Seleno-l-methionine and l-ascorbic acid differentiate the biological activity of doxorubicin and its metal complexes as a new anticancer drugs candidate.

The most important problems of anti-cancer therapy include the toxicity of the drugs applied to healthy cells and the multi-drug cells resistance to chemotherapeutics. One of the most commonly used anticancer drugs is doxorubicin (DOX) used to treat certain leukemias and non-Hodgkin's lymphomas, as well as bladder, breast, stomach, lung, ovarian, thyroid, multiple myeloma and other cancers. Preliminary studies showed that metal complex with DOX improve its cytostatic activity with changes in their molecular structure and distribution of electrons, resulting in a substantial change of its biological activity (including antitumor activity). Thus, there is a chance to receiving derivatives of DOX with low toxicity for the healthy body cells, thus increasing its therapeutic selectivity. In the present study we examined the influence of Mn, Mg, Fe, Co and Ni, seleno-l-methionine and vitamin C on biological activity of DOX in prokaryotic model - Escherichia coli RFM443, with plasmid transcriptional fusion of recA promoter and luxCDABE as a reporter gene. Cytotoxic potency of tested chemicals was calculated on the basis of the bacteria culture growth inhibition (GI%) values. Genotoxic properties were calculated on the basis of the fold increase (FI) of relative luminescence units (RLU) values compared to control. Obtained results showed that doxorubicin metal complexes particularly with Ni, Co and Fe increased the cyto- and genotoxic activities of DOX. Bacteria culture supplemented with SeMet and vitamin C differentiate the DOX and its metal complexes toxicity. It seems, that DOX-Ni, DOX-Fe and DOX-Co complexes could be potent cytostatic drug candidates. Moreover, we noticed different sensitivity of recA::luxCDABE for 3 h and 24 h cultures of bacteria strain. It suggests, that the potency of genetic construct reactivity- recA::luxCDABE in E. coli depends on the growth-phase of bacterial culture.

IN VITRO EVALUATION OF BIOLOGICAL ACTIVITY OF CINNAMIC, CAFFEIC, FERULIC AND CHLOROGENIC ACIDS WITH USE OF ESCHERICHIA COLI K-12 RECA::GFP BIOSENSOR STRAIN.

Cinnamic acid and its derivatives are important and promising compounds in cancer therapy, because of its broad spectrum of anicancer and antioxidative ability, and with high potential for development into new generation drugs. The aim of this study was to compare the cyto- and genotoxic effects of cinnamic acid and its derivatives with the use of4Escherichia coli K-12 recA::gfp microbial biosensor strain with plasmid fusion of recA promoter and gfp gene as reporter. Obtained results indicate that recA::gfpmut2 genetic system was a sensitive biosensor to the most chemicals tested in our experiments. The cinnamic acid and its derivatives modulated the reactivity of wcA promoter in relation to control sample and significantly inhibited bacteria cells growth. In the light of our results only chlorogenic and ferulic acids at higher concentrations demonstrated cyto and genotoxic activity toward to E. coli K-12 mcA::gfp cells.

Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs.

The main group of enzymes responsible for the collagen and other protein degradation in extracellular matrix (ECM) are matrix metalloproteinases (MMPs). Collagen is the main structural component of connective tissue and its degradation is a very important process in the development, morphogenesis, tissue remodeling, and repair. Typical structure of MMPs consists of several distinct domains. MMP family can be divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other non-classified MMPs. MMPs and their inhibitors have multiple biological functions in all stages of cancer development: from initiation to outgrowth of clinically relevant metastases and likewise in apoptosis and angiogenesis. MMPs and their inhibitors are extensively examined as potential anticancer drugs. MMP inhibitors can be divided into two main groups: synthetic and natural inhibitors. Selected synthetic inhibitors are in clinical trials on humans, e.g. synthetic peptides, non-peptidic molecules, chemically modified tetracyclines, and bisphosphonates. Natural MMP inhibitors are mainly isoflavonoids and shark cartilage.

CYTOTOXIC AND GENOTOXIC STUDIES OF QUERCETIN, QUERCETIN SODIUM SALT AND QUERCETIN COMPLEXES WITH NICKEL (II) AND ZINC (II).

The aim of this study was to compare the cyto- and genotoxic effects of quercetin, quercetin sodium salt as well as its complexes with nickel (II) and zinc (II) with the use of Escheiichia coli K-12 recA::gfp microbial biosensor strain containing transcriptional fusion between DNA-damage genotoxin-inducible recA promoter involved in the SOS regulon response and fast folding GFP (green fluorescent protein) variant reporter gene - gfpnmt2. Obtained results indicate that recA::gfpmut2 genetic system was a sensitive biosensor to the most of tested chemicals. The complex of quercetin with sodium, nickel and zinc increased (and in some cases modulated) the reactivity of ircA promoter in relation to control sample. The results indicated that E. coli K-12 iecA::gfp mut2 strain could be potentially useful for monitoring of cytotoxic and genotoxic effect of some biological natural compounds, potentially used in anticancer chemoprevention and therapy.

Impact of polymorphism of the regulatory subunit of the µ-calpain (CAPN1S) on the proteolysis process and meat tenderness of young cattle.

The objective of this study was to estimate the impact of the polymorphism of µ-calpain (CAPN1S) gene on protein changes of the cattle muscle tissue and its tenderness during 10-day cold storage. The analysis was performed on the longest dorsal and lumbar muscles collected from 76 bulls 6 to 12 months of age. Polymorphism identification of the above-mentioned gene was conducted using the PCR-RFLP technique. Its effect on the course of the proteolysis process was assessed by monitoring changes in proportions of tissue proteins during 10-day process of meat ageing. Special attention was focused on changes in native titin (T1) share and products of its degradation (proteins of molecular weight (m.w.) of 2400 and 200 kDa), α-actinin and protein of 37 kDa as well as myosin heavy chains (MHC). In the case of the last proteins, their polymorphism was evaluated as well. Meat tenderness was estimated measuring the value of shear force and sensorially. The highest tenderness was ascertained for the heterozygote. Its improvement was associated with a significant decrease in proportions of proteins of molecular weight of approximately 37 kDa accompanied by an increase of those with 200 kDa molecular weight. Muscles derived from cattle of CT genotype were characterised by the highest proportions of type 2a MHC isoform. Value differences between proportions determined for the heterozygote and CC and TT homozygotes of the CAPN1S gene were statistically significant. Therefore, it can be presumed that the process of meat tenderisation was especially connected with MHC polymorphism.

A novel polymorphisms in intron 12 of the bovine calpastatin gene.

Calpastatin (CAST) is a specific inhibitor of the ubiquitous calcium-dependent proteases-mu-calpain and m-calpain, found in mammalian tissues. This proteolytic system plays a key role in the tenderization process that occurs during post-mortem storage of meat under refrigerated conditioning. Fragments of the bovine CAST gene including intron 12 were amplified and subjected to SSCP analysis. Four new SNPs were found within intron 12 of the CAST gene: a transition T/C at position 3893+155* A/G at position 3893+163, a transversion T/A at position 3893+223 and a substitution A/G at position 3893+428 (consensus sequence--GenBank AY834771). The genetic variants in the bovine CAST gene can be analyzed with RFLP method and was studied in 375 bulls of six breeds, including Hereford, Aberdeen-angus, Simmental, Charolaise, Limousine and Polish Black-and-White (BW; Fresian) breeds.

Bovine mu-calpain (CAPN1) gene: new SNP within intron 14.

The calpain system originally comprised molecules: two Ca2+-dependent proteases, mu-calpain and m-calpain, and a third polypeptide, calpastatin, whose only known function is to inhibit the two calpains. This proteolytic system plays a key role in the tenderisation process that occurs during post-mortem storage of meat under refrigerated conditioning. Their polymorphism is examined from the point of view of their effect on corresponding production traits. The calpain genes are investigated as potential candidate genes for a quantitative trait locus (QTL) affecting meat tenderness. In this study a new single nucleotide polymorphism (SNP) was found within intron 14 of the bovine CAPN1 gene, being transition C --> T at position 4685 nt (consensus sequence - GenBank No. AF 248054), as this mutation creates a new FokI restriction site detected with PCR-RFLP analysis. This sequence fragment of the SNP position has already been deposited in the GenBank database under accession No. AY639597. The RFLP-FokI polymorphism was studied in 141 bulls of seven breeds, including the native Polish Red (PR, preserved), and Polish Black-and White (BW) breed. The frequency of alleles T and C varied between the breeds considered, the mean reaching 0.38 and 0.62, respectively. Associations between CAPN1/FokI gene polymorphism and meat production traits were studied in BW (n = 84) young bulls. In the animals of the TT genotype the lean share in valuable cuts (%) was found more favourable than in CC animals.

Noninvasive fluorescent screening of microinjected bovine embryos to predict transgene integration.

Most transgenic domestic animals are generated by direct microinjection of DNA fragments into zygote pronuclei. It has generally been assumed that the majority of integration events should occur prior to the first round of chromosomal DNA replication. The aim of this study was to investigate the expression of GFP in bovine preimplantation embryos by using a gfp reporter gene consisting of chicken beta-actin promoter, the CMV-IE enhancer, gfp cDNA (EGFP) (732 bp) and rabbit beta-globin polyadenylation sequences. In five experiments 302 bovine zygotes were injected while 75 served as a control. The fluorescence intensity was detected at 72 and 168 h following fertilization in bovine embryos injected with 3 ng/microl in experiments 1-3, and injected with 5 ng/microl in experiments 4-5. Eight embryos were considered as expressing green fluorescence protein; 2 of them were 100% fluorescent after microinjection of a higher dose of the DNA; one was 75%, two--50%, and three 25% transgenic. The mosaicism was assumed to be at 75%. The results indicated that the fluorescence could be analyzed at any time of bovine embryo development. It was therefore concluded, that chicken beta-actin promoter together with the CMV-IE enhancer would confer a strong expression of the gfp reporter gene in preimplantation bovine embryos. Therefore, using GFP that could be simply detected in live bovine (transgenic) embryos would be very promising in establishing transgenic lines of domestic animals producing in their fluids human therapeutic proteins.

Green fluorescent protein as a molecular marker in microbiology.

Molecular markers such as: lacZ (b-galactosidase), xylE (catechol 2,3-dioxygenase), lux (bacterial luciferase), luc (insect luciferase), phoA (alkaline phosphatase), gusA and gurA (beta-glucuronidase), gfp (green fluorescent protein), bla (beta-lactamase) and other antibiotic resistance markers, heavy metals resistance genes are commonly used in environmental microorganisms research (Errampaii et al., 1998; Kohler et al., 1999). Most of these markers require one or more substrates, complex media and/or expensive equipment for detection. The gfp gene is widely used as a marker because of its very useful properties such as high stability, minimal toxicity, non-invasive detection and the ability to generate the green light without addition of external cofactors and without application of expensive equipment. Various applications of that reporter gene were showed starting from monitoring of microorganism's survival in complex biological systems such as activated sludge to biodegradation of chemical compounds in soil. GFP allowed the detection, determination of spatial location and enumeration of bacterial cells from diverse environmental samples such as biofilm and water. The gfp as a biomarker was very useful in monitoring of gene expression and protein localisation in bacterial cells, too. The techniques with using gfp marker promise to supply a better understanding of environmental processes. It can make possible to use that knowledge in designing more effective and more efficient methods of biodegradation of toxic compounds from different environments.

Expression of microinjected reporter gene lacZ during first cleavages of rabbit embryos.

The growing use of reporter genes in a model transgenic system has been a fundamental approach of biology, but the strategy of transgenic embryo selection prior to transfer to foster mothers may greately increase the efficiency of transgenic livestock production. This study was conducted to assess the possibility of beta-galactosidase (beta-gal)-labeled transgenic rabbit embryo production. Rabbit zygotes were obtained from superovulated females after mating. Zygotes were microinjected into male pronuclei with pCMV-lacZ or SV40-lacZ constructs; while some embryos were co-injected with the scaffold attachment sequences--SAR. Embryos from control non-injected and microinjected groups were cultured in vitro. After 24, 48, 72, or 96 h of culture the embryos were stained with X-gal for beta-galactosidase. Transgenic embryos produced by pronuclear injection showed a discrete pattern of beta-galactosidase expression. The percentage of transgenesis with pCMV-lacZalone was 1.5, but with SAR sequences it increased to 4.2. In the case of SV40-lacZ construct, the efficiency of transgenesis was 2.3% and 4.1%, respectively. The mosaicism was 66.7% for all embryos injected with both constructs with or without SAR. The highest numbers of 100%-transgenic (non-mosaic) embryos were found in the group co-injected with SV40-lacZ and SAR. Transgenesis was seen as early as 24 h after injection, in four-cell embryos. Most of the microinjected embryos showed delayed development as compared with control. It was concluded that lacZ may serve as a reliable reporter for early transgenic embryo selection in order to produce transgenic animals.