PPARbeta regulates vitamin A metabolism-related gene expression in hepatic stellate cells undergoing activation

Activation of cultured hepatic stellate cells correlated with an enhanced expression of proteins involved in uptake and storage of fatty acids (FA translocase CD36, Acyl-CoA synthetase 2) and retinol (cellular retinol binding protein type I, CRBP-I; lecithin:retinol acyltransferases, LRAT). The increased expression of CRBP-I and LRAT during hepatic stellate cells activation, both involved in retinol esterification, was in contrast with the simultaneous depletion of their typical lipid-vitamin A (vitA) reserves. Since hepatic stellate cells express high levels of peroxisome proliferator activated receptor beta (PPARbeta), which become further induced during transition into the activated phenotype, we investigated the potential role of PPARbeta in the regulation of these changes. Administration of L165041, a PPARbeta-specific agonist, further induced the expression of CD36, B-FABP, CRBP-I, and LRAT, whereas their expression was inhibited by antisense PPARbeta mRNA. PPARbeta-RXR dimers bound to CRBP-I promoter sequences. Our observations suggest that PPARbeta regulates the expression of these genes, and thus could play an important role in vitA storage. In vivo, we observed a striking association between the enhanced expression of PPARbeta and CRBP-I in activated myofibroblast-like hepatic stellate cells and the manifestation of vitA autofluorescent droplets in the fibrotic septa after injury with CCl4 or CCl4 in combination with retinol.     

Folding units govern the cytochrome c alkaline transition

The alkaline transition of cytochrome c is a model for protein structural switching in which the normal heme ligand is replaced by another group. Stopped flow data following a jump to high pH detect two slow kinetic phases, suggesting two rate-limiting structure changes. Results described here indicate that these events are controlled by the same structural unfolding reactions that account for the first two steps in the reversible unfolding pathway of cytochrome c. These and other results show that the cooperative folding-unfolding behavior of protein foldons can account for a variety of functional activities in addition to determining folding pathways.     

B7-H1 (programmed death-1 ligand) on dendritic cells is involved in the induction and maintenance of T cell anergy

In an effort to identify immunoregulatory molecules on dendritic cells (DC), we generated and screened for mAbs capable of modulating the T cell stimulatory function of DC. A particularly interesting mAb was mAb DF272. It recognizes monocyte-derived DC, but not blood monocytes or lymphocytes, and has profound immunomodulatory effects on DC. Treatment of DC with intact IgG or Fab of mAb DF272 enhanced their T cell stimulatory capacity. This effect on DC was accompanied by neither an up-regulation of costimulatory molecules such as B7.1 (CD80), B7.2 (CD86), and MHC class II molecules nor by an induction of cytokine production, including IL-1, TNF-alpha, IL-10, and IL-12. Moreover, the well-established inhibitory function of IL-10-treated DC could be reverted with mAb DF272. Even T cells, anergized because of stimulation with IL-10-treated DC, could be reactivated and induced to proliferate upon stimulation with mAb DF272-treated DC. Furthermore, mAb DF272-treated DC favored the induction of a type-1 cytokine response in T cells and inhibited IL-10 production. By using a retrovirus-based cDNA expression library generated from DC, we cloned and sequenced the mAb DF272-defined cell surface receptor and could demonstrate that it is identical with B7-H1 (programmed death-1 ligand), a recently identified new member of the B7 family of costimulatory molecules. Our results thus demonstrate that the mAb DF272-defined surface molecule B7-H1 represents a unique receptor structure on DC that might play a role in the induction and maintenance of T cell anergy.     

A survey of the gastrointestinal nematodes of Spanish ibex (Capra pyrenaica) in a high mountain habitat

We analyzed the content of the abomasum (n = 79) and small intestine (n = 83) of Spanish ibex from Sierra Nevada Natural Park, southern Spain. Fifteen species of trichostrongylid nematodes were identified, 4 of which were found for the first time in this host, i.e., Nematodirus fillicollis, N. oiratianus, Ostertagia lyrata, and O. ostertagi. Teladorsagia circumcincta and Marshallagia marshalli were the most abundant abomasal species, whereas N. abnormalis, N. davtiani, and N. oiratianus were dominant in the small intestine. Counts of both abomasal and intestinal nematodes were generally low (year-round-median = 292 and 94 worms, respectively), and significantly lower numbers of M. marshalli, N. davtiani, and N. oiratianus were found in summer. No sex-related differences in helminth abundance were found, but young ibex harbored significantly more N. davtiani and N. oiratianus than adults. The presence of scabies was not related to increased nematode counts.     

The pseudorabies virus UL11 protein is a virion component involved in secondary envelopment in the cytoplasm

Homologs of the small tegument protein encoded by the UL11 gene of herpes simplex virus type 1 are conserved throughout all herpesvirus subfamilies. However, their function during viral replication has not yet been conclusively shown. Using a monospecific antiserum and an appropriate viral deletion and rescue mutant, we identified and functionally characterized the UL11 protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL11 encodes a protein with an apparent molecular mass of 10 to 13 kDa that is primarily detected at cytoplasmic membranes during viral replication. In the absence of the UL11 protein, viral titers were decreased approximately 10-fold and plaque sizes were reduced by 60% compared to wild-type virus. Intranuclear capsid maturation and nuclear egress resulting in translocation of DNA-containing capsids into the cytoplasm were not detectably affected. However, in the absence of the UL11 protein, intracytoplasmic membranes were distorted. Moreover, in PrV-DeltaUL11-infected cells, capsids accumulated in the cytoplasm and were often found associated with tegument in aggregated structures such as had previously been demonstrated in cells infected with a PrV triple-mutant virus lacking glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Thus, the PrV UL11 protein, like glycoproteins E, I, and M, appears to be involved in secondary envelopment.     

Virus-neutralizing activity mediated by the Fab fragment of a hemagglutinin-specific antibody is sufficient for the resolution of influenza virus infection in SCID mice

Antibodies (Abs) contribute to the control of influenza virus infection in vivo by reducing progeny virus yield from infected cells (yield reduction [YR]) and by inhibiting progeny virus from spreading the infection to new host cells (virus neutralization [VN]). Previous studies showed that the infection could be resolved in severe combined immunodeficiency (SCID) mice by treatment with hemagglutinin (HA)-specific monoclonal antibodies (MAbs) that exhibit both VN and YR activities but not by MAbs that exhibited only YR activity. To determine whether virus clearance requires both activities, we measured the therapeutic activity of an HA-specific MAb (VN and YR) and its Fab fragment (VN) by intranasal (i.n.) administration to infected SCID mice. Immunoglobulin G (IgG) and Fab cleared the infection with i.n. 50% effective doses (ED(50)s) of 16 and 90 pmol, respectively. To resolve an established infection solely by VN activity, Fab must be present in the respiratory tract at an effective threshold concentration until all infected cells have died and production of virus has ceased. Because IgG and Fab had different half-lives in the respiratory tract (22 and 8 h, respectively) and assuming that both operated mainly or solely by VN, it could be estimated that clearance was achieved 24 h after Ab treatment when both reagents were present in the respiratory tract at approximately 10 pmol. This dose was approximately 200 times larger than the respiratory tract-associated Ab dose resulting from administration of the intraperitoneal ED(50) (270 pmol) of IgG. This indicated that our procedure of i.n. administration of Ab did not make optimal use of the Ab's therapeutic activity.     

The processing of eIF4GI by human rhinovirus type 2 2A(pro): relationship to self-cleavage and role of zinc

The 2A proteinase (2A(pro)) of human rhinoviruses (HRVs) is a cysteine protease containing a structurally important zinc ion. In the viral polyprotein, the enzyme cleaves between the C terminus of VP1 and its own N terminus. 2A(pro) also processes the two isoforms of the cellular protein, eukaryotic initiation factor 4G (eIF4G). We have shown that mature HRV2 2A(pro), when translated in vitro in rabbit reticulocyte lysates, efficiently cleaves eIF4GI, although the enzyme was not immediately active upon synthesis. Here, we examine the relationship between self-processing and eIF4GI cleavage. The onset of both reactions first occurred at least 10 min after initiation of protein synthesis. Furthermore, when self-processing was prevented by a specific mutation between VP1 and 2A(pro), the VP1-2A(pro) precursor was essentially unable to cleave eIF4GI, implying that self-processing is a prerequisite for eIF4GI cleavage. 2A(pro) synthesized in the presence of a potent zinc chelator is inactive; however, upon addition of excess zinc, HRV2 2A(pro) rapidly gained activity. Finally, the presence of the zinc chelator in the culture medium can protect HeLa cells from HRV infection.     

Biochemical characterization of a CDC6-like protein from the crenarchaeon Sulfolobus solfataricus

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function.     

Analysis of DNA replication intermediates suggests mechanisms of repeat sequence expansion

We previously developed a system to investigate the mechanism of repeat sequence expansion during eukaryotic Okazaki fragment processing. Upstream and downstream primers were annealed to a complementary template to overlap across a CAG repeat region. Annealing by the competing primers lead to structural intermediates that ligated to expand the repeat segment. When an equal number of repeats overlapped on the upstream and downstream primers, a 2-fold expansion was expected, but no expansion occurred. We show here that such substrates do not expand irrespective of their repeat length. To reveal mechanism, we tested different hairpin loop intermediates expected to form and facilitate ligation. Substrates configured to form large loops in either the upstream or downstream primer alone allowed expansion. Large or small fixed position single loops allowed expansion when located at least six nucleotides up- or downstream of the nick. Fixed loops in both primers, simulating a double loop intermediate, allowed expansion as long as each loop was nine nucleotides from the nick. Thus, neither the double loop configuration required to form with equal length overlaps nor the large single loop configuration are fundamental structural impediments to expansion. We propose a model for the expansion mechanism based on the relative stabilities of single loop, double loop, hairpin, and flap intermediates that is consistent with the observed expansion efficiency of equal and unequal overlap substrates. The model suggests that the equilibrium concentration of double loop intermediates is so vanishingly small that they are not likely contributors to sequence expansion.