Vipoxin and its components: structure-function relationship.

The neurotoxin Vipoxin has been of growing research interest since the time of its isolation from the venom of the Bulgarian viper Vipera ammodytes meridionalis. Vipoxin is a heterodimeric postsynaptic ionic complex composed of two protein subunits-a basic and strongly toxic His48 secretory phospholipase A(2) (sPLA(2)) enzyme and an acidic, enzymatically inactive and nontoxic component, originally named Inhibitor. When separated, sPLA(2) enzyme loses its toxicity in 3-4 days and catalytic activity in 2 weeks. After the establishment of the high degree of sequence homology (62%) and crystal structure of the subunits, Vipoxin was served as an example of molecular evolution of a toxic but unstable sPLA(2) into an inhibitor subunit which stabilizes the enzyme and preserves its pharmacological activity. Beginning our research on Vipoxin, intrigued by the unique relationship-structure-function and based on the previous experience, we were more than surprised to establish the lack of so-called inhibitory function of the acidic subunit on the toxicity and catalytic activity of basic sPLA(2). On the contrary, the acidic subunit activated the sPLA(2) enzyme in vitro. Our studies undoubtedly proved that is more correctly to present Vipoxin as a heterodimeric complex composed of one basic catalytic subunit and one acidic regulatory subunit. Their interaction in a common quaternary protein structure is more than a noncovalent association between the two subunits. It allows pharmacological sites to be targeted and biological functions to be potentiated. We attempt to present the previous studies and new findings on Vipoxin and its components.

Stability of diazepam in blood samples at different storage conditions and in the presence of alcohol.

Diazepam is one of the mostly used benzodiazepines and it is frequently analyzed in different biological samples, especially blood samples. The diazepam stability in the sample matrices is an important factor regarding reliable data obtaining. The storage is the main factor determining the stability of diazepam in blood samples and it is the object of the study presented. Remaining diazepam amount in spiked whole blood and plasma samples were tested at different storage temperatures, in the absence or presence of sodium fluoride as stabilizer as well as the influence of ethanol on diazepam stability was evaluated. The results of the study indicated that the temperature is the main storage factor affecting diazepam stability. In the fluoride stabilized blood samples the amount of diazepam decreases up to 85% of initial level when stored at -20° C for the period of testing (12 weeks). The presence of low (0.5 g/L) or high (3g/L) ethanol concentrations influences the stability of diazepam at -20 °C. In whole blood samples, the combination of sodium fluoride and ethanol decreases additionally (15-25%) the concentration of the analyte. Freeze-thaw experiments of whole blood samples show about 5-9% decrease in diazepam concentration after the first cycle. The freeze-thaw experiments on plasma samples, containing ethanol and/or fluoride show insignificant decreases of analyte concentration. Further experiments on benzodiazepines stability at different storage conditions or in combination of different factors should be undertaken in forensic toxicology to ensure the data quality, their reliability and reproducibility.

Hematological changes in case of chronic cadmium intoxication and monensin detoxication. Relationship with rheological variables.

The study evaluated the affect of chronic cadmium (Cd) and monensin treatment on some hematological parameters and its relationship with the rheological variables. Adult male mice were subjected to chronic treatment with cadmium acetate [Cd(CH3COO)2 × 2H2O] (group 1), Cd(CH3COO)2 × 2H2O followed by treatment with low dose monensin (group 2) and Cd(CH3COO)2 × 2H2O followed by high dose monensin treatment (group 3). Cd(CH3COO)2 × 2H2O and deprotonated monensin were dissolved in distilled water and given daily to the experimental animals. Mice drinking distilled water served as a control group (group 4). Hematological parameters and erythrocyte morphology were evaluated in parallel with whole blood viscosity (WBV). Cd treatment reduced Hb and increased RDW. The addition of high dose monensin significantly improved erythrocytic indices compared to the control. Erythrocyte anisocytosis was observed in blood smears of Cd-treated mice corresponding to the increased RDW. WBV was significantly elevated in the experimental groups in the whole range of shear rates compared to the control group and in groups 2 and 3 was lower than in group 1 but remained higher compared to group 4. Correlations were found between WBV and RBC, Hb, Hct, MCV and RDW. The results suggest that hemorheological parameters such as WBV should be monitored in parallel with the hematological parameters when monensin is applied and heavy metal intoxication is suspected.

Severe coagulopathy after Vipera ammodytes ammodytes snakebite in Bulgaria: a case report.

The case report presents a severe coagulopathy in a 56-year-old man following envenomation by the snake (Vipera ammodytes ammodytes) on his left hand. Initially the man was in shock, with an extremely low blood pressure and tachycardia. Local signs included a painful blister formation on the envenomation site. Twenty-four hours later, the man developed acute thrombocytopenia (platelets number 10 x 10(9)/l) and ecchimoses formation on the affected limb and on the left side of his body due to a disseminated intravascular coagulation syndrome, which lasted 13 days and required repeated administration of blood products, antivenin and supportive treatment. The patient was discharged from the hospital after 18 days in a good condition. The case report indicates that the coagulopathy may be a serious life-threatening complication after V. ammodytes ammodytes snakebite.

Hemolytic and anticoagulant study of the neurotoxin vipoxin and its components--basic phospholipase A2 and an acidic inhibitor.

In the present study, we demonstrate for the first time that the potent neurotoxin vipoxin from the venom of Vipera ammodytes meridionalis exhibits hemolytic and anticoagulant properties. By investigating the effects of phospholipids and calcium ions on hemolysis, we established that the phospholipase A2 (PLA2) enzyme activity is responsible for the hemolytic properties. This was confirmed by chemical modification of the PLA2 active-site histidine residue with p-bromophenacylbromide. Applying different clotting assays, we show that the PLA2 is a weakly anticoagulant enzyme, which affects intrinsic tenase complex by the hydrolysis of procoagulant phospholipids, rather than by nonenzymatic mechanisms (binding to specific coagulation factors). The whole complex--vipoxin--does not affect the coagulation system.

Effect of organic selenium on turkey semen quality during liquid storage.

The aim of the present study was to evaluate the effect of dietary organic selenium on the turkey semen during storage. Twenty males (BUT, Big 6, 40 weeks of age) were divided into control (n=10) and experimental group (n=10). The turkeys in the both groups were fed with a commercial diet containing 0.1 ppm Se in the form of sodium selenite. The experimental birds were additionally supplied with 0.3 ppm organic Se in the form Sel-Plex (Alltech, Inc.). After 30 days of feeding, the semen samples were collected twice a week for the 3 weeks of the study and diluted 1+1(v/v) with TUR-2 diluent, and stored in a water bath (+10 to 15 degrees C) for 6 h. The percentage of motile spermatozoa, the sperm viability (live/dead spermatozoa), total lipids, phospholipids and total cholesterol were assessed in fresh and stored semen. The fertilizing ability of semen was assessed by artificial insemination of 30 hens per group with dose containing 200x10(6) spermatozoa weekly. After 6 h of semen storage, the motility of spermatozoa decreased significantly in the control group (by 8.7 relative percent, P<0.05) and only by four relative percent (P>0.05) in experimental group reflecting a protective effect of dietary Se supplementation. The proportion of live spermatozoa was higher in fresh semen and significantly lower in stored semen. The positive effect of Se supplementation was observed on the lipid composition of stored semen: the concentration of the total lipids and phospholipids in the seminal plasma from control group significantly increased, while in the experimental group remained constant. Better semen integrity in the experimental group was associated with an improved fertilizing ability of spermatozoa: the fertility rate of stored spermatozoa in the control group was 88%, while in the experimental group was 90.5%.

Comparison of biochemical parameters of Muscovy drake semen diluted and stored at 4 degrees C in three buffers.

A comparison study of biochemical parameters of semen from Muscovy drakes diluted and stored at 4 degrees C in three buffers--IMV-buffer (France), HIA-1 and AU (Bulgaria) was carried out. The ejaculates were collected twice a week from ten 1-year-old Muscovy drakes using laying Muscovy females as teaser. Semen was diluted immediately, respectively, with IMV-buffer, HIA-1 and AU, and cold-stored (4 degrees C) for 1, 3 and 6h. The intensities of oxygen uptake at the third hour in semen diluted, respectively, with IMV-buffer (200+/-1.6 nAO/10(9) spermcellsmin), with HIA (224+/-44 nAO/10(9) spermcellsmin) and with AU (238+/-48 nAO/10(9) spermcellsmin) were highly significant in comparison with neat semen (75+/-0.7 nAO/10(9)spermcellsmin). The observed intensity of fructolysis was highest when using AU, followed by HIA-1 and IMV-buffer. During the first hour of storage the level of pyruvic acid was significantly lower in semen diluted with Bulgarian extenders, and this stability for AU referred to the entire period. For lactic acid, the differences were not statistical significant. Our investigations do not show significant differences concerning the dynamics of inorganic phosphate and total lipids after dilution with all tested extenders. On the contrary, high increase of cholesterol efflux from spermatozoa to seminal plasma-diluents were obtained after 6 h of storage. All extenders, IMV-buffer (France), HIA-1 and AU (Bulgaria) for diluting and short time storage of semen from Muscovy drakes at 4 degrees C maintain the necessary comfort of energy metabolism of the spermatozoa.

Vipoxin specificity studied by gas chromatographic determination of enzymatic reaction products. Influence of Ca2+, Mg2+ and Sr2+

Gas chromatographic procedure with mass spectrometric detection was applied to quantitatively determine the enzymatic specificity and activity of vipoxin (a neurotoxin from the Vipera ammodytes meridionalis venom) as well as the influence of Ca2+, Mg2+ and Sr2+ on these properties.

[Intracranial hemorrhages in patients being treatment with anticoagulants]. / Intrakranialni khemoragii pri bolni na lechenie s antikoagulanti.

Over the period 1994-1998, five patients presenting coagulation status impairment in the course of sintrom treatment are hospitalized in the Clinic of Emergency Neurology and Neurosurgery. Three of them are with craniocerebral trauma (2 severe forms--score 5 by the Glasgow Coma Rating scale, and one mild form--score 15), one has spontaneous intracerebral hematoma, and one--acute hydrocephalus against the background of ruptured aneurysm of the basilar artery (score 4). Three patients are given sintrom treatment because of rheumatic heart disease (2 with mitral valve prosthetic replacement and one in the post-commissurotomy period), and two--because of absolute arrhythmia, complicated by systemic emboli in the extremities and/or pulmonary artery. Four patients are subjected to operation--2 with craniocerebral injury, one with intracerebral hematoma and one with acute hydrocephalus of which in two cases the outcome is fatal. One female patient presenting serious craniocerebral trauma is not operated (refusal by the relatives) and dies. The therapeutic approach to this contingent of patients is comprehensively discussed.

[Clinical studies of patients with spinal cord metastases]. / Klinichni prouchvaniia pri bolni s grubnachnomozuchni metastazi.

Eighty-one patients with spinal-cord tumors are hospitalized in the Clinic of Emergency Neurology and Neurosurgery over the period 1988 through 1997. Of the total number, 74 patients of which 25 presenting metastases from neoplasms with primary location out of the central nervous system, are subjected to operation. There are 4 tumors involving the cervical segment, 1--cervicothoracic, 17--thoracic, 1--thoracolumbar, and 2--lumbar segment. Histological verification is done in all patients operated on. The indications for undertaking surgery in this particular contingent of patients are discussed.

[Clinical assessment in patients presenting brain metastases]. / Klinichni prouchvaniia pri bolni s moz'chni.

Over the period January 1988 to January 1998, in the clinic of emergency neurology and neurosurgery of the University Hospital "Queen Giovanna"--Sofia a total of 348 patients with brain tumors are hospitalized. Of them 329 cases are subjected to operation, and in 19 no surgery is undertaken. Forty-four patients with metastatic brain tumors are operated, and in 14 no operation is done. Of those operated 25 (56.8%) are men, and 19 (43.2%)--women. The mean age of male patients is 53.9 years, and of female patients--54.4 years. In 35 cases the metastases are supratentorially situated, and in eight--subtentorially (a female patient has both supra- and subtentorial metastases). Reoperation is performed in three patients, and one dies after the operation. In 13 cases (29.5%) the primary focus of lesion is known prior to operation. Of all patients included in the series 6 present pulmonary carcinoma (46%), 5--melanoma (30.8%), 2--breast carcinoma (15.4%), and one--carcinoma of the gastrointestinal tract (7.8%).

[Practical guidelines for the management of closed spinal cord injuries]. / Praktichni belezhki za povedenieto pri zakriti grubnachnomozuchni travmi.

The principles underlying the necessity of operative management in handling closed spinal cord trauma, its indications and types of operative interventions are comprehensively discussed. Under separate headings are described the peculiarities of the approach to injuries involving cervical, thoracic and lumbal segments of the vertebral column, with a summarization of currently used methods and procedures, contributing to the more complete functional recovery of the spine, preservation and creation of optimal conditions for regaining the functions of the spinal cord affected which in turn, renders easier the performing of daily living activities, precludes the development of a variety of complications in the body as a whole, and first and foremost, allows for early initiation of rehabilitation measures.

[The basic principles of diagnosis and treatment in emergency surgery]. / Osnovni printsipi na diagnostika i lechenie v speshnata khirurgiia.

A review of the literature is made, in the attempt to establish the principles of the diagnosis and treatment in emergency surgery. They are listed as follows: 1. To preserve patient life and evaluate the functional capacity of the organism; 2. To establish emergency diagnosis; 3. To choose the method of treatment; 4. Method of operation. The guidelines in emergency surgery is to develop and perfect new methods of operation and new technical facilities, to raise the professional skill of the surgeon and to improve the organization of work in surgical clinics and departments.

[Biliary ileus]. / Vurkhu biliarniia ileus.

This is a report on nine cases presenting biliary ileus. All patients undergo operative management. Two female patients present Bouveret's syndrome. The inference is reached that gallstone induced intestinal obstruction is characterized by atypical clinical patterns, conditioning in turn the diagnostic difficulties encountered, and the necessity to undertake operative treatment against the background of advanced ileus. The latter is one of the rarest forms of intestinal obstruction. Biliary ileus treatment consists in the removal of small intestinal impatency. The concretion is pushed in oral direction, and its evacuation is successfully accomplished by enterotomy, duodenotomy or gastrotomy. Two fatal outcomes due to severe advanced form of ileus are reported.