Pathogenesis of porphyrin-sensitized lethal and sub-lethal photo-injury

By means of morphological methods the mechanisms of thanatogenesis were studied in 57 white mice after the hematoporphyrin derivative administration and xenon lamp radiation imitating the sun spectrum. In dependence of integrative doses of sensitized photoradiation and individual resistance of the organism there was observed immediate (hours), retained (days) and remote (weeks) death of animals. In the first two groups death came as a result of photocoagulation of plasma proteins and toxemic shock with liver, lung and brain injury, and in the third group--as a result of suppurative resorptive intoxication against the background of ulcerous skin necrosis.     

State of coagulation hemostasis and fibrinolysis processes in dynamics of rapidly progressing forms of botulinal intoxication

Phase disturbances of coagulation blood potential were revealed in experiments on white rats in dynamics of rapidly progressing form of botulinic intoxication, intoxication being caused by intraperitoneal injection of type C toxin. In preclinical period of intoxication activation of procoagulant and anticoagulant parts of hemostasis system, as well as fibrinolysis system, was noted. Similar shifts were revealed in the developmental period of generalized pareses of skeletal musculature. Only in the terminal stage of intoxication insufficiency of mechanism in formation of prothrombinase activity developed by simultaneous activation of anticoagulant mechanism and fibrinolysis system.     

Effect of nitrobenzoic acid on methemoglobin content in blood and activity of antioxidative enzymes in erythrocytes]

Intraperitoneal administration of meta-nitrobenzoic, 3,5-dinitrobenzoic, 2,4,6-trinitrobenzoic and 3,5-dinitro-4-methylbenzoic acids to the white mice in a dose equal to LD50 has induced an increase in the methaemoglobin content in their blood. Total activity of dehydrogenases of pentosephosphate pathway, content of 2,3-diphosphoglycerate increase in response to the intoxication evoked by the mentioned acids. The acute intoxication does not practically change the activity of the key enzymes of antioxidant protection: superoxide-dismutase, catalase and glutathione peroxidase.     

Functional piglet model for the clinical syndrome and postmortem findings induced by staphylococcal enterotoxin B

Staphylococcal enterotoxin (SE) B causes serious gastrointestinal illness, and intoxication with this exotoxin can lead to lethal toxic shock syndrome. In order to overcome significant shortcomings of current rodent and nonhuman primate models, we developed a piglet model of lethal SEB intoxication. Fourteen-day-old Yorkshire piglets were given intravenous SEB, observed clinically, and sacrificed at 4, 6, 24, 48, 72, or 96 hrs posttreatment. Clinical signs were biphasic with pyrexia, vomiting, and diarrhea within 4 hrs, followed by terminal hypotension and shock by 96 hrs. Mild lymphoid lesions were identified as early as 24 hrs, with severe lymphadenopathy, splenomegaly, and prominent Peyer's patches found by 72 hrs. Widespread edema-most prominent in the mesentery, between loops of spiral colon, and in retroperitoneal connective tissue-was found in animals at 72 hrs. Additional histologic changes included perivascular aggregates of large lymphocytes variably present in the lung and brain, circulating lymphoblasts, and lymphocytic portal hepatitis. Preliminary molecular investigation using gene array has uncovered several gene profile changes that may have implications in the pathophysiology leading to irreversible shock. Five genes were selected for further study, and all showed increased mRNA levels subsequent to SEB exposure. The use of this piglet model will continue to elucidate the pathogenesis of SEB intoxication and facilitate the testing of new therapeutic regimens that may better correlate with human lesions.     

Modifications of liver content of cyclic nucleotides in the rat poisoned with white phosphorus]

In rat liver following white phorphorus poisoning a biphasic increase in cyclic AMP concentration was observed. After a lag period of 1 hour the cyclic AMP content rose to a first peak at 4 hours and to a second peak at 12 hours of intoxication. The cyclic AMP level fell to normal after 24 hours, by which time the cyclic nucleotide concentration was approaching control values. On the contrary, cyclic GMP content was found to the normal level during the different stages of intoxication. Only at 36 hours the cyclic GMP amount appeared significantly increased above the control values. Serum activity of alanine- and aspartate-amino transferases was found changed from 8 hours to 24 hours after poisoning. The serum level of the two enzymes was overlapping the control values after 36 hours. These results are discussed in relation to hepatocyte necrosis following white phosphorus intoxication.     

Distribution and replenishing of riboflavin in albino mice with acute alcoholic intoxication

It is shown that acute alcohol intoxication in the tissues of white mice accelerates riboflavin metabolism. In this case arrival of the exogenous vitamin with accompanying depletion of its endogenous resources is observed to intensify.     

Stress analgesia in experimental burn shock

Experiments with curarized and slightly anesthetised adult cats with burn shock have demonstrated pronounced depression of excitation transmission in associative and nonspecific afferent systems during somatic, sound and light stimulation. Effects controlling the activity of the cardiovascular system were facilitated in efferent systems. In white rats, burn shock led to an increase in the somatic and visceral pain threshold during the first 5 days. Rausedyl and naloxone reduced stress analgesia caused by burn shock in white rats. Burn shock enhanced opiate-like activity (beta-endorphine, beta-lipotropin) of the white rat forebrain as shown by radioimmunoassay. The data suggest that stress analgesia associated with experimental burn shock is likely to be accounted for by the increased production of endogenous opiates.     

Transition between Acute and Chronic Hepatotoxicity in Mice Is Associated with Impaired Energy Metabolism and Induction of Mitochondrial Heme Oxygenase-1

The formation of protein inclusions is frequently associated with chronic metabolic diseases. In mice, short-term intoxication with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to hepatocellular damage indicated by elevated serum liver enzyme activities, whereas only minor morphological changes are observed. Conversely, chronic administration of DDC for several weeks results in severe morphological damage, characterized by hepatocellular ballooning, disruption of the intermediate filament cytoskeleton, and formation of Mallory-Denk bodies consisting predominantly of misfolded keratins, Sqstm1/p62, and heat shock proteins. To evaluate the mechanistic underpinnings for this dichotomy we dissected the time-course of DDC intoxication for up to 10 weeks. We determined body weight change, serum liver enzyme activities, morphologic alterations, induction of antioxidant response (heme oxygenase-1, HO-1), oxidative damage and ATP content in livers as well as respiration, oxidative damage and the presence and activity of HO-1 in endoplasmic reticulum and mitochondria (mtHO-1). Elevated serum liver enzyme activity and oxidative liver damage were already present at early intoxication stages without further subsequent increase. After 2 weeks of intoxication, mice had transiently lost 9% of their body weight, liver ATP-content was reduced to 58% of controls, succinate-driven respiration was uncoupled from ATP-production and antioxidant response was associated with the appearance of catalytically active mtHO-1. Oxidative damage was associated with both acute and chronic DDC toxicity whereas the onset of chronic intoxication was specifically associated with mitochondrial dysfunction which was maximal after 2 weeks of intoxication. At this transition stage, adaptive responses involving mtHO-1 were induced, indirectly leading to improved respiration and preventing further drop of ATP levels. Our observations clearly demonstrate principally different mechanisms for acute and chronic toxic damage.     

Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis

Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes.     

Complex pathogenetic therapy of experimental botulism

The study of survival and life span of mice and white rats upon the administration of LD50 of the toxin has shown that an antihypoxic agent--gutimine (50-200 mg/kg)--had a protecting effect in type C botulinum intoxication. A combined use of gutimine and 4-aminopyridine (1-5 mg/kg), facilitating a transmitter release in synapses, had a more marked protecting effect in botulinum intoxication. Due to the potentiation of the drugs effect during their combined application, the doses of each drug in the combination could be reduced.     

Specific intoxication in anthrax infection

The pathomorphological picture of experimental B. anthracis infection in white rats (strain Fisher-344) essentially corresponds to experimental anthracic intoxication with very moderately pronounced morphological manifestation of B. anthracis invasion. This indicates that specific anthracic intoxication is an essential component of the pathological process in B. anthracis infection.     

Behaviour of cyclic nucleotides and Ca2+ levels in liver tissue of rats poisoned by white phosphorus and trichlorobromomethane

The content of hepatic cyclic AMP was increased soon after intoxication by white phosphorus. Its level reached a maximum 4 h after poisoning, but in subsequent phases tended to return to normal. In contrast, the cyclic GMP concentration was altered only 24 and 36 h after treatment with the same hepatotoxin. Similar modifications of cAMP and cGMP content were also detected after poisoning by trichlorobromomethane (CBrCl3). As a consequence, an altered cGMP/cAMP ratio was found in both experimental conditions. Further, the modification of cAMP content after white phosphorus was detected prior to liver damage (steatosis and necrosis), while the highest concentration of the cyclic nucleotide in CBrCl3-poisoned rats was found when fatty liver was already evident. In addition, in phosphorus-poisoned rats, the hepatic content of Ca2+ was found to be unmodified during all phases of the intoxication, while after CBrCl3 a phasic increase of the Ca2+ level was observed at 4, 24 and 36 h.     

Changes in Na+, K+-adenosinetriphosphatase and 5'-nucleotidase activities in cell membrane isolated from rat liver after acute white phosphorus poisoning

Na+, K+-ATPase and 5'-Nucleotidase activities in rat liver plasmamembranes after "in vivo" intoxication with a single dose of white phosphorus (10 mg/kg b.w. "per os") are investigated. Na+, K+-ATPase activity is significantly increased 1 hour and inhibited 12 hours after intoxication. 5'-Nucleotidase is strongly increased at 1, 2 and 4 hours after poisoning and is significantly decreased at 12 hours. The enhancement of both the enzymatic activities is evident prior to triglyceride accumulation in rat liver. Our results suggest that lipid fluidity of cell membrane is early and mildly affected during white phosphorus poisoning.     

The content of reduced glutathione in rat liver after poisoning with white phosphorus

The content of hepatic GSH was evaluated in rats after poisoning with white phosphorus. In addition, liver damage following the administration of the hepatotoxin was assessed by determining hepatic triglyceride accumulation. Experiments in parallel were carried out in an attempt to evaluate the enhanced susceptibility of hepatic tissue to peroxidative decomposition of unsaturated lipids 'in vitro', as measured by the production of TBA-reacting substances. Our data indicate that only in the early stage of intoxication is it possible to detect a slight decrease of GSH content in the liver, while during the subsequent stages the concentration of GSH was unaffected. At 6 hours of intoxication the level of hepatic triglycerides was significantly increased. Pretreatment with GSH was followed by an amelioration of fatty infiltration, but the content of hepatic GSH was unchanged. The production of TBA-reacting products was found enhanced only at 6 hours of intoxication. These results are discussed in relation to the role of lipid peroxidation in liver injury by white phosphorus.     

Differential expression profiling of the proteomes and their mRNAs in porcine white and red skeletal muscles

Skeletal muscle is an heterogeneous tissue with various biochemical and physical properties of several fiber types. In this study, we carried out the comparative study of protein expression patterns in white and red muscles using two-dimensional gel electrophoresis (2-DE). From more than 500 protein spots detected on each 2-DE gel, we screened five proteins that were differentially expressed between white and red muscles. Using peptide mass fingerprint and tandem mass spectrometry analysis these proteins were identified as myoglobin, two slow-twitch isoforms of myosin light chain and two small heat shock proteins (HSP20 and HSP27). The protein levels of myoglobin, myosin light chain and HSP20 were higher in red muscle, whereas HSP27 was higher in white muscle. In addition, genes of the identified proteins were cloned and their mRNAs were examined. Positive correlations between protein content and their mRNA levels were observed in white and red muscle. These results may provide us with valuable information to understand the different expression profiling between white and red muscle at the protein level.     

Study of the restoration and balance of thiamine in the tissues of albino mice in alcoholic intoxication

Acute inhalative alcoholic intoxication has been studied for its effect on the influx and level of free and bound thiamine in tissues of white mice. It is established that acute inhalative ethanol intoxication increases 35S-thiamine incorporation in tissues and decreases the level of endogenic free and bound thiamine. The results obtained permit a conclusion on intensification of the thiamine renewal in tissues with its sufficient influx from outside as affected by the ethanol narcosis.     

Possibilities of pharmacologic correction of Na, K-ATPase activity in the spinal cord in botulism

The experiments on white rats have shown that gutimin is capable of reactivating Na, K-ATPase of the synaptosomes of the jugular spinal cord in type C botulinic intoxication. Serotonin prevented Na, K-ATPase activity inhibition only in preclinical period of intoxication. Parmidin injection did not prevent suppression of Na, K-ATPase activity either in preclinical period or in skeletal muscle paresis.     

Histochemical and cytologic changes in the liver in experimental poisoning and subsequent pregnancy]

In 275 white rats chronic intoxication was produced by injection of amidopyrine acetaldehyde and chloraldehyde derivatives for 4 months. Histochemical and electron microscopic investigations of the liver under the experimental conditions and subsequent pregnancy revealed certain changes in nucleic metabolism, glycogenolisis, in the content of sulfhydryl, disulfide, carboxylic groups of protein molecules. Decrease in activity of enzymes of Krebs cycle and pentosophosphate cycle proved the deep changes in hepatocytes at the level of oxidation-reduction processes and protein synthesis. Ultramicroscopic changes in the nucleus, in cytoplasmic network, mitochondria and local degenerative alterations determined the level of morphologic reconstructions in connection with the intoxication. As the liver performs a number of vital functions and is closely connected with regulatory systems of the organism, morphofunctional shifts in the organ affect unfavourably the system mother--fetus.     

Proteolytic signaling mechanisms in skeletal muscles in patients with alcohol-induced muscle disease

Chronic alcoholic myopathy is one of the most numerous and profound manifestations of chronic alcohol intoxication. This disease is characterized by the pronounced atrophy of the locomotor muscles, which involves fibers expressing predominantly type II (fast) myosin isoforms. In early experiments with rats receiving alcohol and studies of patients, the impairment of the anabolic intracellular signaling pathways and decrease in protein synthesis rate were shown. We were the first to analyze the signaling pathways involved in the pathogenesis of alcoholic myopathy with different fiber atrophy levels. At the early stages of pathogenesis, we observed also a sufficient increase in mRNA of E3 ubiquitin ligases. However, the ubiquitinylation level was not altered in patients as compared to the control subjects. This phenomenon could be related to an increased expression of heat shock proteins known for their protective action.     

Molecular identification and expression of heat shock cognate 70 (HSC70) in the Pacific white shrimp Litopenaeus vannamei

Heat shock protein 70s (HSP70s) are fundamental chaperone proteins that are indispensable to most living organisms. In order to investigate the function of HSP70 and heat shock response in shrimp, a heat shock cognate (HSC70) gene of the white shrimp (Litopenaeus vannamei), containing a 1959-bp open reading frame, was cloned and characterized. The amino acid sequence, 71.5 kDa of molecular weight, shares 80-99.6% homology with 12 diverse species' HSP70s and HSC70s. In fact, some segments of the eukaryotic HSC70 sequence, such as ATP/GTP-binding site, cytoplasmic HSP70 C-terminal sequence, and GGMP/GAP repeats, are also found in the putative shrimp HSC70. Moreover, multi-tissue RT-PCR was performed to assay the basal expressions of HSC70 in the heart, gill, hepatopancreas, stomach, gut, and muscle. The results demonstrate that the basal expressions of HSC70 in theses organs are similar to that of beta-actin. Furthermore, quantitative real-time experiments showed that HSC70 was up-regulated in hepatopancreas (4.6-fold), stomach (5.9-fold), gut (2.6-fold), and muscle (3.5-fold) but not in the heart (1.7-fold) and gill (1.6-fold) after 2 h of heat shock. Nevertheless, the HSC70 was found to be highly expressed in the heart and gill following 6 h of heat shock. This suggests that HSC70 in white shrimp possess both short-term and long-term responses to heat shock stress, indicating this HSC70 may be a heat-dependent HSC70 member. Finally, we constructed an expression vector to generate HSC70 in Escherichia coli BL21, which displayed immune cross-reactivity with mouse HSP70 antibody. In conclusion, the identification and expression of white shrimp HSC70 gene present useful data for studying the molecular mechanism of heat shock response and the effect of heat shock proteins in shrimps' cytoprotection.