Purification and partial characterization of goose ceruloplasmin

The preparation and properties of ceruloplasmin from goose blood plasma are described. Ammonium sulfate was used to precipitate the crude protein followed by adsorption and elution from DEAE-Sephadex A-50. Further treatment with an ethanol-chloroform mixture and Sephadex G-200 yielded an intensely blue protein possessing a high degree of chemical purity and biological activity. Goose ceruloplasmin, existing in two forms, appears to be a single polypeptide, apparent Mr121,300, with an A610/A280 ratio of 0.07. Copper represented 0.32%, which corresponded to six atoms of copper per protein molecule. Although the amount of EPR-detectable copper was the same as in mammalian ceruloplasmins there were some differences in EPR parameters, mainly in A parallel. Goose ceruloplasmin's amino acid composition, although similar in many residues to human ceruloplasmin, was lower in tyrosine, cystine/cysteine, and acidic amino acids. Valine was found as the N-terminal amino acid. Hexose, hexosamine, sialic acid, and fucose accounted for 6.65% of the weight. Goose protein contained only half the sialic acid of human ceruloplasmin. Two values for Km using either p-phenylenediamine (0.64 and 0.053 mM) or o-dianisidine (0.76 and 0.15 mM) were evaluated from Lineweaver-Burk plots. EPR studies on reactions with water radiolysis products at cryogenic temperatures allowed us to discover that goose ceruloplasmin, like human and bovine ceruloplasmins, possesses superoxide dismutase activity.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

Effect of Solcoseryl on nerve tissue under in vitro conditions]

Explants from trigeminal ganglia and skin of chick embryos and hippocampus from fetal rats were cultivated in Maximow assembly in the presence of Solcoseryl (Solco AG, Basel), a blood extract of calf. Solcoseryl in vitro did not influence the regeneration of nerve fibers from CNS explants. A stimulatory effect of Solcoseryl in vitro by 1% concentration on the outgrowth of new processes in explants of PNS was demonstrated. It is discussed: under optimal concentration Solcoseryl may be important for the influence of the composition of the medium in which explants of the nerve system and skin are cultivated.     

Nitric oxide complexes in the interaction between primary and secondary tumor of L5178Y lymphoma.

Heme and non-heme Fe-NO complexes were observed in regard to the growth of primary and secondary solid tumors and ascites of murine L5178Y lymphoma. The complexes were detected by electron paramagnetic resonance spectroscopy at liquid nitrogen temperature. Primary solid tumors and secondary solid tumors or ascites were inoculated on the same day, or with a delay. The primary tumor inhibited growth of the secondary solid tumor only if the latter was inoculated with a delay, which did not correlate with the change of the types, nor with the increase in the level of Fe-NO complexes detected in the tissue, suggesting a "non-immunological" character of this inhibition. In some animals with solid tumors, spontaneous ascites developed. This process resulted in a marked decrease in the level of Fe-NO complexes in the solid tumor tissue. The primary solid tumor, however, did not influence the growth of secondary ascites, but intensified NO generation in the ascites of animals with partial removal of ascitic fluid. This experimental group survived 2.2 days longer than the control group without primary solid tumor. Our research revealed that the presence of Fe-NO complexes in the interaction between primary and secondary tumor strongly depends on the form of the tumor: solid or ascitic, and that murine L5178Y lymphoma may serve as a convenient model for the research on "concomitant immunity" against in vivo growing tumors. This is the first EPR study on "concomitant immunity" in regard to tumor-tumor and tumor-ascites interactions in vivo.     

X-band and S-band EPR detection of nitric oxide in murine endotoxaemia using spin trapping by ferro-di(N-(dithiocarboxy)sarcosine)

Ammonium salt of N-(dithiocarboxy)sarcosine (DTCS) chelated to ferrous salt was tested as an NO-metric spin trap at room temperature for ex vivo measurement of (.)NO production in murine endotoxaemia. In a chemically defined in vitro model system EPR triplet signals of NO-Fe(DTCS)(2) were observed for as long as 3 hours, only if samples were reduced with sodium dithionite. This procedure was not necessary for the ex vivo detection of (.)NO in endotoxaemic liver homogenates at X-band or in the whole intact organs at S-band, whereas only a weak signal was observed in endotoxaemic lung. These results suggest that in endotoxaemia not only high level of (.)NO, but also the redox properties of liver and lung might determine the formation of complexes of (.)NO with a spin trap. Nevertheless, both S- and X-band EPR spectroscopy is suitable for (.)NO-metry at room temperature using Fe(DTCS)(2) as the spin trapping agent. In particular, S-band EPR spectroscopy enables the detection of (.)NO production in a whole organ, such as murine liver.     

Implication of gastrin in cyclooxygenase-2 expression in Helicobacter pylori infected gastric ulceration

Gastroduodenal ulcerations have worldwide distribution and the infection with Helicobacter pylori (HP) has been implicated in pathogenesis of this disease. The HP infection is usually accompanied by hypergastrinemia and enhanced generation of prostaglandins (PG), both implicated in the pathogenesis of peptic ulcerations but no study has been undertaken to assess the relationship between the HP infection and coexpression of gastrin and cyclooxygenases (COX), the rate limiting enzymes in the PG production. Since HP infection, usually accompanying peptic ulcerations, results in increased release of gastrin, a potent gastric mitogen that might be capable to induce COX-2 and to generate PG, we decided 1) to compare the seroprevalence of HP and its cytotoxic protein, CagA, in gastric ulcer patients with those in age- and gender-matched controls; 2) to determine the gene expression of gastrin and its receptors (CCK(B)-R) at the margin of gastric ulcer and in the mucosa of antrum and corpus before and after successful eradication of HP, 3) to assess the plasma levels and gastric luminal contents of gastrin before and after HP eradication and 4) to examine the mRNA and enzyme protein expression of COX-1 and COX-2 as well as the PGE2 generation in ulcer margin tissue and gastric antral and fundic mucosa before and after the HP eradication. The trial material included 20 patients with gastric ulcer and 40 age- and gender-matched controls. Anti-HP and anti-CagA IgG seroprevalence was estimated by specific antisera using ELISA tests. Gene expressions of gastrin, CCK(B)-R, COX-1 and COX-2 were examined using RT-PCR with beta-actin as a reference and employing Western blotting for COX-2 expression, while gastrin and PGE2 were measured by RIA. All gastric ulcers were located at smaller curvature within the antral mucosal area. The seroprevalence of HP, especially that expressing CagA, was significantly higher in gastric ulcers (85%) than in controls (62.5%). Both gastrin and CCK(B)-R mRNA were detected by RT-PCR in ulcer margin and gastrin mRNA was overexpressed in remaining antral mucosa, while CCK(B)-R mRNA was overexpressed in fundic mucosa of HP infected patients. Similarly, COX-2 mRNA and protein were found in margin of gastric ulcer and in the HP infected antral and fundic mucosa but not in the mucosa of HP eradicated patients in whom ulcers completely healed and gastrin was expressed only in antrum, CCK(B)-R only in corpus, while COX-1 was detected both in antrum and corpus. HP positive gastric ulcer patients showed about three times higher levels of plasma immunoreactive gastrin and about 50% higher luminal gastrin contents than the HP negative controls and this increased plasma and luminal gastrin was normalized following the HP eradication. A significant fall in gastrin and CCK(B)-R mRNA expression was noticed six weeks after HP eradication in gastric antral and fundic mucosa, while COX-2 mRNA completely disappeared after this treatment. We conclude that 1) HP infected gastric ulcer margin coexpresses gastrin, its receptors (CCK(B)-R), and COX-2; 2) HP infection may be implicated in gastric ulceration via increased release of gastrin that could be responsible for the overexpression of COX-2 that in turn could help ulcer healing through the stimulation of mucosal cell growth, restoration of the glandular structure and angiogenesis in the ulcer area and 3) gastrin produced in HP infected antral mucosa seems to be involved in the induction of COX-2 and PG production by this enzyme and this may contribute to the ulcer healing.     

Bacteria in cancer therapy: a novel experimental strategy

Resistance to conventional anticancer therapies in patients with advanced solid tumors has prompted the need of alternative cancer therapies. Moreover, the success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity to normal tissues. Several decades after Coley's work a variety of natural and genetically modified non-pathogenic bacterial species are being explored as potential antitumor agents, either to provide direct tumoricidal effects or to deliver tumoricidal molecules. Live, attenuated or genetically modified non-pathogenic bacterial species are capable of multiplying selectively in tumors and inhibiting their growth. Due to their selectivity for tumor tissues, these bacteria and their spores also serve as ideal vectors for delivering therapeutic proteins to tumors. Bacterial toxins too have emerged as promising cancer treatment strategy. The most potential and promising strategy is bacteria based gene-directed enzyme prodrug therapy. Although it has shown successful results in vivo yet further investigation about the targeting mechanisms of the bacteria are required to make it a complete therapeutic approach in cancer treatment.     

Antigens HLA-G, sHLA- G and sHLA- class I in reproductive failure

It can be supposed that relation between HLA-G polymorphism and sHLA-G protein expression are associated with successful embryo implantation and pregnancy maintenance. The aim of the study was the estimation specific differences in expression of sHLA-G and sHLA- class I antigens in women with reproductive failure in comparison with fertile women. The study sample enrolled 80 women, divided into 2 groups. The study group (B) enrolled 60 women with reproductive failure including 20 women with 3 recurrent spontaneous abortions in the first trimester of pregnancy (RSA), 20 women with empty sac (ES) and 20 women with 3 consecutive in-vitro fertilization failures (IVFf). The control group (C) enrolled 20 fertile women with at least 2 children. Soluble HLA- class I antigens (sHLA-I) and soluble HLA-G (sHLA-G) were determined using ELISA test kits from IBio Vendor Labolatory Medicine, Inc. HLA-G allele found in individuals in our study were identified by comparing the obtained bp sequences of exon 2., 3. and 4. with bp sequences of HLA-G antigen published at the Nolan Research Institute website. The highest concentration of sHLA-I is noted among women with HLA-G 10401 allele which differed significantly for the mean sHLA-I concentration calculated for all the remaining alleles (p<0.0001). The most prevalent alleles were: HLA-G 10101, 10102 and 10108 with sHLA-I concentrations among women bearing those alleles significantly lower in comparison to the HLA-G 10401 carriers (p<0.001). Allele 10101 and 10102 was related to the lower significantly plasma sHLA-I concentrations than 10108 allele (p<0.02). Lowest mean sHLA-G values were observed in the IVFf group with significant difference from the remaining groups (p<0.05). To conclude, sHLA-G molecules is associated to certain HLA-G alleles and imply that sHLA-G levels are under genetic control. Low concentration sHLA-G seems to be prognostically important in IVF failure.     

A Highlights from MBoC Selection: Peptide TFP5/TP5 derived from Cdk5 activator P35 provides neuroprotection in the MPTP model of Parkinson’s disease

Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. Recent evidence indicates that cyclin-dependent kinase 5 (Cdk5) is inappropriately activated in several neurodegenerative conditions, including PD. To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity. Previously we reported that TFP5 peptide has neuroprotective effects in animal models of Alzheimer’s disease. Here we show that TFP5/TP5 selective inhibition of Cdk5/p25 hyperactivation in vivo and in vitro rescues nigrostriatal dopaminergic neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) in a mouse model of PD. TP5 peptide treatment also blocked dopamine depletion in the striatum and improved gait dysfunction after MPTP administration. The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflammation and apoptosis. Here we show selective inhibition of Cdk5/p25 ­hyperactivation by TFP5/TP5 peptide, which identifies the kinase as a potential therapeutic target to reduce neurodegeneration in Parkinson’s disease.