Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B region.     

Effects of fibroin microcarriers on inflammation and regeneration of deep skin wounds in mice

The process of tissue regeneration following damage takes place with direct participation of the immune system. The use of biomaterials as scaffolds to facilitate healing of skin wounds is a new and interesting area of regenerative medicine and biomedical research. In many ways, the regenerative potential of biological material is related to its ability to modulate the inflammatory response. At the same time, all foreign materials, once implanted into a living tissue, to varying degree cause an immune reaction. The modern approach to the development of bioengineered structures for applications in regenerative medicine should be directed toward using the properties of the inflammatory response that improve healing, but do not lead to negative chronic manifestations. In this work, we studied the effect of microcarriers comprised of either fibroin or fibroin supplemented with gelatin on the dynamics of the healing, as well as inflammation, during regeneration of deep skin wounds in mice. We found that subcutaneous administration of microcarriers to the wound area resulted in uniform contraction of the wounds in mice in our experimental model, and microcarrier particles induced the infiltration of immune cells. This was associated with increased expression of proinflammatory cytokines TNF, IL-6, IL-1β, and chemokines CXCL1 and CXCL2, which contributed to full functional recovery of the injured area and the absence of fibrosis as compared to the control group.     

Selection of RNA replicons capable of persistent noncytopathic replication in mammalian cells

The natural life cycle of alphaviruses, a group of plus-strand RNA viruses, involves transmission to vertebrate hosts via mosquitoes. Chronic infections are established in mosquitoes (and usually in mosquito cell cultures), but infection of susceptible vertebrate cells typically results in rapid shutoff of host mRNA translation and cell death. Using engineered Sindbis virus RNA replicons expressing puromycin acetyltransferase as a dominant selectable marker, we identified mutations allowing persistent, noncytopathic replication in BHK-21 cells. Two of these adaptive mutations involved single-amino-acid substitutions in the C-terminal portion of nsP2, the viral helicase-protease. At one of these loci, nsP2 position 726, numerous substitution mutations were created and characterized in the context of RNA replicons and infectious virus. Our results suggest a direct correlation between the level of viral RNA replication and cytopathogenicity. This work also provides a series of alphavirus replicons for noncytopathic gene expression studies (E. V. Agapov, I. Frolov, B. D. Lindenbach, B. M. Prágai, S. Schlesinger, and C. M. Rice, Proc. Natl. Acad. Sci. USA 95:12989-12994, 1998) and a general strategy for selecting RNA viral mutants adapted to different cellular environments.     

Epitope of the catalytic subunit of viscumin, exposed on its interaction with a lipid bilayer

It was previously shown that the catalytic subunit of the plant toxin viscumin induces aggregation of small unilamellar liposomes and this process is inhibited by the mab_TA7 monoclonal antibody produced to the denatured catalytic subunit of viscumin (Agapov, I.I. et al., FEBS Lett., 1999, vol. 464, pp. 63-66). The interaction of the synthetic F101-T105 and A96-T105 fragments of the viscumin catalytic subunit with the mab_TA7 monoclonal antibody was studied by 1H NMR spectroscopy. The results of this study demonstrated that only the A96-T105 fragment is capable of binding to mab_TA7. A nuclear Overhauser effect observed in the antigen-antibody complex and registered on the resonances of the free peptide and exchanging between the free state and the antibody-bound state was analyzed; the mab_TA7 antigen determinant (H99-T105) was identified; and its conformation and orientation within the complex with the antibody were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Carboranyl-Chlorin e6 as a Potent Antimicrobial Photosensitizer

Antimicrobial photodynamic inactivation is currently being widely considered as alternative to antibiotic chemotherapy of infective diseases, attracting much attention to design of novel effective photosensitizers. Carboranyl-chlorin-e₆ (the conjugate of chlorin e₆ with carborane), applied here for the first time for antimicrobial photodynamic inactivation, appeared to be much stronger than chlorin e₆ against Gram-positive bacteria, such as Bacillus subtilis, Staphyllococcus aureus and Mycobacterium sp. Confocal fluorescence spectroscopy and membrane leakage experiments indicated that bacteria cell death upon photodynamic treatment with carboranyl-chlorin-e₆ is caused by loss of cell membrane integrity. The enhanced photobactericidal activity was attributed to the increased accumulation of the conjugate by bacterial cells, as evaluated both by centrifugation and fluorescence correlation spectroscopy. Gram-negative bacteria were rather resistant to antimicrobial photodynamic inactivation mediated by carboranyl-chlorin-e₆. Unlike chlorin e₆, the conjugate showed higher (compared to the wild-type strain) dark toxicity with Escherichia coli ΔtolC mutant, deficient in TolC-requiring multidrug efflux transporters.     

Boronated derivatives of chlorin <Emphasis Type="Italic">e</Emphasis><Subscript>6</Subscript> and fluoride-containing porphyrins as penetrating anions: a study using bilayer lipid membranes

Boronated derivatives of porphyrins are studied extensively as promising compounds for boron-neutron capture therapy and photodynamic therapy. Understanding of the mechanism of their permeation across cell membranes is a key step in screening for the most efficient compounds. In the present work, we studied the ability of boronated derivatives of chlorin e ₆ and porphyrins, which are mono-, di-, and tetra-anions, to permeate through planar bilayer lipid membranes (BLM). The translocation rate constants through the hydrophobic part of the lipid bilayer were estimated for monocarborane and its conjugate with chlorin e ₆ by the method of electrical current relaxation. They were similar, 6.6 and 6.8 sec⁻¹, respectively. Conjugates of porphyrins carrying two and four carborane groups were shown to permeate efficiently through a BLM although they carry two charges and four charges, respectively. The rate of permeation of the tetraanion estimated by the BLM current had superlinear dependence on the BLM voltage. Because the resting potential of most mammalian cells is negative inside, it can be concluded that the presence of negatively-charged boronated groups in compounds should hinder the accumulation of the porphyrins in cells.     

Monitoring in vivo changes in lung microstructure with ³He MRI in Sendai virus-infected mice

Recently, a Sendai virus (SeV) model of chronic obstructive lung disease has demonstrated an innate immune response in mouse airways that exhibits similarities to the chronic airway inflammation in human chronic obstructive pulmonary disease (COPD) and asthma, but the effect on distal lung parenchyma has not been investigated. The aim of our study is to image the time course and regional distribution of mouse lung microstructural changes in vivo after SeV infection. (1)H and (3)He diffusion magnetic resonance imaging (MRI) were successfully performed on five groups of C57BL/6J mice. (1)H MR images provided precise anatomical localization and lung volume measurements. (3)He lung morphometry was implemented to image and quantify mouse lung geometric microstructural parameters at different time points after SeV infection. (1)H MR images detected the SeV-induced pulmonary inflammation in vivo; spatially resolved maps of acinar airway radius R, alveolar depth h, and mean linear intercept Lm were generated from (3)He diffusion images. The morphometric parameters R and Lm in the infected group were indistinguishable from PBS-treated mice at day 21, increased slightly at day 49, and were increased with statistical significance at day 77 (p = 0.02). Increases in R and Lm of infected mice imply that there is a modest increase in alveolar duct radius distal to airway inflammation, particularly in the lung periphery, indicating airspace enlargement after virus infection. Our results indicate that (3)He lung morphometry has good sensitivity in quantifying small microstructural changes in the mouse lung and that the Sendai mouse model has the potential to be a valid murine model of COPD.     

Dipeptidyl peptidase I-dependent neutrophil recruitment modulates the inflammatory response to Sendai virus infection

The role of innate immunity in the pathogenesis of asthma is unclear. Although increased presence of neutrophils is associated with persistent asthma and asthma exacerbations, how neutrophils participate in the pathogenesis of asthma remains controversial. In this study, we show that the absence of dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease found in neutrophils, dampens the acute inflammatory response and the subsequent mucous cell metaplasia that accompanies the asthma phenotype induced by Sendai virus infection. This attenuated phenotype is accompanied by a significant decrease in the accumulation of neutrophils and the local production of CXCL2, TNF, IL-1beta, and IL-6 in the lung of infected DPPI-/- mice. Adoptive transfer of DPPI-sufficient neutrophils into DPPI-/- mice restored the levels of CXCL2 and enhanced cytokine production on day 4 postinfection and subsequent mucous cell metaplasia on day 21 postinfection. These results indicate that DPPI and neutrophils play a critical role in Sendai virus-induced asthma phenotype as a result of a DPPI-dependent neutrophil recruitment and cytokine response.