Effects of divalent metal ions on the alphaB-crystallin chaperone-like activity: spectroscopic evidence for a complex between copper(II) and protein

AlphaB-crystallin is a small heat shock protein, showing chaperone-like activity, that is expressed in the lens and in several other tissues. The role of some metal ions in the alphaB-crystallin biology starts to be well documented. In some neuro-degenerative pathologies, like Parkinson and Alzheimer's diseases, alphaB-crystallin is expressed at high levels. In the same pathologies an accumulation of divalent metal cations is observed. In order to investigate the interactions between human alphaB-crystallin and divalent metal ions, the effect of copper, zinc and calcium on the chaperone-like activity of the protein has been studied. Copper and zinc at concentrations 0.1 and 1 mM significantly increase the chaperone-like activity, whereas calcium 1 mM completely inhibits activity. Electron paramagnetic resonance (EPR) and circular dichroism (CD) spectra indicate the possible complex formation between Cu(II) and protein at physiological pH. Molecular modeling calculations, carried out for the probable Cu(II) binding site, suggest that a complex with three histidine residues is possible.     

Secretion of recombinant human fibrinogen by the murine mammary gland

Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the A, B and fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 g/ml, with total secretion of subunits approaching 700 g/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the B and chains were rate limiting. Both the B and chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for chains over B chains. Also, the subunit complexes ₂, A₂ and the individual subunits A, B and were found as secretion products. When the B was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of B chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.     

Transgenic Mice Expressing Recombinant Human Protein C Exhibit Defects in Lactation and Impaired Mammary Gland Development

To determine if the production of recombinant human protein C (rHPC) could be increased in milk, we created two lines of mice homozygous for the mouse whey acidic protein (WAP)/human protein C (HPC) transgene. Females of both lines had normal growth, activity and fertility, but failed to lactate normally and were unable to raise litters. Histological analyses of mammary glands from lactating homozygous females showed barely distended alveoli filled with dense-staining milk. Epithelial cells within these alveoli had distinct, centrally located nuclei and contained intracellular lipid droplets. Hemizygous animals derived from these lines were able to lactate and raised normal sized litters. Northern blot analysis showed that the 6.4 homozygous (6.4H) line expressed the transgene at higher levels then corresponding hemizygous (6.4) animals, but the 4.2 homozygous (4.2H) line expressed the transgene at lower levels than the 4.2 hemizygous line. The 6.4H line also had increased rHPC levels in the milk as revealed by western blot analysis. The 4.2H, 6.4, and 6.4H lines showed decreased and/or delayed expression of WAP, -casein, and -lactalbumin mRNA's compared to wild type animals during lactogenesis. The 4.2 line showed decreased mRNA expression for -casein and -lactalbumin, but normal or higher expression of WAP during lactogenesis. Elevated levels of some proteins were detected in the milk of transgenic mice. From these results, it is concluded that expression of rHPC induced a lactational phenotype that involves abnormal morphological, biochemical, and functional differentiation of mammary epithelial cells. However, the induction of this phenotype does not appear to be directly related to the level of rHPC mRNA expression, thus suggesting that the basis of this phenotype may involve secondary, rather than primary, effects of rHPC on mammary gland development.Deceased.     

Synthesis and induction of apoptosis in B cell chronic leukemia by diosgenyl 2-amino-2-deoxy-beta-D-glucopyranoside hydrochloride and its derivatives

2-Acetamido-2-deoxy-D-glucose hydrochloride (D-glucosamine hydrochloride) has been used for the preparation of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-trifluoroacetamido-beta- (4) and 2-tetrachlorophthalimido-alpha,beta-D-glucopyranose (6), which have been transformed into the appropriate bromides and the chloride. Both bromo and chloro sugars were used as a glycosyl donors for the glycosylation of diosgenin [(25R)-spirost-5-en-3beta-ol]. These condensations were conducted under mild conditions, using silver triflate as a promoter, and gave diosgenyl glycosides 9 and 12. Each of them was converted into diosgenyl 2-amino-2-deoxy-beta-D-glucopyranoside hydrochloride (11) and N-acylamido derivatives. The structures of all new glycosides were established by 1H and 13C NMR spectroscopy. These diosgenyl glycosides are the first saponins containing the D-glucosamine residue that have been synthesized. These compounds show promising antitumor activities. The synthetic saponins increase the number of apoptotic B cells, in combination with cladribine (2-CdA), that are isolated from chronic lymphotic leukemia (B-CLL) patients.     

Spatial interactions between grey wolves and Eurasian lynx in Białowieża Primeval Forest, Poland

Various species of large predators are reported to influence each other through interference or exploitation competition that may affect demography and survival of the subordinate species. We analyzed spatial relationships between grey wolf (Canis lupus) and Eurasian lynx (Lynx lynx) in Białowieża Primeval Forest (BPF, eastern Poland) to determine how they partitioned the space. The wolves (n = 8) and lynx (n = 14) were radio-tracked in 1991–1999. Three wolves and seven lynx were radio-tracked simultaneously in 1994–1996. Territories of wolf packs and home ranges of lynx overlapped considerably (76% of wolf territories and 50% of lynx home ranges, on average). In three cases, their core areas were also overlapping. Wolf-lynx dyads with overlapping home ranges were simultaneously located at distances from 0 to 28 km from each other. We found neither avoidance nor attraction between wolves and lynx occupying the same areas. We concluded that in BPF, the two large predators coexist due to specialization on different preferred prey and heterogeneous habitat.     

Construction of physical map and mapping of chromosomal virulence genes of the biovar 3 Agrobacterium (Rhizobium vitis) strain K-Ag-1

Most plant pathogenic Agrobacterium strains have been classified into three biovars, "biovar 1 (A. tumefaciens; Rhizobium radiobacter), biovar 2 (A. rhizogenes; R. rhizogenes) and biovar 3 (A. vitis; R. vitis)". The bacteria possess diverse types of genomic organization depending on the biovar. Previous genomic physical maps indicated difference in location of rDNA and chromosomally-coded virulence genes between biovar 1 and 2 genomes. In order to understand biovar 3 genome and its evolution in relation to the biovar 1, 2 and 3 genomes, we constructed physical map of a pathogenic biovar 3 strain K-Ag-1 in this study. Its genome consisted of two circular chromosomes (3.6 and 1.1 Mbp in length), and three plasmids (560, 230 and 70 kbp). Gene mapping based on the physical map showed presence of two rDNA loci in the larger chromosome and at least one rDNA locus in the smaller chromosome. Six chromosomal virulence genes, namely chvA, chvD, chvE, glgP, exoC and ros were found in the larger chromosome and not in the smaller chromosome. The location of rDNA loci is similar with that of biovar 1 genome, whereas the location of chromosomal virulence genes is similar with that of biovar 2 genome despite of the closer 16S-rRNA based phylogenetic relation of biovar 3 with biovar 1 than with biovar 2. Genomic PFGE RFLP analysis revealed that the K-Ag-1 strain, which was isolated on a kiwifruit plant in Japan, has the closest intra-species relation with two strains isolated from grapevine plants in Japan among eight biovar 3 strains examined. This datum suggests that the line of the strain is a major one in biovar 3 in Japan. Evolution of the genome of the strain is discussed based on the data.     

Evaluation of TLR4 expression and chosen parameters of oxidative-antioxidative balance in young children with food allergy

The authors evaluated mRNA TLR4 expression on neutrophils and the chosen parameters of oxidative-antioxidative balance in blood of 35 children with food allergy (17 of them with IgE-dependent allergy and 18 with IgE-independent allergy) and 15 healthy children without any allergy. The age of these children ranged from 1 to 36 months. Children with food allergy in comparison with healthy children were found to have lower mRNA TLR4 expression, higher average value of chemiluminescence (CL) and its increase after stimulation by fMLP, PMA and OZ as well as lower TAS values. Disturbances of oxidative-antioxidative balance were found in children with food allergy. We suggest that natural immunity is involved in the development of food allergy mechanisms. Moreover, chemiluminescence can be used as an additional diagnostic test.     

Anti-neoplastic effect of protein kinase CK2 inhibitor, 2-dimethylamino-4,5,6,7-tetrabromobenzimidazole (DMAT), on growth and hormonal activity of human adrenocortical carcinoma cell line (H295R) in vitro

Several studies indicate the involvement of protein kinases in the progression of various malignancies. Kinase inhibitors are therefore becoming important anticancer drugs. CK2 kinase (casein kinase-2) has been suggested to be a constituent of a neoplastic milleu, and its inhibition might represent a new approach to cancer therapy. Adrenocortical carcinomas (ACCs) are highly malignant neoplasms with poor overall prognosis. We have examined the effects of 2-dimethylamino-4,5,6,7-tetrabromobenzimidazole (DMAT), a potent CK2 inhibitor, on the H295R human adrenocortical cancer cell line. Treatment with DMAT decreases the secretion of aldosterone, dehydroepiandrosterone sulfate, and androstendione and results in an accumulation of 17-OH-progesterone. Cell growth as measured by the MTT and 5-bromo-2′-deoxyuridine incorporation assays is inhibited, and cell cycle analysis has revealed a slight induction of apoptosis. Thus, CK2 kinase activity is probably involved in human ACC endocrine activity and growth.