Hemodynamic Changes Provoked through Intravascular Injection of the Echis carinatus Venom in Rats.

Echis carinatus (E. carinatus) is known for its hematological and nephrotoxic properties in the envenomed patients. Based on the limited data upon the cardiovascular changes associated with this dangerous venomous snake in Iran, the current study purposed to evaluate the venom-induced hemodynamic manifestations in rats. Venom (120 µg/kg) was administered intravenously within one minute through the left femoral vein, and the hemodynamic parameters were continuously recorded using a pressure transducer (MLT844, ADInstruments, Australia). The venom caused prominent hypotension leading to death a few minutes after a transient uprise in blood pressure. It also induced a decrease in heart and pulmonary rates, yet it had no arrhythmogenic properties. Additionally, pre-treatment with the pepsin-derived Iranian polyvalent antivenom (30 µl/Kg) completely neutralized the hemodynamic responses but had no effect when instilled two minutes after venom injection. Heparin (300 IU/kg) and epinephrine (1.5 µg/kg) prevented dramatic hypotension when used 10 minutes before venom instillation; however, atropine (1 mg/kg), dexamethasone (1 mg/kg), and ketorolac (10 mg/ml) had no effects. All treated rats were killed post-injection. Histologically, the lung was the most vulnerable organ with mononuclear infiltration, microcystic formation, and significant capillary congestion. Prominent renal pathological deterioration also occurred, including mesangial cell infiltration and diffuse bleeding, leading to acute tubular necrosis. Modest portal inflammation and vascular congestion were observed in the hepatic tissue of the envenomed rats. The crude venom of Iranian Echis carinatus caused hypotension leading to bradycardia, a decrease in pulmonary rate, and death without significant histological changes to the heart.

Experimental Evaluation of Mouse Hind Paw Edema Induced by Iranian Naja oxiana Venom.

Iranian Naja oxiana (the Elapidae family) known as cobra snake inhabits in the northwestern part of Iran. This study aimed to evaluate the edematogenic potency of the crude venom with intraplantar injection into mice. Additionally, the inhibitory effects of three different drugs (i.e., promethazine, dexamethasone, and piroxicam) on paw edema were examined. Moreover, the gelatinase activity of this venom was assessed using the zymography method. Paw edema was induced by the intraplantar injection of different concentrations of the venom (0.5-5 μg dissolved in 50 μl of normal saline) into the mice (six in each group). It was estimated through the measurement of the increase in the paw thickness (%) with a digital caliper. The paws were pretreated and the rate of changes was measured after the venom injection. Pathological findings in the treated paws were evaluated with hematoxylin and eosin staining. Paw thickness reached its maximum amount within 5 min and resolved after 1 h. This venom had no gelatinase activity using the zymography method ruling out its role in edema. It caused non-hemorrhagic diffuse edema with the infiltration of inflammatory cells (i.e., leukocytes and lymphocytes) in the dermis. Intraperitoneal pretreatment with drugs significantly inhibited the venom-induced (1 μg/paw) edema; however, all the mice died unexpectedly a day after piroxicam injection. This in vitro and in vivo preliminary study demonstrated for the first time that N. oxiana venom-induced non-hemorrhagic edema in a short time. Dexamethasone (phospholipase A2 inhibitor; 1 mg/kg) and promethazine (H1 inhibitor; 5 mg/kg) decreased the venom-induced edema (p <0.001). It is suggested to carry out further studies to identify different mediators in venom-induced edema formation.

Comparison of clinical symptoms after Helicobacter pylori eradication in functional dyspepsia patients based on endoscopic view of antral gastropathy.

Functional dyspepsia is a common gastric disease that can be associated with Helicobacter pylori infection. The aim of this study is to evaluate antral endoscopy of individuals who presented with functional dyspepsia, H. pylori infection status and the effects of eradication therapy on the symptoms. Following the diagnosis of dyspepsia as per Rome III criteria, 260 individuals who were eligible for the study underwent upper gastrointestinal endoscopy and were divided into four groups of 65 according to the endoscopic view, grades I, II, III and IV (negative). Stool antigen test was also performed for all patients to identify H. pylori infection. The early signs of dyspepsia were assessed by a standard questionnaire. In all groups, omeprazole, amoxicillin, clarithromycin and metronidazole were used for eradication treatment, and 1 month after the treatment, a faecal antigen test was repeated to evaluate the eradication of H. pylori. There was no statistically significant difference between the groups in terms of clinical symptoms before treatment. The highest response to eradication treatment was seen in individuals with antral gastropathy grade III (66.2%) and the lowest response was in patients without antral gastropathy Grade IV (32.3%). This difference was statistically significant. There was no statistically significant relationship between the participants in terms of family history, age, gender and response to treatment. Eradicating H. pylori reduces the symptoms of dyspepsia. The response of eradication therapy was greatest among the patients with grade III antral gastropathy.

Expression and characterization of a recombinant single-domain monoclonal antibody against MUC1 mucin in tobacco plants.

A promising alternative to conventional antibodies is the single-domain antibody fragment of the Camelidae (V(HH)), which (because of features such as small length, high expression, solubility, and stability) is preferred to other antibody derivatives. In this report, a recombinant single-domain antibody (V(HH)) against MUC1 mucin in the tobacco plant, which may be considered as a suitable and economical alternative expression system, was produced. This antibody was expressed under the control of a strong constitutive promoter, CaMV35S, and NOS terminator. A plant high-expression sequence (Kozak sequence) was linked at the 5' end for overexpression of the V(HH) gene. The constructed cassette (pBIV(HH)) was transferred to agrobacterium, and the VHH gene was inserted into the plant genome by agrobacterium-mediated transformation. Transgenic lines were selected on kanamycin (100 mg/L) and maintained in soil, and subsequent generations were obtained. The presence and expression of the transgene was confirmed in the transformants by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot. Tobacco transgenic lines leave expressed V(HH) at levels varying from 1.12% to 1.63% of the total soluble protein. This report examines the transformation and expression of recombinant single-domain antibody (V(HH)) against antigen-associated tumor in tobacco plants.

Rubella virus glycoprotein interaction with the endoplasmic reticulum calreticulin and calnexin.

Very little is known about the cellular factors that are required for the maturation of rubella virus glycoproteins (E2 and E1) in the endoplasmic reticulum of the infected cell. In the present study, we established the interaction of the ER chaperone proteins, calreticulin and calnexin, with the RV E1 and E2 proteins in cells stably expressing the viral proteins. The interaction between E2 and calnexin was significantly higher than with calreticulin. In pulse-chase experiments, the half-life of the E2-calnexin was >45 min, whereas the half-life of the calreticulin-E2 interaction was approximately 10 min. Tunicamycin and castanospermine treatments altered the mobilities of intracellular E1 and E2, due to either lack of oligosaccharide ligand addition or trimming of terminal glucose residues, respectively. Further, the drug treatments resulted in a loss of E1 and E2 interaction with calreticulin or calnexin, thereby demonstrating that the interaction is through monoglucosylated forms of RV proteins. These studies suggest that the interaction of RV glycoproteins with the ER chaperone proteins is essential for their maturation in the endoplasmic reticulum.

Rubella virus E2 signal peptide is required for perinuclear localization of capsid protein and virus assembly.

The rubella virus (RV) structural proteins capsid, E2, and E1 are synthesized as a polyprotein precursor. The signal peptide that initiates translocation of E2 into the lumen of the endoplasmic reticulum remains attached to the carboxy terminus of the capsid protein after cleavage by signal peptidase. Among togaviruses, this feature is unique to RV. The E2 signal peptide has previously been shown to function as a membrane anchor for the capsid protein. In the present study, we demonstrate that this domain is required for RV glycoprotein-dependent localization of the capsid protein to the juxtanuclear region and subsequent virus assembly at the Golgi complex.

Uncontrolled reoxygenation by initiating cardiopulmonary bypass is associated with higher protein S100 in cyanotic versus acyanotic patients.

BACKGROUND: The systemic reoxygenation injury produced by initiating cardiopulmonary bypass (CPB) in infants with cyanotic heart disease may be associated with cerebral dysfunction and injury. Increased protein S100 (S100) serum levels may indicate cerebral and blood brain barrier damage as well as inflammatory changes, therefore serving to quantify these changes. The present clinical study assessed S100 in cyanotic patients undergoing CPB with normoxic versus hyperoxic paO2 in acyanotic cases and in controls without CPB. METHODS: 43 patients with congenital heart disease aged 5 days to 15 years (mean 4.4 years) were enrolled consecutively and divided in four groups: (1) Cyanotic infants undergoing controlled normoxic reoxygenation on CPB (n = 12), (2) cyanotic infants undergoing uncontrolled hyperoxic reoxygenation on CPB (n = 9), (3) acyanotic infants operated with CPB (n = 16) and (4) patients operated without CPB (n = 6). Blood samples were collected after induction of anesthesia (A), up to 4 hours after surgery (B) and at postoperative day one (C). RESULTS: Preoperative S100 serum levels [microg/l] in all groups were below clinical relevance. S100 increased markedly after surgery in groups 1 and 2. Differences in postoperative S100 levels were significant between groups 1 (0.45 +/- 0.13) and 3 (0.35 +/- 0.09; p = 0.018), between groups 2 (1.41 +/- 0.47) and 3 (p = 0.01), and between groups 2 and 4 (0.29 +/- 0.09; p = 0.045). There were no significant differences in postoperative S100 levels (B) between groups 1 and 2 (p = 0.05), groups 1 and 4 (p = 0.05), or groups 3 and 4 (p = 0.93). CONCLUSION: Uncontrolled hyperoxic reoxygenation on CPB for surgical correction of congenital heart defects is associated with higher S100 levels in cyanotic infants as compared to acyanotic patients undergoing comparable operations.

Rubella virus capsid protein induces apoptosis in transfected RK13 cells.

Rubella virus is an enveloped positive-strand RNA virus that can cause mild to severe birth defects or death in an infected fetus. RV induction of programmed cell death, demonstrated in cell culture, has been implicated in the pathogenesis. The timing of apoptosis, 48 h p.i., suggested that accumulation of RV structural proteins might induce cell death in infected cells. Expression of RV structural proteins, capsid, envelope glycoproteins E1 and E2, in transiently transfected RK13 cells was as potent an inducer of cell death as RV infection. Immunofluorescence microscopy revealed that RV structural protein transfected cells exhibited the condensed nuclei typical of apoptotic cell death. Transfection with the capsid protein construct, but not E2 and E1, resulted in as much cell death as joint expression of all three RV structural proteins. Capsid required a membrane-anchoring domain to induce cell death, but a heterologous polypeptide fused to the capsid membrane anchor did not cause apoptosis. Deletion mutants demonstrated that the apoptosis-inducing activity resides in the N-terminal 170 amino acids of capsid. Though apoptosis-inducing capsid constructs appear to have an ER sub-cellular localization, disruption of the ER calcium storage capacity does not correlate with cell death. Mechanisms consistent with these results are discussed.

Rubella virus-induced apoptosis varies among cell lines and is modulated by Bcl-XL and caspase inhibitors.

Rubella virus (RV) causes multisystem birth defects in the fetuses of infected women. To investigate the cellular basis of this pathology, we examined the cytopathic effect of RV in three permissive cell lines: Vero 76, RK13, and BHK21. Electron microscopy and the TUNEL assay showed that the cytopathic effect resulted from RV-induced programmed cell death (apoptosis) in all three cell lines, but the extent of apoptosis varied among these cells. At 48 h postinfection, the RK13 cell line showed the greatest number of apoptotic cells, the Vero 76 cell line was approximately 3-fold less, and BHK21 had very few. An increased multiplicity of infection and longer time postinfection were required for the BHK21 cell line to reach the level of apoptotic cells in Vero 76 at 48 h. Purified RV induced apoptosis in a dose-dependent fashion, but not UV-inactivated RV or virus-depleted culture supernatant. Specific inhibitors of the apoptosis-specific proteases caspases reduced RV-induced apoptosis and led to higher levels of RV components in infected cells. To address the role of regulatory proteins in RV-induced apoptosis, the antiapoptotic gene Bcl-2 or Bcl-XL was transfected into RK13 cells. Although a high level of Bcl-2 family proteins was expressed, no protection was observed from apoptosis induced by RV, Sindbis virus, or staurosporine in RK13 cells. In BHK21 cells, however, increased expression of Bcl-XL protected cells from apoptosis. The observed variability in apoptotic response to RV of these cell lines demonstrates that programmed cell death is dependent on the unique properties of each cell and may be indicative of how selective organ damage occurs in a congenital rubella syndrome fetus.

Mutations in the conserved C-terminal sequence in thyroid hormone receptor dissociate hormone-dependent activation from interference with AP-1 activity.

A short C-terminal sequence that is deleted in the v-ErbA oncoprotein and conserved in members of the nuclear receptor superfamily is required for normal biological function of its normal cellular counterpart, the thyroid hormone receptor alpha (T3R alpha). We carried out an extensive mutational analysis of this region based on the crystal structure of the hormone-bound ligand binding domain of T3R alpha. Mutagenesis of Leu398 or Glu401, which are surface exposed according to the crystal structure, completely blocks or significantly impairs T3-dependent transcriptional activation but does not affect or only partially diminishes interference with AP-1 activity. These are the first mutations that clearly dissociate these activities for T3R alpha. Substitution of Leu400, which is also surface exposed, does not affect interference with AP-1 activity and only partially diminishes T3-dependent transactivation. None of the mutations affect ligand-independent transactivation, consistent with previous findings that this activity is mediated by the N-terminal domain of T3R alpha. The loss of ligand-dependent transactivation for some mutants can largely be reversed in the presence of GRIP1, which acts as a strong ligand-dependent coactivator for wild-type T3R alpha. There is excellent correlation between T3-dependent in vitro association of GRIP1 with T3R alpha mutants and their ability to support T3-dependent transcriptional activation. Therefore, GRIP1, previously found to interact with the glucocorticoid, estrogen, and androgen receptors, may also have a role in T3R alpha-mediated ligand-dependent transcriptional activation. When fused to a heterologous DNA binding domain, that of the yeast transactivator GAL4, the conserved C terminus of T3R alpha functions as a strong ligand-independent activator in both mammalian and yeast cells. However, point mutations within this region have drastically different effects on these activities compared to their effect on the full-length T3R alpha. We conclude that the C-terminal conserved region contains a recognition surface for GRIP1 or a similar coactivator that facilitates its interaction with the basal transcriptional apparatus. While important for ligand-dependent transactivation, this interaction surface is not directly involved in transrepression of AP-1 activity.

A unique thyroid hormone response element in the human immunodeficiency virus type 1 long terminal repeat that overlaps the Sp1 binding sites.

Long terminal repeat (LTR) of human immunodeficiency virus (HIV) type 1 is activated by thyroid hormone (T3) receptor alpha (T3R alpha) in the absence of ligand. Addition of T3 reverses this effect. This activity is mediated by a high affinity T3 response element (T3RE) within the HIV-1 LTR, termed the HIV-T3RE (bases -74 to -50), which coincides with the Sp1 element as demonstrated by mobility shift, DNaseI footprinting, and methylation interference analyses. HIV-T3RE mediates ligand-independent activation of transcription by T3R alpha when linked to a heterologous promoter. In addition, the viral transactivator Tat synergizes with T3R alpha to activate the HIV-1 LTR in the absence of T3, which is relieved in its presence. These findings have implications for the possible control of HIV-1 LTR activity by T3.