Platelet plasminogen activator inhibitor: purification and characterization of interaction with plasminogen activators and activated protein C

Plasminogen activator inhibitor (PAI) was purified in active form from porcine platelets under nondenaturing conditions. The purified inhibitor (Mr 47,000) reacts with tissue-type plasminogen activator (t-PA), urokinase (UK), and activated protein C (APC) to yield both SDS-stable complexes and a modified PAI of slightly reduced molecular weight. The second-order rate constants for the inhibition of t-PA and UK by PAI are 3.5 X 10(7) and 3.4 X 10(7) M-1 s-1, respectively. Activated protein C reacts with PAI with a second-order rate constant of 1.1 X 10(4) M-1 s-1. This rate is not accelerated by protein S, phospholipid, and calcium, or heparin. It is concluded that (1) PAI can function as both inhibitor and substrate of its target proteases, (2) if APC promotes fibrinolysis via inactivation of PAI, then APC must be present in concentrations several orders of magnitude greater than t-PA, or the interaction of APC and PAI must be accelerated by presently unknown mechanisms, and (3) in the absence of heparin, platelet PAI is the most rapid inhibitor of APC yet described.     

Effect of micelle diameter on tryptophan dynamics in an amphipathic helical peptide in phosphatidylcholine

The effect of dimyristoylphosphatidylcholine (DMPC) on the conformation and environment of the single tryptophan residue of a model amphipathic helical polypeptide has been investigated by fluorescence quenching with a water-soluble, neutral quencher (acrylamide) and multiple-frequency phase fluorometry. The peptide H-Ser-Ser-Ala-Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Ly s-Glu- Ala-Phe-Ser-Ser-Ser-OH [18As; Kanellis, P., Romans, A.Y., Johnson, B.J., Kercret, H., Chiovetti, R., Jr., Allen, T.M., & Segrest, S.P. (1980) J. Biol. Chem. 255, 11464] was synthesized by solid-phase techniques. Peptide was incubated at 26 degrees C with DMPC at various peptide:lipid weight ratios. The diameter of the resulting disk-shaped micelles increases with increasing lipid concentration from 12.0 +/- 0.4 nm at a 1:1 weight ratio of peptide to lipid to a maximum of 48.7 +/- 1.0 nm at a 1:13 ratio. At a weight ratio of 1:5, the average diameter is 22.7 +/- 0.6 nm. Decreasing the peptide:lipid ratio of the micelle resulted in a blue-shift in the fluorescence emission maximum (from 337 nm at 1:1 to 334 nm at 1:5), an increase in the fluorescence lifetime of the tryptophan measured by the phase shift method at 18 MHz (from 3.12 ns at 1:1 to 3.61 ns at 1:5), a decrease in the rate of fluorescence quenching by acrylamide (from 0.87 x 10(9) M-1 s-1 at 1:1 to 0.42 x 10(9) M-1 s-1 at 1:5), and an increase in the activation energy for quenching (from 6.7 kcal/mol at 1:1 to 12.7 kcal/mol at 1:5).(ABSTRACT TRUNCATED AT 250 WORDS)     

An assessment of short-term depletion of stream macroinvertebrate benthos by drift

A study was conducted to determine if emigration of drifting macroinvertebrates from a stream riffle which was blocked for one week from immigration by upstream colonists significantly reduced the abundance of drift collected from the tail of the riffle. The head of a 9 m long riffle of a 2nd order stream in Maryland (USA) was blocked from incoming drift by a 250 m mesh weir. Upstream immigration of invertebrates into the riffle was largely prevented by a partition placed at the tail of the riffle which held the drift nets. Benthos and drift samples were collected from the riffle prior to weir placement and following its removal, and drift was collected at dusk on each day. No difference in drift or in benthic abundance between the beginning and end of the study was observed. This is largely attributed to recruitment of immature insects (primarily hatching of eggs present at the outset), particularly of Dolophilodes distinctus and species of Tanytarsini, from within the riffle. Results suggest that recruitment of riffle species is of sufficient magnitude to more than compensate for short-term riffle depletion due to drift. Samples of drifting and non-drifting (benthic) animals were held without food for 12 h after collection and mortality within each group was determined. The mortality of drifting animals was three-fold that of benthic animals.     

Plasmid content and homology of 16 strains of filamentous,nonheterocystous cyanobacteria

We have developed several strain-specific, rapid, small-scale plasmid isolation procedures in order to characterize the plasmid profiles of 16 filamentous, nonheterocstous cyanobacteria. At least one distinct plasmid was found in eight strains, with seven of these containing two or more different plasmids. Eight strains were found to be without plasmid DNA. Both the large, 12.9 kb, and the small, 1.6 kb, plasmids fromPlectonema boryanum 581 were isolated, purified, and cloned. Southern blots of plasmid DNAs from the eight strains were probed with these cloned DNAs and also with ultra-pure plasmid DNA fromPhormidium liridum 426. Four strains ofP. boryanum (485, 581, 594, 1542) andP. luridum 426 have identical plasmid profiles, and plasmid homology is extensive.     

Production of the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) by cell-free extracts from Streptomyces clavuligerus

Abstract Glycerol-stabilised cell extracts of Streptomyces clavuligerus contain an enzyme activity which synthesises ACV from the individual amino acids L -α-aminoadipic acid, L -cysteine and L -valine. Enzyme activity was optimum in reaction mixtures containing 1 mM ATP together with an ATP regenerating system. The ACV synthetase enzyme formed ACV analogs when provided with L - carboxymethylcysteine in place of L -α-aminoadipic acid or when provided with L - allo isoleucine or L -α-aminobutyrate in place of L -valine. Multistep conversion of individual amino acids to penicillin and cephalosporin antibiotics was restricted as a result of the inhibitory effects of L -α-aminoadipic acid and L -cysteine on isopenicillin N synthetase.     

Nanoplanktonic coccolithophorids (Prymnesiophyceae, Haptophyceae) from the Weddell Sea, Antarctica

The examination of whole mounts prepared for transmission electron microscopy has resulted in the finding of thirteen taxa of nanoplanktonic coccolithophorids from the Weddell Sea, Antarctica. The material was collected as part of the AMERIEZ programme, March 1986. Cold-water adapted nanoplanktonic coccolithophorids have previously been shown to constitute a recurrent plankton element at subarctic and arctic localities. Three of the Weddell Sea species, Wigwamma annulifera, W. arctica , and Papposphaera sagittifera , are conspecific with northern hemisphere material, while two species, Calciarcus alaskensis and Turrisphaera arctica , are possibly identical with previously described arctidsubarctic material. Six taxa new to science have been described from the Weddell Sea, Wigwamma antarctica, W. triradiata, Trigonaspis melvillea, Pappomonas weddellensis, Papposphaera obpyramidalis , and P. simplicissima . The cooccurrence of identical forms at the two poles, and the fact that the species described are allocated to "arctic" genera, indicate a geologically relatively recent exchange of biological material between the poles.     

Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA

Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation of infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component of 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.     

Detection of a non-structural protein of M r 11 000 encoded by the virion DNA of maize streak virus

A polypeptide of approximately 11 000 daltons (11 kDa protein) encoded by an open reading frame (10.9 ORF) from the virion sense of maize streak virus (MSV) DNA has been detected among the products of in vitro translation reactions programmed with RNA from infected maize plants and also in total protein extracts from infected leaves. The 11 kDa protein has not been detected in virions and is therefore proposed to have a nonstructural role.Viral DNA with an additional in-frame translation stop codon in the 10.9 ORF was not infectious when transmitted to maize plants via Agrobacterium tumefaciens agroinfection, suggesting that the 10.9 ORF may be essential for virus function. Computer comparison data show that equivalent ORFs in wheat dwarf virus (WDV) and digitaria streak virus (DSV) have some sequences in common with the 10.9 ORF of MSV. Further-more, the absence of similar sequences in geminiviruses which infect dicotyledonous plants suggests that the 11 kDa protein and its putative homologs in WDV and DSV have a function necessary only for those geminiviruses which infect the Gramineae.The significance of the 11 kDa protein in relation to expression of the virion sense DNA of MSV is discussed.