Matrix plasminogen activator inhibitor. Modulation of the extracellular proteolytic environment

We have previously demonstrated that plasminogen activator inhibitor (PAI-1) is associated with the extracellular matrix of cultured bovine smooth muscle cells (Knudsen, B.S., Harpel, P.C., Nachman, R.L. (1987) J. Clin. Invest. 80, 1082-1089). In this report we describe the physiologic role of PAI-1 during the interaction of the tissue plasminogen activator (t-PA) secreting Bowes human melanoma cell line with endothelial extracellular matrices. In addition we have characterized the t-PA.PAI complexes formed during this interaction in the presence and absence of plasminogen. In the absence of plasminogen, a 104-kDa complex between Bowes t-PA and PAI-1 appears in the supernatant. In the presence of plasminogen, PAI initially prevents plasmin formation on the matrix and protects the matrix from degradation by plasmin. The 104-kDa t-PA.PAI complex is degraded into a 68 and a 47-kDa complex by small amounts of plasmin generated from secreted Bowes t-PA and plasminogen. Analysis of these complexes revealed that t-PA is rapidly cleaved by plasmin within the complex whereas complexed PAI-1 is not further degraded. Matrix-associated PAI-1 may play an important role in the protection of extracellular matrices from remodeling and degradation by cellular t-PA and plasminogen.     

Isolation, identification and synthesis of locustamyoinhibiting peptide (LOM-MIP), a novel biologically active neuropeptide from Locusta migratoria.

A novel peptide termed locustamyoinhibiting peptide (LOM-MIP) was isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. The primary structure of this nonapeptide has been determined Ala-Trp-Gln-Asp-Leu-Asn-Ala-Gly-Trp-NH2. LOM-MIP suppresses the spontaneous contractions of the hindgut and oviduct of Locusta migratoria and of the hindgut of Leucophaea maderae. This novel peptide is, however, structurally different from leucomyosuppressin, a hindgut suppressing peptide isolated from Leucophaea maderae heads. LOM-MIP has a Gly-TrpNH2 carboxy-terminal in common with APGWamide, a penis retractor muscle inhibiting peptide isolated from the snail, Lymnea stagnalis. In addition, it shows carboxy-terminal sequence similarities with locust AKH II which ends in AGWamide. No sequence similarities were found with other vertebrate or invertebrate peptides. Synthetic LOM-MIP showed biological as well as chemical characteristics indistinguishable from those of native LOM-MIP.     

Active fragments and analogs of the insect neuropeptide leucopyrokinin: structure-function studies

Evaluation of analogs of the blocked insect myotropic neuropeptide leucopyrokinin (LPK) has demonstrated its relative insensitivity to amino acid substitution in the N-terminal in contrast to the C-terminal region. Truncated analogs of LPK without the first, second, and third N-terminal amino acids retain a significant 144%, 59% and 30% of the activity of the parent octapeptide, respectively. The [2-8]LPK analog is the first fragment of an insect neuropeptide to exhibit greater activity than the parent hormone. In contrast, truncated analogs of the insect myotropic, proctolin, exhibit little or no activity. The pentapeptide fragment Phe-Thr-Pro-Arg-Leu-NH2 has been identified as the active core of LPK.     

Activation of immobilized plasminogen by tissue activator. Multimolecular complex formation

Ternary complex formation of tissue plasminogen activator (TPA) and plasminogen (Plg) with thrombospondin (TSP) or histidine-rich glycoprotein (HRGP) has been demonstrated using an enzyme-linked immunosorbent assay, an affinity bead assay, and a rocket immunoelectrophoresis assay. The formation of these complexes was specific, concentration dependent, saturable, lysine binding site-dependent, and inhibitable by fluid phase plasminogen. Apparent Kd values were approximately 12-36 nM for the interaction of TPA with TSP-Plg complexes and 15-31 nM with HRGP-Plg complexes. At saturation the relative molar stoichiometry of Plg:TPA was 3:1 within the TSP-containing complexes and 1:1 within HRGP-containing complexes. The activation of Plg to plasmin by TPA on TSP- and HRGP-coated surfaces was studied using a synthetic fluorometric plasmin substrate (D-Val-Leu-Lys-7-amino-4-trifluoromethyl coumarin). Kinetic analysis demonstrated a marked increase in the affinity of TPA for plasminogen in the presence of surface-associated TSP or HRGP. Compared to fluid phase activation or activation on fibronectin- or Factor VIII-related antigen-coated surfaces there was a 35-fold increase in efficiency of plasmin generation. A substantial amount (up to 71%) of the plasmin formed remained surface-associated and was found to be protected from inhibition by alpha 2-plasmin inhibitor. Greater than 200-fold increase in inhibitor concentration was required to effect 50% inhibition. Complex formation of locally released tissue plasminogen activator with Plg immobilized on TSP or HRGP surfaces may thus play an important role in effecting proteolytic events in nonfibrin-containing microenvironments.     

Ordered water structure in an A-DNA octamer at 1.7 A resolution

The crystal structure of the deoxyoctamer d(G-G-Br U-A-BrU-A-C-C) was refined to a resolution of 1.7 A using combined diffractometer and synchrotron data. The analysis was carried out independently in two laboratories using different procedures. Although the final results are identical the comparison of the two approaches highlights potential problems in the refinement of oligonucleotides when only limited data are available. As part of the analysis the positions of 84 solvent molecules in the asymmetric unit were established. The DNA molecule is highly solvated, particularly the phosphate-sugar back-bone and the functional groups of the bases. The major groove contains, in the central BrU-A-BrU-A region, a ribbon of water molecules forming closed pentagons with shared edges. These water molecules are linked to the base O and N atoms and to the solvent chains connecting the O-1 phosphate oxygen atoms on each strand. The minor groove is also extensively hydrated with a continuous network in the central region and other networks at each end. The pattern of hydration is briefly compared with that observed in the structure of a B-dodecamer.     

Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function

Calcium-activated protease (CAP) was purified from the cytosol fraction of homogenized human platelet concentrates using a combination of gel filtration chromatography and affinity chromatography on antipain aminohexyl-Sepharose and activated thiol-Sepharose 4B. Purified CAP is composed of two different polypeptides of Mr = 80,000 and 27,000. Half-maximal protease activity was observed at 0.52 mM Ca2+, and all activity was inhibited by antipain, leupeptin, and N-ethylmaleimide. Activated CAP showed a time-dependent inactivation in the presence of 1 mM Ca2+ with only 5% of the control protease activity remaining after a 1-h exposure to calcium. Preincubation of washed platelets with varying amounts of CAP (0.2-0.4 units) significantly interfered with thrombin-induced platelet aggregation. In addition, ristocetin-induced platelet agglutination in the presence of von Willebrand factor was completely inhibited by 0.4 units of CAP. Concomitant with these protease-induced changes in platelet function, a decrease was observed in a major glycoprotein band of Mr = 150,000 present in platelet membranes and presumed to be glycoprotein Ib. In addition to these effects on platelets, CAP inhibited thrombin-induced production of prostacyclin by cultured human endothelial cells in a dose-dependent manner when the cells were pretreated with CAP. Thus platelet CAP can modulate membrane functions in both platelets and endothelial cells and may thus contribute to the regulation of hemostasis.     

Reproductive responses of Iphiseius degenerans and Neoseiulus teke (Acari: Phytoseiidae) to changes in the density of the cassava green mite, Mononychellus tanajoa (Acari: Tetranychidae)

We assessed the reproductive responses of adult female Iphiseius degenerans and Neoseiulus teke to increasing density of three stages of their prey, Mononychellus tanajoa, on cassava leaf discs under laboratory conditions. The oviposition rates increased with number of prey consumed per predator per day with a maximum of approximately two eggs per day for I. degenerans and four eggs per day for N. teke. The oviposition rate of N. teke was higher when consuming eggs than other prey stages. Neoseiulus teke was more efficient than I. degenerans in converting consumed prey into egg production. The data were adequately described by simple mathematical models.     

The dynamics of the cassava green mite Mononychellus tanajoa in a seasonally dry area in Kenya

The population dynamics of the cassava green mite Mononychellus tanajoa was studied on cassava during 35 weeks (early March to first of November 1989) in an experimental field near Lake Victoria in Western Kenya. The mite population peaked at the onset of the long dry season with 1,100 mites/leaf, declined sharply to a level of about 300 individuals/leaf, not to increase again until the next rainy season commenced. An indigenous phytoseiid predator Iphiseius degenerans was abundant during the dry spell with a maximum about 9 predators/leaf.A nonlinear regression analysis revealed that food depletion in combination with I. degenerans predation limited the population growth of the mites, whereas rain intensity had no effect. The predator exhibited no aggregative response to high densities of M. tanajoa and stayed mainly in the lower part of the canopy while the spider mites preferred the top, indicating that I. degenerans is a generalist predator without capacity to control M. tanajoa alone. However, in combination with another density dependent factor, such as food depletion, the predator may have prevented the spider mites from causing complete defoliation during the dry season.     

The effect of the cassava green mite Mononychellus tanajoa on the growth and yield of cassava Manihot esculenta in a seasonally dry area in Kenya

The effect of the cassava green mite Mononychellus tanajoa on the growth and yield of cassava Manihot esculenta was studied over a 10-month period in two field trials near Lake Victoria in Kenya. One plot was maintained free of mites by means of acaricide, while the other was artificially infested.The highest population density of M. tanajoa occurred during the dry season. A maximum leaf area index (LAI) of about 2 was reached at the onset of the dry season. The total leaf area of mite infested plants was reduced compared with uninfested plants during the dry spell. During the following rainy season infested plants recovered and attained the same leaf area as uninfested plants. A multiple regression model predicting the leaf area showed that 58% of the seasonal variation could be explained by plant age, soil water, and leaf injury.The net growth rate of infested plants was lower than that of uninfested plants. Maximum values of 21 (infested plants) and 49 (uninfested plants) g m^-2 week^-1 were attained at the onset of the second rainy season. No difference was found between uninfested and infested plants with respect to net assimilation rates per unit leaf area during the dry season. The net assimilation rates reached a maximum almost at the same time as the growth rates, but the infested plants peaked slightly earlier and at a lower level than the uninfested plants. M. tanajoa did not affect the relative allocation of dry matter into stems and storage roots, but the absolute allocation of dry matter declined with increasing mite injury. Thus, after 10 months the dry matter of infested plants was reduced by 29% and 21% for storage roots and stems, respectively, compared with the uninfested plants.     

Patterns of DNA Variability at X-Linked Loci in Mus Domesticus

Introns of four X-linked genes (Hprt, Plp, Glra2, and Amg) were sequenced to provide an estimate of nucleotide diversity at nuclear genes within the house mouse and to test the neutral prediction that the ratio of intraspecific polymorphism to interspecific divergence is the same for different loci. Hprt and Plp lie in a region of the X chromosome that experiences relatively low recombination rates, while Glra2 and Amg lie near the telomere of the X chromosome, a region that experiences higher recombination rates. A total of 6022 bases were sequenced in each of 10 Mus domesticus and one M. caroli. Average nucleotide diversity (π) for introns within M. domesticus was quite low (π = 0.078%). However, there was substantial variation in the level of heterozygosity among loci. The two telomeric loci, Glra2 and Amg, had higher ratios of polymorphism to divergence than the two loci experiencing lower recombination rates. These results are consistent with the hypothesis that heterozygosity is reduced in regions with lower rates of recombination, although sampling of additional genes is needed to establish whether there is a general correlation between heterozygosity and recombination rate as in Drosophila melanogaster.