation assay.

Enhanced metabolism as a common feature of cancer plasticity.

Cancer cells often rely on glycolytic metabolism in order to fulfill high demands of ATP and macromolecules for the sustained growth and proliferation. However, glycolysis is not necessarily the main source of energy for all cancer cells. Some of them rather depend on glutamine or lactate that favor the utilization of oxidative metabolic pathway. Different employment rate of metabolism creates variable products that participate in the formation of environmental milieu, which in turn triggers broad spectrum of cellular signaling pathways leading to migration, invasion, or proliferation. In this review we discuss different metabolic pathways promoted in tumor cells and describe the possibilities of their targeting as therapeutic strategies.

Ovine herpesvirus 1 (OVHV-1) thymidine kinase locus sequence analysis: evidence that OVHV-1 belongs to the Macavirus genus of the Gammaherpesvirinae subfamily.

The HindIII-HincII fragment of the 5.5 kbp H11 HindIII clone of ovine herpesvirus 1 (OvHV-1) was cloned and its primary structure was determined by preparation of nested deletion subclones and their sequencing. Sequence analysis of the overlapping clones revealed that 3239 bp OvHV-1 fragment contains complete thymidine kinase (TK) gene, a partial open reading frame of ORF20 and that encoding glycoprotein H (gH). The conserved OvHV-1 TK displayed the highest similarity to homologous TK proteins encoded by members of the Macavirus genus of the Gammaherpesvirinae subfamily. These data including our previous analysis of the partial sequence of VP23 homologue might serve as further evidence that OvHV-1 should be categorized within the genus Macavirus of the Herpesviridae family.

Antioxidant enzymes in barley green biomass.

Green biomass of young barley plants exhibited statistically significant higher activity of superoxide dismutase (SOD) and catalase (CAT) at sampling I (in the phase of plant development DC 29) compared to the later sampling II (DC 31). Significant effects of varieties, years and interactions of the studied factors on the activity of the studied antioxidants were determined. During the experiment period (2005-2007), the variety Sebastian provided statistically significant higher average SOD activity (486 U.g-1) versus the variety Malz (416 U.g-1 dry matter) and line KM1910 (418 U.g-1 dry matter). No statistically significant difference was recorded between the latter two varieties. Average catalase activity of the varieties did not show any significant difference. Significantly higher CAT activity in the sampling I was recorded on average of years and locations in the variety Sebastian and hull-less line KM1910 (935 and 907 U.g-1) compared to the variety Malz (675 U.g-1). We can state that green biomass of young spring barley plants taken during the growth phase DC 29 was a significant source of enzymes catalase and superoxide dismutase in the course of the experiment (2005-2007).

Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2.

Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance.

Alternative splicing variant of the hypoxia marker carbonic anhydrase IX expressed independently of hypoxia and tumour phenotype.

CA IX is a hypoxia-induced, cancer-associated carbonic anhydrase isoform with functional involvement in pH control and cell adhesion. Here we describe an alternative splicing variant of the CA9 mRNA, which does not contain exons 8-9 and is expressed in tumour cells independently of hypoxia. It is also detectable in normal tissues in the absence of the full-length transcript and can therefore produce false-positive data in prognostic studies based on the detection of the hypoxia- and cancer-related CA9 expression. The splicing variant encodes a truncated CA IX protein lacking the C-terminal part of the catalytic domain. It shows diminished catalytic activity and is intracellular or secreted. When overexpressed, it reduces the capacity of the full-length CA IX protein to acidify extracellular pH of hypoxic cells and to bind carbonic anhydrase inhibitor. HeLa cells transfected with the splicing variant cDNA generate spheroids that do not form compact cores, suggesting that they fail to adapt to hypoxic stress. Our data indicate that the splicing variant can functionally interfere with the full-length CA IX. This might be relevant particularly under conditions of mild hypoxia, when the cells do not suffer from severe acidosis and do not need excessive pH control.

Sequence analysis of the regions flanking terminal repeats of the genome of umava isolate of murine gammaherpesvirus.

The umava isolate of murine gammaherpesvirus (MHV-umava) slightly differs from Murine gammaherpesvirus 68 (MHV-68) and two other isolates of murine gammaherpesvirus (MHV), MHV-76 and MHV-72 in some biological properties. To identify the region(s) in the MHV-umava genome responsible for this phenomenon, we compared the sequences flanking terminal repeats (TRs) of the MHV-umava genome with those of MHV-68, MHV-76 and MHV-72. Restriction and sequence analyses revealed in MHV-umava as compared to MHV-68 approximately 9.3 kbp deletion at the left end of the genome and approximately 1.5 kbp deletion at the right end of the genome. While the approximately 9.3 kbp deletion was similar to that in MHV-76, the approximately 1.5 kbp deletion was unique for MHV-umava.

Natural inactivation of phosphorus by aluminum in atmospherically acidified water bodies.

Atmospheric acidification of catchment-lake ecosystems may provide natural conditions for the in-lake control of P cycling. This process is based on the elevated transport of aluminum from acidified soils and its subsequent precipitation in the water body and is described for strongly acidified forest lakes, acidified and circumneutral reservoirs, and a moderately acidified alpine lake. In water bodies with episodically or permanently acidified inflows a pH gradient develops between lake water and tributaries due to: (i) neutralization of acidic inflows after mixing with waters with undepleted carbonate buffering system, and/or (ii) the in-lake alkalinity generation dominated by biochemical removal of NO3- and SO4(2-). With the pH increasing towards neutrality, ionic Al species hydrolyze and form colloidal Al hydroxides (Al(part)) with large specific surfaces and strong ability to bind orthophosphate from the liquid phase. Moreover, Alpart settles and increases the P sorption capacity of the sediment. The presence of Al(part) on the bottom reduces orthophosphate release from sediments after its liberation from ferric oxyhydroxides during anoxia because Al(part) is not sensitive to redox changes. Consequently, the natural in-lake P inactivation may be expected in any water body with elevated Al input and a pH gradient between its inlet and outlet.

PrP fragment 106-126 is toxic to cerebral endothelial cells expressing PrP(C).

A hydrophobic, fibrillogenic peptide fragment of human prion protein (PrP106-126) had in vitro toxicity to neurons expressing cellular prion protein (PrP(C)). In this study, we proved that primary cultures of mouse cerebral endothelial cells (MCEC) express PrP(C). Incubation of MCEC with PrP106-126 (25-200 microM) caused a dose-dependent toxicity assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase release, bis-benzimide staining for nuclear morphology, and trypan blue exclusion test. Pentosan polysulphate (50-100 microg/ml), a drug effective in scrapie prophylaxis, dose-dependently attenuated the injury. MCEC cultures from mice homogenous for the disrupted PrP gene were resistant to the toxicity of PrP106-126. In conclusion, cerebral endothelium expressing PrP(C) may be directly damaged during spongiform encephalopathies.

Physiological expression of the gene for PrP-like protein, PrPLP/Dpl, by brain endothelial cells and its ectopic expression in neurons of PrP-deficient mice ataxic due to Purkinje cell degeneration.

Recently, a novel gene encoding a prion protein (PrP)-like glycoprotein, PrPLP/Dpl, was identified as being expressed ectopically by neurons of the ataxic PrP-deficient (PRNP(-/-)) mouse lines exhibiting Purkinje cell degeneration. In adult wild-type mice, PrPLP/Dpl mRNA was physiologically expressed at a high level by testis and heart, but was barely detectable in brain. However, transient expression of PrPLP/Dpl mRNA was detectable by Northern blotting in the brain of neonatal wild-type mice, showing maximal expression around 1 week after birth. In situ hybridization paired with immunohistochemistry using anti-factor VIII serum identified brain endothelial cells as expressing the transcripts. Moreover, in the neonatal wild-type mice, the PrPLP/Dpl mRNA colocalized with factor VIII immunoreactivities in spleen and was detectable on capillaries in lamina propria mucosa of gut. These findings suggested a role of PrPLP/Dpl in angiogenesis, in particular blood-brain barrier maturation in the central nervous system. Even in the ataxic Ngsk PRNP(-/-) mice, the physiological regulation of PrPLP/Dpl mRNA expression in brain endothelial cells was still preserved. This strongly supports the argument that the ectopic expression of PrPLP/Dpl in neurons, but not deregulation of its physiological expression in endothelial cells, is involved in the neuronal degeneration in ataxic PRNP(-/-) mice.

Characterization of RKZ isolate of ovine herpesvirus 1.

Cytopathic effect (CPE) characterized mainly by foci of rounded cells was observed in cultures of primary plexus choroideus cells from healthy lamb following cryopreservation. It was possible to transmit the infectious agent to other primary cells of ovine origin by co-cultivation with infected cells. By indirect immunofluorescence microscopy it was found that high percentage of sheep (65-80% in 3 different herds from Slovakia) are infected with this infectious agent. Electron microscopy of cells with CPE revealed the presence of herpesvirus particles. Viral DNA was isolated from infected cells using pulse-field gel electrophoresis and further used as probe in Southern blot analysis. The probe reacted specifically only with DNA from cells infected with Ovine herpesvirus 1 (OvHV-1) but not with DNA of other ruminant herpesviruses. Some of the HindIII restriction fragments of DNA of the obtained OvHV-1 isolate denominated RKZ were cloned. Part of the H9 clone was sequenced identifying a gene that encoded a polypeptide homologous to conserved herpesvirus VP23 structural protein. From comparison of the sequence of this clone with VP23 sequences of other herpesviruses it was deduced that OvHV-1 might be classified within the Rhadinovirus genus of the Gammaherpesvirinae subfamily. The sequencing of the H9 clone of DNA of RKZ isolate enabled establishment of sensitive and highly specific polymerase chain reaction (PCR) assay for detection of OvHV-1.

Glycoprotein gD of MDV lacks functions typical for alpha-herpesvirus gD homologues.

Glycoprotein D (gD) belongs to family of conserved structural proteins of alpha-herpesviruses. During productive infection of cells by herpes simplex virus 1 (HSV-1) gD has several important functions, is involved in virus penetration to and release from infected cells and is one of main targets of neutralizing antibodies. Similar functions are shared also by other alpha-herpesvirus gD homologues. Surprisingly, in previous studies it was found that MDV gD expression could not be detected during infection in vitro using immunological methods. In this study we have analyzed expression of MDV gD and its biological consequences. In vitro expression using rabbit reticulocyte lysate and/or overexpression in transfected cells showed that the second ATG codon is required for synthesis of mature, glycosylated gD. In addition, it was found that gD overexpression is neither toxic for transfected cells nor is involved in membrane fusion. After MDV infection of a proprietary cell line stably transfected with plasmid overexpressing MDV gD, no viral particles could be found in culture. On the other hand, cells overexpressing the MDV gD were sensitive to MDV infection in similar way as parental, non-transfected cells. From our study and results of other authors we propound the following conclusions: (i) MDV gD expression is blocked during in vitro infection at transcription level; (ii) MDV gD is lacking many important functions characteristic for other alpha-herpesvirus gD homologues; (iii) overexpression of single MDV gD does not result in production of mature infectious MDV particles.

Identification and characterization of a cDNA clone derived from the Marek's disease tumour cell line RPL1 encoding a homologue of alpha-transinducing factor (VP16) of HSV-1.

We have identified and sequenced a 2.3 kb cDNA clone RPL(N.S) 6 derived from the Marek's disease virus (MDV)-transformed cell line RPL1, which contained open reading frames (ORFs) homologous to UL49 (VP22) and UL48 (VP16) of herpes simplex virus. Northern blot hybridization identified a 2.5 kb transcript corresponding to this cDNA clone in the total RNA from MSB1 lymphoblastoid cells, but not in RNA from the original RPL1 cells, most probably due to the very low level of its transcription. In vitro translation demonstrated that both MDV UL49 and UL48 can be expressed from a single mRNA.

The 132 bp repeats are present in RNA transcripts from 1.8 kb gene family of Marek disease virus-transformed cells.

Marek's disease virus (MDV) is an oncogenic, lymphotropic herpesvirus of chickens: Loss of its tumourigenic potential is believed to be associated with amplification of the 132 bp repeats from BamHI-D and BamHI-H fragments. We prepared cDNA libraries from RPL1 and MSB1 cell line and from the latter we identified a clone which spanned the 132 bp repeats within the BamHI-H fragment. By sequencing and Northern blot analysis we confirmed the presence of the 132 bp repeats. The analysis by PCR made on the total RNA revealed two 132 bp repeats in MDV transcripts from RPL1 and two to three repeats in transcripts from MSB1 cells. These results show that sequences within the 132 bp repeats are transcribed and are not spliced out as previously reported.