Substrate specificity of the human matrix metalloproteinase stromelysin and the development of continuous fluorometric assays.

To probe the specificity of the metalloendoproteinase stromelysin toward peptide substrates, we determined kc/Km values for the stromelysin-catalyzed hydrolyses of peptides whose design was based loosely on the structure of a known SLN substrate, substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2, hydrolysis at Gln-Phe, kc/Km = 1700 M-1 s-1). Several noteworthy points emerge from this study: (i) Catalytic efficiency is dependent on peptide chain length with N-terminal truncation of substance P resulting in more pronounced rate-constant reductions than C-terminal truncation. These results suggest the existence of an extended active site for stromelysin. (ii) Preferences at positions P3, P2, P1, P1', and P2' are for the hydrophobic amino acids Pro, Leu, Ala, Nva, and Trp, respectively. (iii) Investigation of specificity at P3' supports our earlier hypothesis that SLN has a requirement for a hydrogen-bond donor at this position in its substrates. Based on these observations, we designed and had synthesized the fluorogenic substrate N-(2,4-dinitrophenyl)Arg-Pro-Lys-Pro-Leu-Ala-Nva-TrpNH2, whose stromelysin-catalyzed hydrolysis can be monitored continuously (kc/Km = 45,000 M-1 s-1).     

N-α-biotinylated-neuropeptide Y analogs: Syntheses, cardiovascular properties, and application to cardiac NPY receptor visualization

Two monobiotinylated analogs of neuropeptide Y (NPY) were synthesized by coupling the N-hydroxysuccinimidyl esters of biotin and (6-biotinylamido)-hexanoic acid, respectively, to the free alpha-NH2 group of the side chain protected NPY peptide resin. Crude peptides obtained by HF cleavage were purified by RPLC and their integrities were confirmed by amino acid and mass spectral analysis. As with NPY, both biotinylated analogs inhibited 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes in a dose-dependent manner. N-alpha-[(6-biotinylamido)-hexanoyl]-NPY exhibited potencies comparable to that of NPY whereas N-alpha-biotinyl-NPY was slightly less potent. In the in vivo experiments, however, both the biotinylated analogs exhibited responses comparable to NPY in increasing arterial blood pressure and decreasing heart rate in anesthetized rats. The responses of the biotinyl analogs were longer lasting than those of NPY. Histochemical studies revealed that N-alpha-[(6-biotinylamido)-hexanoyl]-NPY could label the NPY receptors in rat cardiac ventricular tissues. This labeling was specific since intact NPY inhibited the staining. These studies show that biotinyl-NPY analogs exhibit biological potencies comparable to intact NPY and can therefore be used to further probe the NPY-receptor interaction.     

Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon.

One potential approach for characterizing uncultivated prokaryotes from natural assemblages involves genomic analysis of DNA fragments retrieved directly from naturally occurring microbial biomass. In this study, we sought to isolate large genomic fragments from a widely distributed and relatively abundant but as yet uncultivated group of prokaryotes, the planktonic marine Archaea. A fosmid DNA library was prepared from a marine picoplankton assemblage collected at a depth of 200 m in the eastern North Pacific. We identified a 38.5-kbp recombinant fosmid clone which contained an archaeal small subunit ribosomal DNA gene. Phylogenetic analyses of the small subunit rRNA sequence demonstrated it close relationship to that of previously described planktonic archaea, which form a coherent group rooted deeply within the Crenarchaeota branch of the domain Archaea. Random shotgun sequencing of subcloned fragments of the archaeal fosmid clone revealed several genes which bore highest similarity to archaeal homologs, including large subunit ribosomal DNA and translation elongation factor 2 (EF2). Analyses of the inferred amino acid sequence of archaeoplankton EF2 supported its affiliation with the Crenarchaeote subdivision of Archaea. Two gene fragments encoding proteins not previously found in Archaea were also identified: RNA helicase, responsible for the ATP-dependent alteration of RNA secondary structure, and glutamate semialdehyde aminotransferase, an enzyme involved in initial steps of heme biosynthesis. In total, our results indicate that genomic analysis of large DNA fragments retrieved from mixed microbial assemblages can provide useful perspective on the physiological potential of abundant but as yet uncultivated prokaryotes.     

A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation.

Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce actin accumulation beneath adherent bacteria. We found that EPEC adherence to epithelial cells mediates the formation of fingerlike pseudopods (up to 10 microm) beneath bacteria. These actin-rich structures also contain tyrosine phosphorylated host proteins concentrated at the pseudopod tip beneath adherent EPEC. Intimate bacterial adherence (and pseudopod formation) occurred only after prior bacterial induction of tyrosine phosphorylation of an epithelial membrane protein, Hp90, which then associates directly with an EPEC adhesin, intimin. These interactions lead to cytoskeletal nucleation and pseudopod formation. This is the first example of a bacterial pathogen that triggers signals in epithelial cells which activates receptor binding activity to a specific bacterial ligand and subsequent cytoskeletal rearrangement.     

Role of phosphoglucomutase in lipooligosaccharide biosynthesis in Neisseria gonorrhoeae.

A region of pSG30 that complements the pyocin-derived gonococcal lipooligosaccharide (LOS) mutants 1291d and 1291e was characterized by DNA sequence analysis and an open reading frame of 1,380 bases was identified that is 89% similar and 56% identical over 452 amino acids to the algC gene product from Pseudomonas aeruginosa that encodes phosphomannomutase. Enzymatic analysis of gonococcal crude protein extracts demonstrated that pSG30 encodes phosphoglucomutase (PGM) and phosphomannomutase activity. This activity is absent in 1291d and 1291e but is restored upon introduction of pSG30. PGM encoded by pSG34, a subclone of pSG30, was able to complement Escherichia coli PGM1, a strain deficient in PGM, as determined by bacteriophage C21 plaque formation. A revertant of 1291d that binds monoclonal antibody 2-1-L8 (specific for a 3.6-kDa LOS component) was isolated. The construction of a site-specific deletion of this region in the chromosome of 1291 confirms the role of this open reading frame in LOS biosynthesis.     

TGFβ alters growth and differentiation related gene expression in proliferating osteoblasts in vitro,preventing development of the mature bone phenotype

This study examines the mechanism by which TGF-β1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre-osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c-fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF-β1-treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF-β1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF-β1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and α1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF-β1 response throughout the course of osteoblast differentiation. The initiation of a TGF-β1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF-β1. These in vitro results indicate that TGF-β1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml. © 1994 Wiley-Liss, Inc.     

Differential effects of warfarin on mRNA levels of developmentally regulated vitamin K dependent proteins,osteocalcin, and matrix Gla protein in vitro

The role of the vitamin K dependent proteins, osteocalcin which is bone specific and matrix Gla protein (MGP) found in many tissues, has been studied by inhibition of synthesis of their characteristic amino acid, γ-carboxyglutamic acid (Gla) with the anticoagulant sodium warfarin. The effect of sodium warfarin on expression of these proteins, and other phenotypic markers of bone and cartilage during cellular differentiation and development of tissue extracellular matrix, was examined in several model systems. Parameters assayed include cell growth (reflected by histone gene expression) and collagen types I and II, osteopontin, alkaline phosphatase, and mineralization. Studies were carried out in calvarial bone organ cultures, normal diploid rat osteoblast and chondrocyte cultures, and rat osteosarcoma cell lines ROS 17/2.8 and 25/1. In normal diploid cells, warfarin consistently stimulated cell proliferation (twofold). In osteoblast cultures, MGP mRNA levels were generally increased (three to tenfold). Notably, MGP mRNA levels were not affected in chondrocyte cultures, either with chronic or acute warfarin treatments. Osteocalcin mRNA levels and synthesis were decreased up to 50% in ROS 17/2.8 cells and in chronically treated (1 and 5 μg/ml sodium warfarin) rat osteoblast cultures after 22 days. Early stages of osteoblast phenotype development from the proliferation period to initial tissue formation (nodules) appeared unaffected; while after day 14, further growth and mineralization of the nodule areas were significantly decreased in warfarin-treated cultures. In summary, warfarin has opposing effects on the expression of two vitamin K dependent proteins, MGP and osteocalcin, in osteoblast cultures and MGP is regulated differently between cartilage and bone as reflected by cellular mRNA levels. Additionally, warfarin effects expression of nonvitamin K dependent proteins which may reflect the influence of warfarin on endoplasmic reticulum associated enzymes. © 1994 Wiley-Liss, Inc.