Both the identity and source of the rodent collagenase(s) that mediates matrix remodeling in liver fibrosis remain elusive. We have recently demonstrated an unequivocal role for scar-associated macrophages (SAMs) in the spontaneous resolution of liver fibrosis and sought to determine whether SAMs are the source of matrix metalloproteinase (MMP) 13 (collagenase 3), considered to be the primary interstitial collagenase in rodents. In this study, we demonstrate an association between MMP13 expression and the presence of SAMs in the regression of experimental liver fibrosis. mmp13 gene expression was restricted to regions of fibrosis that were rich in SAMs. Both MMP13 mRNA and protein colocalized to large phagocytes within and directly apposed to hepatic scars. Using the CD11b-DTR-transgenic mouse to deplete SAMs in a model of chronic CCl4 injury, we found that SAM depletion resulted in a 5-fold reduction in mmp13 message (p = 0.005). Furthermore, resolution of CCl4-induced fibrosis was retarded in MMP13-deficient mice. Thus, SAMs selectively, during resolution of fibrosis induce and use the major collagenase MMP13 to mediate the resorption of interstitial matrix and successfully remodel the fibrotic liver.

A mutation in the maize Ms45 gene results in a male-sterile phenotype due to the absence of pollen. Cytological examination of microspores in ms45 mutant plants reveals that the cell wall of the developing male gametophyte is poorly formed, while genetic analysis indicates that mutations at Ms45 are sporophytic. The Ms45 gene has been isolated by transposon tagging and transcription was shown by RNA hybridization analysis to be tassel-specific. In this report, an efficient system for molecular complementation is described and used to confirm that a transformed copy of the wild-type gene was able to fully restore fertility to ms45 mutant maize and to characterize MS45 protein expression in maize anthers. The MS45 protein was localized to the tapetum and expressed maximally during the early-vacuolate microspore stage of development. Based on these observations, phenotypic complementation of ms45 was used as an assay to determine tapetal cell-layer activity of other anther-specific and constitutive promoters.

The red clover necrotic mosaic virus (RCNMV) genome is split between two single-stranded RNA species termed RNA-1 and RNA-2. RNA-1 directs the synthesis of 88-kDa (p88), 57-kDa (p57), 37-kDa (p37), and 27-kDa (p27) polypeptides and RNA-2 a 35-kDa (p35) polypeptide in vitro. The coding order of the RNA-1 products was determined to be 5'-p27-p57-p37-3'. Antibodies to synthetic peptides representing the carboxyl terminal portions of p27 and p57 immunoprecipitated their respective polypeptides in addition to p88, suggesting that p88 is a fusion protein. A frameshift heptanucleotide sequence element has been identified in RCNMV RNA-1. In addition, a stable stem-loop secondary structure adjacent to the heptanucleotide sequence is predicted. Together, these sequence elements suggest that a ribosomal frameshifting event occurs which allows translational readthrough of the p27 open reading frame into the p57 open reading frame, generating the observed p88 product. An RNA-1 expression construct fusing the p57 and the CP open reading frame was engineered to investigate the ribosomal frameshifting event. CP antibodies immunoprecipitated a fusion protein of the predicted size containing the carboxyl portion of CP. Site-directed mutagenesis of the frameshift element indicates that in vitro, p88 can also be expressed alternatively by suppression of an amber termination codon. Based on these data, we propose that the putative RCNMV RNA polymerase is an 88-kDa polypeptide expressed by a ribosomal frameshifting mechanism similar to those utilized by retroviruses.

The 3'-terminal region of wheat streak mosaic virus (WSMV) genomic RNA was cloned and a cDNA sequence of 1809 nucleotides upstream of the poly(A) tract was determined. The sequence contains a single open reading frame of 1662 nucleotides and a 3' untranslated region of 147 nucleotides. Translation products from WSMV RNA and WSMV cDNA transcripts were immunoprecipitated by WSMV capsid protein antiserum, indicating that the 3'-terminal region of WSMV RNA encodes the capsid protein.Five potential N-terminal capsid protein protease cleavage sites were identified, which would yield proteins ranging from 31-7K to 46.8K. Alignment of the deduced amino acid sequence of the WSMV capsid protein with those of other potyviruses showed significant, but limited, identity as compared to the alignment of two or more aphid-transmitted potyviruses. Although WSMV has characteristics distinct from potyviruses, because of its particle morphology, translation strategy apparently based on polyprotein processing, the ability to form cytoplasmic cylindrical inclusions and the degree of capsid protein homology with aphidtransmitted potyviruses, it should be considered a member of the potyvirus group.

A key feature of recovery from liver fibrosis is hepatic stellate cell (HSC) apoptosis, which serves the dual function of removing the major source of neomatrix and tissue inhibitors of metalloproteinases thereby facilitating matrix degradation. The mechanisms regulating HSC apoptosis remain undefined but may include the interaction of nerve growth factor (NGF) with its receptor, p75, on HSC. In this study, by TaqMan polymerase chain reaction in situ hybridization and immunohistochemistry, we demonstrate that NGF is expressed by hepatocytes during fibrotic injury. Peak hepatocyte expression of NGF (48 hours after CCl(4) injection) coincides with maximal rate of apoptosis of HSC by terminal dUTP nick-end labeling staining. Addition of recombinant NGF to HSC in tissue culture causes a dose-dependent increase in apoptosis. NGF regulates nuclear factor (NF)-kappaB activity, reducing p50/p65 binding detected by electromobility shift assay and reduced NF-kappaB CAT reporter activities from both basal unstimulated levels and after NF-kappaB induction by tumor necrosis factor. In each case, a relative reduction in NF-kappaB binding was associated with a significant increase in caspase 3 activity. These data provide evidence that NGF is expressed during fibrotic liver injury and may regulate number of activated HSCs via induction of apoptosis.