Prothrombin cleavage by human vascular smooth muscle cells: A potential alternative pathway to the coagulation cascade

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R¹⁵⁵-S¹⁵⁶. Cleavage at R²⁷¹-T²⁷² generated fragment 1.2 and prethrombin 2 whilst cleavage at R²⁸⁴-T²⁸⁵ yielded truncated prothrombin 2 (prethrombin 2′). However, cleavage at R³²⁰-I³²¹ which, during prothrombin activation produces two-chain α-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1–3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R²⁷¹-S²⁷² and by α-thrombin at R¹⁵⁵-S¹⁵⁶ and R²⁸⁴-T²⁸⁵. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30–40 kDa. © 1995 Wiley-Liss, Inc.     

Effect of a membrane interactive peptide on plant cells of canola (Brassica napus) and two fungal pathogens

Summary A membrane interactive peptide was toxic to microspores, pollen and protoplasts of canola in the 1–5 µM concentration range. Similarly, at 5.0 µM the peptide completely inhibited germination of conidia ofVerticillium albo-atrum; however, when tested with conidia of a virulent isolate of blackleg (Leptosphaeria maculens), a fungal pathogen of canola, much higher levels (>30 µM) of the peptide were required to reduce or arrest germination and growth of the conidia. When testing the relative toxicities of novel peptides on plant cells and their pathogens, pollen germination is a simple, rapid and reliable alternative to protoplasts.     

Gypsy moth cell lines divergent in viral susceptibility

Summary A series of cell lines unique in insect virus susceptibility pattern have been isolated from the ovaries of the gypsy moth (Lymantria dispar: Lepidoptera: Lymantriidae) on a synthetic medium with mammalian and avian serum supplementation. Growth curves showed the poorest growth occurring on peptone-based media with somewhat better growth on amino-acid-based media. The best growth was obtained with combined media. Serological study distinguished the present cell lines from one another and from cell lines derived from other insect species grown routinely in the same laboratory. Baculovirus susceptibility among the new lines varied from no response to a specific complete replication response upon challenge by the homologous (gypsy moth) nuclear polyhedrosis virus. This research was funded in part through a reimbursable agreement with the U.S. Forest Service.     

Incorporation von [D-4,5-3H]-Ieucine und [2-14C]-Acetat in die photosynthetischen Pigmente und Chinone von Chlorella pyrenoidosa

Der Metabolismus der photosynthetischen Pigmente und Chinone von Chlorella wurde mit Hilfe von ¹⁴CO₂, [2-¹⁴C]-Acetat und [D-4,5-³H]-Leucin als Vorstufen der Isoprenoidsynthese untersucht. Auch wurde untersucht, ob Leucin als Vorstufe in der Biosynthese der Terpenoide fungiert. Die Verwendung der Doppelmarkierung sollte bestehende Unterschiede in der Regulation und Biosynthese der Isoprenoide der Chloroplasten, Mitochondrien und des Zytoplasmas aufdecken. Neben der Verwendung von Radioisotopen wurde die Inkorporation von Deuterium in die Carotinoide verwendet, um den turnover der Prenyl-Lipide direkt nachzuweisen. In tracer-kinetischen Untersuchungen mit ¹⁴CO₂ konnte gezeigt werden, daß ¹⁴-C-α-Carotin zu ¹⁴C-Lutein und ¹⁴C-ß-Carotin zu ¹⁴C-Zeaxanthin umgesetzt wird. Eine Vorstufenfunktion läßt sich auch für Plastohydrochinon-9 nachweisen, das in Plastochinon-9 umgesetzt wird. Die Markierungskinetik der einzelnen Prenyl-Lipide deutet auf einen schnellen turnover hin. Anhand der ¹⁴C-Inkorporationskinetik wurden Halbwertszeiten im Bereich von 30 min bis 220 min ermittelt, während sich aus den Dekorporationskinetiken Zeiten von 9 bis 113 h ergeben. Prenyl-Lipide wie die Carotine, Chlorophyll a und Plastochinon-9, die einerseits Vorstufenfunktion besitzen, andererseits aber auch direkt an der Photosynthese beteiligt sind, werden schneller umgesetzt als ihre Folgeprodukte. Somit scheint ein direkter Zusammenhang zwischen der Funktion des Prenyl-Lipides im Chloroplasten und seinem Umsatz zu bestehen. Sowohl Acetat wie auch Leucin werden von Chlorella als Vorstufen in der Terpenoidbiosynthese verwendet. Acetat wird aber wesentlich schneller inkorporiert als Leucin, was vielleicht darauf zurückzuführen ist, daß Leucin im Zytoplasma zu Acetat oder Mevalonsäure metabolisiert wird und dann in den Chloroplasten gelangt. Auch im tracer-kinetischen Experiment mit ¹⁴C-Acetat und ³H-Leucin läßt sich eine Vorstufen-Produkt-Beziehung für die Chlorophylle, Carotinoide und Chinone zeigen. Im Vergleich zu den Experimenten mit ¹⁴CO₂ zeigen die tracer-kinetischen Experimente mit ¹⁴C-Acetat und ³H-Leucin, daß die Chloroplasten-Isoprenoide wesentlich langsamer metabolisiert werden und ihre Umsatzraten mehr jenen entsprechen, die aus den ¹⁴C-Dekorporationskinetiken des ¹⁴CO₂-Experimentes resultieren. Es zeigt sich aber auch hier, daß jene Prenyl-Lipide, die direkt an der Photosynthese beteiligt sind, schneller metabolisiert werden. Daß die Chloroplasten-Isoprenoide in autotroph kultivierten Chlorellen einem ständigen turnover unterliegen, läßt sich auch sehr eindrucksvoll mit Hilfe der Deuterium-Inkorporation für die Carotine zeigen. Die aus der Deuterium-Inkorporation erhaltenen Umsätze der Carotine entsprechen in etwa denen, die sich aus ihrer Dekorporationskinetik im tracer-kinetischen Experiment mit [2-¹⁴C]-Acetat und [D-4,5-³H]-Leucin ergeben, zeigen allerdings, daß a-Carotin wesentlich schneller umgesetzt wird als ß-Carotin. Die Untersuchungen wurden durch ein Stipendium der Deutschen Forschungsgemeinschaft im Austausch mit der British Royal Society an K.H.G. unterstützt, wofür wir uns bedanken.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Variation of mitochondrial volume fraction along multiply innervated fibers in rabbit extraocular muscle

Mitochondrial volume fraction was compared among three regions along the length of six multiply innervated fibers (MIFs) in the orbital surface layer of rabbit superior rectus. These MIFs are of about 5 μm diameter toward the middle of their length, and of about 15 μm diameter toward their proximal and distal ends. The region of highest volume fraction (26%) was located toward the proximal end of their segment of minimal diameter, in apparent association with endplate-like nerve junctions. The region of lowest volume fraction (8%) was located at their distal segment of maximal diameter. The region toward the distal end of their segment of minimal diameter displayed an intermediate volume fraction (15%). These mitochondrial volume fractions were further analyzed in terms of the relative contributions of the I-band, the A-band, and the subsarcolemmal mitochondrial clusters. Comparable changes in mitochondrial content occur in both the I-band and A-band: in the fibers' distal segment of maximal diameter, however, the mitochondrial volume fraction in the A-band (5%) is lower than in the I-band (11%). These modifications of mitochondrial content along the fibers' length occur irrespective of the contributions of the subsarcolemmal mitochondrial clusters.     

The carotenoids of Flavobacterium strain R1560

The main carotenoid of Flavobacterium strain R1560 has been identified as (3R,3R)-zeaxanthin. Also present were small amounts of 15-cis-phytoene, phytofluene, -carotene (7,8,7,8-tetrahydro-, -carotene plus 7,8,11,12-tetrahydro-, -carotene), neurosporene, lycopene, -zeacarotene, -carotene, -carotene, -cryptoxanthin, rubixanthin, 3-hydroxy--zeacarotene and several apo-carotenals. Zeaxanthin production was inhibited by nicotine (10 mM), and lycopene and rubixanthin accumulated. The biosynthesis of zeaxanthin is discussed in terms of pathways and also of half-molecule reaction sequences. The presence of zeaxanthin may be a characteristic of a group of Flavobacterium species, and may thus be useful in the taxonomic classification of these organisms.     

Conversion of a 24β-ethyl-25-methylene intermediate into poriferasterol by Trebouxia species

Clerosterol-[26-¹⁴C], a 24β-ethyl-25-methylene sterol [(24S)-24-ethylcholesta-5,25-dien-3β-ol], was incorporated into clionasterol and poriferasterol by cultures of the green algae Trebouxia sp. 213/3 and Trebouxia sp. 219/2. Degradation of the labelled poriferasterol showed that the ¹⁴C retained its identity and was not incorporated as a result of metabolism of the clerosterol-[26-¹⁴C] and randomisation of label. These results are consistent with the proposed production, and subsequent reduction, of a 24β-ethyl-25-methylene intermediate in 24β-ethyl sterol biosynthesis in algae of the order Chlorococcales.     

Monocyte-produced prostaglandin induces Fcγ receptor expression on human T cells

Incubation of human T cells for 18 hr with prostaglandin E₂ (PGE₂ 3 × 10^?6M) causes a slight but significant increase in the percentage of Tγ cells and a reduction in Tμ cells. When PGE was added to “non-Tγ” cells, the increase in the percentage of Tγ cells was more marked (from 1.5% Tγ without PGE to 11% Tγ with PGE₂, P < 0.001). Supernates from cultures of human monocytes also caused an increase in Tγ cells (10% Tγ without supernate to 18% with supernate, P < 0.01), and this increase was blocked if the monocytes were cultured with indomethacin, a prostaglandin synthetase inhibitor (9% Tγ cells). Thus, monocytes may regulate Fcγ receptors on T cells via PGE₂ production.