Determination of the Group Electronegativity of CF3 Group in 3′-O-CF3-Thymidine by 1-NMR

Abstract

The interplay of enthalpy of the gauche effect (ΔH°_GE) of the [X3′-C3′-C4′-O4′] fragment in various 3′-substituted (X) 2′,3′-dideoxythymidine derivatives 1–7 and the inherent anomeric effect drives the two-state North ? South equilibrium in the constituent sugar moiety. The group electronegativity of 3′-OCF₃ substituent in Marriott's, Inamoto's and Mullay's scales has been determined from simple calibration graphs correlating the group electronegativity of various 3′-substituents (X) in 2′,3′-dideoxythymidine derivatives 1–7 with the experimental strength (ΔH°_GE) of the [X3′-C3′-C4′-O4′] gauche effect. ΔH°_GE has been experimentally determined from pseudorotational analyses of temperature-dependent ³J_HH coupling constants, and can be used as an unambiguous tool for direct experimental estimation of the group electronegativity of a specific substituent covalently attached to 3′-carbon of 2′,3′-dideoxythymidine, which can be compared, in turn, with the theoretical estimation carried out according to Marriott's or Inamoto's procedure. Inconsistency found between theoretical values in Marriott's and Inamoto's scales, on the one hand, and between our experimental estimate and the theoretical value in Marriott's scale, on the other, have been solved by refining the electronegativity scale using our experimental data for 1–7.     

Twelve Years of Rabies Surveillance in Sri Lanka, 1999–2010

Background

Rabies is endemic in Sri Lanka, but little is known about the temporal and spatial trends of rabies in this country. Knowing these trends may provide insight into past control efforts and serve as the basis for future control measures. In this study, we analyzed distribution of rabies in humans and animals over a period of 12 years in Sri Lanka.

Methods

Accumulated data from 1999 through 2010 compiled by the Department of Rabies Diagnosis and Research, Medical Research Institute (MRI), Colombo, were used in this study.

Results

The yearly mean percentage of rabies-positive sample was 62.4% (47.6–75.9%). Three-fourths of the rabies-positive samples were from the Colombo, Gampaha, and Kalutara districts in Western province, followed by Galle in Southern province. A high percentage of the rabies samples were from dogs (85.2%), followed by cats (7.9%), humans (3.8%), wild animals (2.0%), and livestock (1.1%). Among wild animals, mongooses were the main victims followed by civets. The number of suspect human rabies cases decreased gradually in Sri Lanka, although the number of human samples submitted for laboratory confirmation increased.

Conclusions

The number of rabid dogs has remained relatively unchanged, but the number of suspect human rabies is decreasing gradually in Sri Lanka. These findings indicate successful use of postexposure prophylaxis (PEP) by animal bite victims and increased rabies awareness. PEP is free of charge and is supplied through government hospitals by the Ministry of Health, Sri Lanka. Our survey shows that most positive samples were received from Western and Southern provinces, possibly because of the ease of transporting samples to the laboratory. Submissions of wild animal and livestock samples should be increased by creating more awareness among the public. Better rabies surveillance will require introduction of molecular methods for detection and the establishment of more regional rabies diagnostic laboratories.     

Evaluation of a New Tumor Necrosis Factor-α-Inducing Membrane Protein of Helicobacter pylori as a Prophylactic Vaccine Antigen

Background: Tumor necrosis factor (TNF)‐α‐inducing protein (Tipα) is a newly identified carcinogenic factor present in Helicobacter pylori. Tipα has the unique function of inducing TNF‐α production by gastric cells in vitro and is assumed to be related with the development of gastritis and gastric cancer. We investigated the effects of vaccination with Tipα against H. pylori infection and analyzed the immune responses. Methods: C57BL/6 mice were immunized via the intranasal route with CpG, recombinant Tipα + CpG, and recombinant del‐Tipα (a mutant of Tipα) + CpG. Eight weeks after the mice were infected with H. pylori (5 × 10⁷ CFU), the number of colonizing bacteria in the stomach was calculated, and the histological severity of gastritis was evaluated. Levels of Tipα‐specific IgG and IgA antibodies in mouse serum were measured by an enzyme‐linked immunosorbent assay (ELISA). Local production of cytokines including Interleukin (IL)‐10, TNF‐α and Interferon (IFN)‐γ in gastric mucosa was also measured by real time‐PCR. Results: Levels of Tipα‐specific antibodies were significantly higher in Tipα‐immunized and del‐Tipα‐immunized mice than in the infection control group. The numbers of colonizing bacteria were significantly reduced in Tipα‐immunized mice (4.29 × 10⁵ CFU/g) and del‐Tipα immunized mice (2.5 × 10⁵ CFU/g) compared with infection control mice (5.7 × 10⁶ CFU/g). The levels of IFN‐γ and IL‐10 were significantly higher in del‐Tipα‐immunized mice than the infection control group. Conclusion: Vaccinations with Tipα and del‐Tipα were effective against H. pylori infection. The inhibition of H. pylori colonization is associated mainly with Th1 cell‐mediated immunity.     

Polycation liposome enhances the endocytic uptake of photosensitizer into cells in the presence of serum

To construct a novel drug delivery carrier that possesses high therapeutic efficacy with low dosage, we designed polyethylenimine-modified liposome (polycation liposome, PCL) and examined the entrapment of photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), for antiangiogenic photodynamic therapy (PDT). Photosensitizer entrapped in PCLs showed enhanced phototoxicity for a human vascular endothelial cell line, ECV304, in comparison with that for nonmodified control liposome. Interestingly, phototoxicity of control liposomal BPD-MA was suppressed in the presence of serum, but PCL maintained the phototoxicity in the presence of serum following PCL-mediated PDT treatment due to the stability of PCL and the reduced detachment of encapsulated photosensitizer from liposome to serum. In fact, PCL enhanced the uptake level of BPD-MA to ECV304 cells despite the presence or absence of serum. Since polycation modification enhances bioavailability of the liposomal photosensitizer and this property is maintained in the presence of serum, PCL would be useful for antiangiogenic PDT.     

Intravascular insulin gene delivery as potential therapeutic intervention in diabetes mellitus

We assessed therapeutic potential of intravascular insulin gene delivery in a diabetic murine model. The rat proinsulin-1 gene cDNA engineered to harbor furin consensus cleavage sequences was inserted into EBV-based plasmid vectors that contained CAG promoter or multimerized rat insulin promoter (RIP). Normal or streptozotocin (STZ)-induced diabetic mice were given an injection of the plasmids via the tail vein under high pressure. Transfection of the CAG-proinsulin construct markedly improved hyperglycemia of diabetic mice, accompanied by a considerable increase in serum insulin concentrations. Although the RIP-plasmid failed to reduce fasting blood glucose, the glucose tolerance test and RT-PCR analysis revealed that insulin production was regulated in the liver in a blood glucose level-dependent manner. The present results suggest a potential therapeutic means of controlling DM.     

Bax interacts with the voltage-dependent anion channel and mediates ethanol-induced apoptosis in rat hepatocytes

Acute ethanol exposure induces oxidative stress and apoptosis in primary rat hepatocytes. Previous data indicate that the mitochondrial permeability transition (MPT) is essential for ethanol-induced apoptosis. However, the mechanism by which ethanol induces the MPT remains unclear. In this study, we investigated the role of Bax, a proapoptotic Bcl-2 family protein, in acute ethanol-induced hepatocyte apoptosis. We found that Bax translocates from the cytosol to mitochondria before mitochondrial cytochrome c release. Bax translocation was oxidative stress dependent. Mitochondrial Bax formed a protein complex with the mitochondrial voltage-dependent anion channel (VDAC). Prevention of Bax-VDAC interactions by a microinjection of anti-VDAC antibody effectively prevented hepatocyte apoptosis by ethanol. In conclusion, these data suggest that Bax translocation from the cytosol to mitochondria leads to the subsequent formation of a Bax-VDAC complex that plays a crucial role in acute ethanol-induced hepatocyte apoptosis.     

A role of anti-verotoxin antibody immunoreactive peptide, Virp5, from rat spinal cord

An anti-verotoxin 2 (VT2) antibody immunoreactive 5-kDa polypeptide (Virp5), has been obtained through screening of the rat spinal cord cDNA library with the aid of anti-VT2 antibody. Virp5 was mainly expressed in the central nervous system, liver and kidney, and localized at glia-like cells and nerve fibers in the central nervous system, vascular endothelial cells and hepatic cells in the liver, as well as epithelial cells of distal tubules in the kidney. Intravenous administration of purified Virp5 elicited a dose-dependent increase in blood pressure. These results suggest that Virp5 commonly exists in the body, being partly playing a role in regulating the blood pressure.     

Method for direct discrimination of intra- and intermolecular hydrogen bonds, and characterization of the G(:A):G(:A):G(:A):G heptad, with scalar couplings across hydrogen bonds

Discrimination of intra- and intermolecular hydrogen bonds in a symmetric multimer has not been accomplished yet, although such discrimination would provide a crucial basis for construction of the multimeric architecture of nucleic acids by NMR. We have developed a direct and unambiguous method for such discrimination involving the use of scalar couplings across hydrogen bonds. The method has been validated with a symmetric dimer of d(GGGCTTTTGGGC), for which the structure including both intra- and intermolecular hydrogen bonds was already reported. This has demonstrated that our method can clearly discriminate these two kinds of hydrogen bonds. Then, the method was applied to a symmetric dimer of d(GGAGGAGGAGGA) and has provided decisive information on its multimeric architecture. Additionally, the values for scalar couplings across hydrogen bonds for G:G and G:A base pairs in the G(:A):G(:A):G(:A):G heptad formed by d(GGAGGAGGAGGA) were determined for the first time. This determination has provided an insight into the nature of the heptad.     

Intracellular interferon triggers Jak/Stat signaling cascade and induces p53-dependent antiviral protection

Intracellular interferons (IFNs) exert biological functions similar to those of extracellular IFNs, but the signal transduction pathway triggered by the intracellular ligands has not been fully revealed. We investigated the signaling cascade by sequence-specific knockdown of signaling molecules by means of the RNA interference. Truncated IFN-beta gene was constructed so that the N-terminal secretory signal sequence was deleted (SD.IFN-beta). Cells transfected with this construct showed phosphorylation and activation of the STAT1 without any detectable secretion of the cytokine. The MHC class I expression was significantly augmented, while the augmentation was suppressed by short interfering RNA duplexes specific for JAK1, TYK2, and IFN-alpha/beta receptor (IFNAR) 1 and 2c chains. The SD.IFN-beta also induced p53 and phosphorylation of p53 at Ser(15). Specific silencing of p53 abrogated the antiviral effect of SD.IFN-beta, suggesting that the tumor suppressor is critically involved in antiviral defense mediated by intracellular IFN.