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.     

A comparison of the levels of hepatocyte growth factor in serum in patients with type 1 diabetes mellitus with different stages of diabetic retinopathy

INTRODUCTION: The aim of this study was to evaluate the blood concentration of hepatocyte growth factor (HGF) in patients at various stages of retinopathy. We hypothesised that the high level of HGF found in diabetic patients may be an important marker of retinopathy progression and that HGF level may be an index of the risk of proliferative retinopathy. MATERIAL AND METHODS: The participants in the study were 76 patients with type 1 diabetes mellitus. Of these, 35 patients were without retinopathy and formed Group 1. Of the remaining 41 patients with retinopathy, 20 patients had non-proliferative diabetic retinopathy (NPDR) and formed Group 2, while 21 patients had proliferative diabetic retinopathy (PDR) and formed Group 3. We evaluated the concentration of HGF In the peripheral blood by an enzyme-linked immunosorbent assay. RESULTS: Mean serum concentrations of HGF in the control group were significantly lower than in the type 1 diabetic patients. We found a significant increase in HGF serum concentrations in diabetic patients with PDR compared with the control group. Mean serum HGF concentrations were significantly higher in diabetic subjects with PDR than in diabetic patients without retinopathy. CONCLUSION: HGF concentration is increased in patients with type 1 diabetes mellitus with proliferative retinopathy, and concentrations increase with the progression of retinopathy, suggesting that HGF plays a role in the pathogenesis of proliferative diabetic retinopathy.     

A distinct sequence in the adenine nucleotide translocase from Artemia franciscana embryos is associated with insensitivity to bongkrekate and atypical effects of adenine nucleotides on Ca2+ uptake and sequestration

Mitochondria isolated from embryos of the crustacean Artemia franciscana lack the Ca(2+)-induced permeability transition pore. Although the composition of the pore described in mammalian mitochondria is unknown, the impacts of several effectors of the adenine nucleotide translocase (ANT) on pore opening are firmly established. Notably, ADP, ATP and bongkrekate delay, whereas carboxyatractyloside hastens, Ca(2+)-induced pore opening. Here, we report that adenine nucleotides decreased, whereas carboxyatractyloside increased, Ca(2+) uptake capacity in mitochondria isolated from Artemia embryos. Bongkrekate had no effect on either Ca(2+) uptake or ADP-ATP exchange rate. Transmission electron microscopy imaging of Ca(2+)-loaded Artemia mitochondria showed needle-like formations of electron-dense material in the absence of adenine nucleotides, and dot-like formations in the presence of adenine nucleotides or Mg(2+). Energy-filtered transmission electron microscopy showed the material to be rich in calcium and phosphorus. Sequencing of the Artemia mRNA coding for ANT revealed that it transcribes a protein with a stretch of amino acids in the 198-225 region with 48-56% similarity to those from other species, including the deletion of three amino acids in positions 211, 212 and 219. Mitochondria isolated from the liver of Xenopus laevis, in which the ANT shows similarity to that in Artemia except for the 198-225 amino acid region, demonstrated a Ca(2+)-induced bongkrekate-sensitive permeability transition pore, allowing the suggestion that this region of ANT may contain the binding site for bongkrekate.     

Leptin impairs myogenesis in C2C12 cells through JAK/STAT and MEK signaling pathways

Reduced lean body mass in genetically obese (ob/ob) or anorectic/cachectic subjects prompted us to verify the hypothesis whether leptin, white adipose tissue cytokine, might be a negative organizer of myogenesis. Recombinant leptin (100 ng/mL) stimulated mitogenesis together with the raise in T^202/Y²⁰⁴P-ERK1/2 protein expression. Concomitantly, it impaired cell viability and muscle fiber formation from C2C12 mouse myoblasts. Detailed acute and chronic studies with the use of metabolic inhibitors revealed that both JAK/STAT3 and MEK/MAPK but not PI3-K/AKT/GSK-3β signaling pathways were activated by leptin, and that STAT3 (Y⁷⁰⁵P-STAT3) and MEK (T^202/Y²⁰⁴P-ERK1/2) mediate these effects. In contrary, insulin evoked PI3-K-dependent phosphorylation of AKT (S⁴⁷³) and GSK-3β (S⁹) and insulin surpassed leptin-dependent inhibition of myogenic differentiation in PI3-K-dependent manner. GSK-3β seems to play dual role in muscle development. Insulin-dependent effect on GSK-3β (S⁹P-GSK-3β) led to accelerated myotube construction. In contrary, leptin through MEK-dependent manner caused GSK-3β phosphorylation (Y²¹⁶P-GSK-3β) with resultant drop in myoblast fusion. Summing up, partially opposite effects of insulin and leptin on skeletal muscle growth emphasize the importance of interplay between these cytokines. They determine how muscle mass is gained or lost.     

The effect of peroxynitrite and some antioxidants on the rate of osmotic hemolysis of bovine erythrocytes

Bovine erythrocytes treated with peroxynitrite (ONOO(-)), a cytotoxic species formed in vivo via the reaction of nitric oxide (NO(.)) and the superoxide anion (O2(-.)), show an increased rate of hemolysis on sudden osmotic stress. The increase in the rate was peroxynitrite concentration dependent. In the presence of some antioxidants (uric acid, ascorbic acid, glutathione, melatonin and albumin), this effect was significantly lower, with ascorbic acid as the most efficient antioxidant.     

Analysis of genetic variability and phylogenetic analysis of selected Czech and French strains of rabbit haemorrhagic disease virus (RHDV)

The objective of this study was to analyse the genetic variability and phylogenetic analysis of six strains of rabbit haemorrhagic disease virus (RHDV), including four Czech strains (CAMPV-351, CAMPV-561, CAMPV-562, CAMPV-558) and two French strains (Fr-1, Fr-2), on the basis of a fragment of the VP60 capsid structural protein-coding gene N-terminal region. The results of our own studies were compared to 26 RHDV strains obtained from GenBank. The analysis of the genetic variability of six RHDV strains indicated that the CAMPV-561 strain is the most genetically variable. Less variable were the Fr-1 and Fr-2 strains, while the least variable was CAMPV-351. In turn, the genetic distance among the six analysed strains and 26 strains obtained from GenBank was the greatest for CAMPV-351 and Whn/China [11.3 % according to the observed divergence (OD) method and 12.2 % according to the maximum likelihood (ML) method], while it was the lowest for CAMPV-351 and FRG (0.8 % in both the OD and ML methods). In turn, the scale of the genetic distances among the six analysed strains and five RHDVa strains (99-05, NY-01, Whn/China, Triptis, Iowa2000) ranged from 9.3–10.3 % in the OD method to 10.3–13.7 % in the ML method. The image of phylogenetic dependencies generated for the strains analysed and those obtained from GenBank revealed their distribution to be in five genetic groups (G1–G5), whereas the analysed strains were included in genetic groups 2 and 3.     

Crystallization and preliminary diffraction analysis of Ca2+-calmodulin-drug and apocalmodulin-drug complexes

Ca²⁺-calmodulin is crystallized with two new and potent drugs: a bisindol derivative (KAR-2, 3”-(β-chloroethyl)-2”,4”-dioxo-3,5”-spiro-oxazolidino-4-deacetoxy-vinblastine) with antitumor activity and an arylalkylamine fendiline analogue (N-(3,3-diphenylpropyl)-N'-[1-(3,4-di-n-butoxy-phenyl)-ethyl]-1,3-diaminopropane) with anticalmodulin activity. The crystals diffract beyond 2.8 Å and differ in unit cell parameters from each other as well as from crystals of Ca²⁺-calmodulin or Ca²⁺-calmodulin-ligand complexes, as reported thus far. Attempts to crystallize Ca²⁺-free calmodulin without drugs failed, in consonance with earlier results; however, single Ca²⁺-free calmodulin crystals diffracting beyond 2.5 Å resolution were grown in the presence of KAR-2. Results indicate that binding of the two drugs to apocalmodulin or Ca²⁺-calmodulin may induce unique novel protein conformers, targets of further detailed X-ray studies. © 1997 Wiley-Liss Inc.     

Purification and analysis of a 5kDa component of enamel matrix derivative

High performance liquid chromatography (HPLC) methods were used to analyse a 5kDa component purified from enamel matrix derivative (EMD), the active ingredient in Emdogain, a commercial product for periodontal tissue regeneration. After initial purification by size-exclusion chromatography (SEC) on a 100 cm x 5 cm column (Bio-Gel P-30 Fine, 280 nm), collected fractions were analysed by size-exclusion HPLC (SE HPLC; TSK-Gel Super SW2000, 220 nm). The fractions containing only the 5kDa component were analysed by reversed-phase high-pressure chromatography (RP HPLC; YMC-Pack ODS-A, 200 nm), revealing four peaks of the 5kDa component. From 1200 mg of EMD (of which 9% is the 5kDa component), approximately 65 mg of lyophilised 5kDa component were obtained, corresponding to a recovery of 60%. The SE HPLC method was mainly suitable for qualitative analysis, whereas the RP HPLC method was appropriate for both qualitative and quantitative analysis.