Effect of diazepam on the metabolic and morphometric parameters of neurons in the dorsal hippocampus in normal and stressed rats

Diazepam (0.5 mg/kg) decreases the swimming-stress-induced activation of the pyramidal neurons in the dorsal hippocamp. This is manifested by a decrease in the stress-induced glycogen consumption, an increase in the RNA content, and (less pronounced) increased in the nucleocytoplasmic ratio.     

Connection between severity of the course of tick-borne encephalitis with the concentration of interleukin-2 and interleukin-6 in blood

AIM: To test IL-6 and IL-2 serum concentrations as indirect prognostic indicators in tick-borne encephalitis (TBE) course. MATERIAL AND METHODS: 13 TBE patients' sera were examined for IL-2 and IL-6. RESULTS: IL-6 was not detected in patients with TBE fever but was detected in meningoencephalytic TBE. CONCLUSION: TBE patients demonstrate a positive correlation between the disease severity and serum IL-6. This criterion is proposed as a prognostic factor of a TBE course.     

The antigenic structure of glycoprotein E2 in the Venezuelan equine encephalomyelitis virus studied using rat monoclonal antibodies]

A collection of 21 rat hybridomas secreting high-affinity monoclonal antibodies to Venezuelan equine encephalomyelitis (VEE) virus was generated. Using a panel of 15 monoclonal antibodies to glycoprotein E2, the antigenic structure of this protein of VEE strains TC-83 and 230 was studied. A competitive radioimmunoassay suggested a new map of the antigenic structure of glycoprotein E2 in which 5 sites including 11 epitopes of monoclonal antibody binding were distinguished. Antibody to E2-2 site neutralized virus infectivity and blocked hemagglutination test and antibody to E2-3 site could only block hemagglutination. Antibodies to other E2 protein sites lacked any biological activity.     

New neutralizing antibody epitopes in hepatitis C virus envelope glycoproteins are revealed by dissecting peptide recognition profiles

One of the greatest challenges to HCV vaccine development is the induction of effective immune responses using recombinant proteins or vectors. In order to better understand which vaccine-induced antibodies contribute to neutralization of HCV the quality of polyclonal anti-E1E2 antibody responses in immunized mice and chimpanzees was assessed at the level of epitope recognition using peptide scanning and neutralization of chimeric 1a/2a, 1b/2a and 2a HCVcc after blocking or affinity elution of specific antibodies. Mice and chimpanzees were immunized with genotype 1a (H77) HCV gpE1E2; all samples contained cross-neutralizing antibody against HCVcc. By functionally dissecting the polyclonal immune responses we identified three new regions important for neutralization within E1 (aa264-318) and E2 (aa448-483 and aa496-515) of the HCV glycoproteins, the third of which (aa496-515) is highly conserved (85-95%) amongst genotypes. Antibodies to aa496-515 were isolated by affinity binding and elution from the serum of a vaccinated chimpanzee and found to specifically neutralize chimeric 1a/2a, 1b/2a and 2a HCVcc. IC50 titres (IgG ng/mL) for the aa496-515 eluate were calculated as 142.1, 239.37 and 487.62 against 1a/2a, 1b/2a and 2a HCVcc, respectively. Further analysis demonstrated that although antibody to this new, conserved neutralization epitope is efficiently induced with recombinant proteins in mice and chimpanzees; it is poorly induced during natural infection in patients and chimpanzees (7 out of 68 samples positive) suggesting the epitope is poorly presented to the immune system in the context of the viral particle. These findings have important implications for the development of HCV vaccines and strategies designed to protect against heterologous viruses. The data also suggest that recombinant or synthetic antigens may be more efficient at inducing neutralizing antibodies to certain epitopes and that screening virally infected patients may not be the best approach for finding new cross-reactive epitopes.     

Deciphering μ-opioid receptor phosphorylation and dephosphorylation in HEK293 cells

BACKGROUND AND PURPOSE

The molecular basis of agonist-selective signalling at the µ-opioid receptor is poorly understood. We have recently shown that full agonists such as [D-Ala²-MePhe⁴-Gly-ol]enkephalin (DAMGO) stimulate the phosphorylation of a number of carboxyl-terminal phosphate acceptor sites including threonine 370 (Thr³⁷⁰) and serine 375 (Ser³⁷⁵), and that is followed by a robust receptor internalization. In contrast, morphine promotes a selective phosphorylation of Ser³⁷⁵ without causing rapid receptor internalization.

EXPERIMENTAL APPROACH

Here, we identify kinases and phosphatases that mediate agonist-dependent phosphorylation and dephosphorylation of the µ-opioid receptor using a combination of phosphosite-specific antibodies and siRNA knock-down screening in HEK293 cells.

KEY RESULTS

We found that DAMGO-driven phosphorylation of Thr³⁷⁰ and Ser³⁷⁵ was preferentially catalysed by G-protein-coupled receptor kinases (GRKs) 2 and 3, whereas morphine-driven Ser³⁷⁵ phosphorylation was preferentially catalysed by GRK5. On the functional level, inhibition of GRK expression resulted in enhanced µ-opioid receptor signalling and reduced receptor internalization. Analysis of GRK5-deficient mice revealed that GRK5 selectively contributes to morphine-induced Ser³⁷⁵ phosphorylation in brain tissue. We also identified protein phosphatase 1γ as a µ-opioid receptor phosphatase that catalysed Thr³⁷⁰ and Ser³⁷⁵ dephosphorylation at or near the plasma membrane within minutes after agonist removal, which in turn facilitates receptor recycling.

CONCLUSIONS AND IMPLICATIONS

Together, the morphine-activated µ-opioid receptor is a good substrate for phosphorylation by GRK5 but a poor substrate for GRK2/3. GRK5 phosphorylates µ-opioid receptors selectively on Ser³⁷⁵, which is not sufficient to drive significant receptor internalization.     

Preparation of monoclonal antibodies specific for the major tegument protein pp65 of human herpesvirus type 5

A panel containing 11 types of monoclonal antibodies (mAb) specific for the recombinant protein pp65 of human herpesvirus type 5 was constructed. Enzyme immunoassay and immunoblotting of the immunochemical properties of mAb indicated that mAb could interact well with both recombinant and native pp65 antigen. Testing the suitability of mAb for an immunocytochemical study established that mAb 5F10 was highly effective in detecting the major tegument protein pp65 in the persistently infected Vero cells. A combination of the immunochemical properties of mAb enables them to be recommended for the immunodiagnosis of human cytomegalovirus infection.