Complex pattern of membrane type 1 matrix metalloproteinase shedding. Regulation by autocatalytic cells surface inactivation of active enzyme

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a type I transmembrane MMP shown to play a critical role in normal development and in malignant processes. Emerging evidence indicates that MT1-MMP is regulated by a process of ectodomain shedding. Active MT1-MMP undergoes autocatalytic processing on the cell surface, leading to the formation of an inactive 44-kDa fragment and release of the entire catalytic domain. Analysis of the released MT1-MMP forms in various cell types revealed a complex pattern of shedding involving two major fragments of 50 and 18 kDa and two minor species of 56 and 31-35 kDa. Protease inhibitor studies and a catalytically inactive MT1-MMP mutant revealed both autocatalytic (18 kDa) and non-autocatalytic (56, 50, and 31-35 kDa) shedding mechanisms. Purification and sequencing of the 18-kDa fragment indicated that it extends from Tyr(112) to Ala(255). Structural and sequencing data indicate that shedding of the 18-kDa fragment is initiated at the Gly(284)-Gly(285) site, followed by cleavage between the conserved Ala(255) and Ile(256) residues near the conserved methionine turn, a structural feature of the catalytic domain of all MMPs. Consistently, a recombinant 18-kDa fragment had no catalytic activity and did not bind TIMP-2. Thus, autocatalytic shedding evolved as a specific mechanism to terminate MT1-MMP activity on the cell surface by disrupting enzyme integrity at a vital structural site. In contrast, functional data suggest that the non-autocatalytic shedding generates soluble active MT1-MMP species capable of binding TIMP-2. These studies suggest that ectodomain shedding regulates the pericellular and extracellular activities of MT1-MMP through a delicate balance of active and inactive enzyme-soluble fragments.     

Failure to relax negative supercoiling of DNA is a primary cause of mitotic hyper-recombination in topoisomerase-deficient yeast cells

In the yeast Saccharomyces cerevisiae, DNA topoisomerases I and II can functionally substitute for each other in removing positive and negative DNA supercoils. Yeast Delta top1 top2(ts) mutants grow slowly and present structural instability in the genome; over half of the rDNA repeats are excised in the form of extrachromosomal rings, and small circular minichromosomes strongly multimerize. Because these traits can be reverted by the extrachromosomal expression of either eukaryotic topoisomerase I or II, their origin is attributed to the persistence of unconstrained DNA supercoiling. Here, we examine whether the expression of the Escherichia coli topA gene, which encodes the bacterial topoisomerase I that removes only negative supercoils, compensates the phenotype of Delta top1 top2(ts) yeast cells. We found that Delta top1 top2(ts) mutants expressing E. coli topoisomerase I grow faster and do not manifest rDNA excision and minichromosome multimerization. Furthermore, the recombination frequency in repeated DNA sequences, which is increased by nearly two orders of magnitude in Delta top1 top2(ts) mutants relative to the parental TOP+ cells, is restored to normal levels when the bacterial topoisomerase is expressed. These results indicate that the suppression of mitotic hyper-recombination caused by eukaryotic topoisomerases I and II is effected mainly by the relaxation of negative rather than positive supercoils; they also highlight the potential of unconstrained negative supercoiling to promote homologous recombination.     

New 3-deoxyanthocyanidins from leaves of Arrabidaea chica

Two new 3-deoxyanthocyanidins, 6,7,3',4'-tetrahydroxy-5-methoxyflavylium and 6,7,4'-trihydroxy-5-methoxyflavylium, and the pigment carajurin, which has been previously identified, were isolated from dried leaves of Arrabidaea chica, a creeper native to the American tropics. The structures of the components were elucidated by 1H- and 13C-NMR spectroscopy and HPLC-MS, including X-ray crystallographic analysis for carajurin.     

Soluble fibrinogen modulates neutrophil functionality through the activation of an extracellular signal-regulated kinase-dependent pathway

The integrin family not only mediates the recruitment of polymorphonuclear leukocytes (PMN) to sites of inflammation but also regulates several effector functions by binding to specific ligands. We have recently demonstrated that soluble fibrinogen (sFbg) is able to trigger an activating signal in PMN through an integrin-dependent mechanism. This activation results in degranulation, phagocytosis enhancement, and apoptosis delay. The aim of the present work was to further elucidate the molecular events that follow sFbg interaction with CD11b in human PMN, and the participation of this signaling pathway in the regulation of neutrophil functionality. We demonstrate that sFbg triggers a cascade of intracellular signals that lead to focal adhesion kinase and extracellular signal-regulated kinase 1/2 tyrosine phosphorylation. The activation of this mitogen-activated protein kinase pathway plays a central role in the sFbg modulation of secondary granule degranulation, Ab-dependent phagocytosis, and apoptosis. However, fibrinogen-induced secretory vesicle degranulation occurs independently of the signaling transduction pathways investigated herein. In the context of an inflammatory process, the intracellular signal pathway activated by sFbg may be an early event influencing the functionality of PMN.     

Direct Organogenesis in Hypocotyl Cultures of Tamarindus Indica

Direct differentiation of shoot buds from hypocotyl segments of 12-d-old seedlings of Tamarindus indica was obtained on Murashige and Skoog (MS) medium with or without growth regulators. The highest regeneration (66 %) and the maximum number of shoots (3 - 4) per explant were obtained from the explants on MS medium containing 6-benzylaminopurine (5 × 10-6 M). A maximum roots per shoot were produced on medium containing 3-indole butyric acid (5 × 10-6 M). The resulting plantlets were hardened and transferred to soil in pots where 75 % of them survived and resumed growth. Histological examination of explants suggests that the shoots were of de novo origin which would make this system suitable for transformation experiments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Messages from ultrahigh resolution crystal structures

The ever growing availability of macromolecular crystal structures determined at atomic resolution has now reached a critical size, making it possible to obtain statistically unbiased data on both protein stereochemistry and the validity of the parameters used in their refinement. Besides the determination of the precise geometry of proteins and their active sites, high resolution structures have made it possible to check the application of normal mode calculations, to calculate charge density distributions and to analyze hydration shells around protein molecules. Even if only a few structures involve protein complexes, either with ligands or prosthetic groups, the information obtained in these cases is of great interest for obtaining the physical parameters of these interactions.     

Biosynthesis of the basal lamina as a result of interaction between thyroid and mesenchymal cells in culture

Porcine thyroid cells were cultured alone or in mixed cultures with mesenchymal cells. The formation of a basal lamina in vitro was investigated ultrastructurally. Follicular reassociation of thyroid cells occurred in both types of culture; however, it was followed by formation of the basal lamina only when mesenchymal cells were present. The present findings suggest an epithelial origin of the basal lamina resulting from an interaction with mesenchymal cells.