Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.     

Reversal by protein kinase C inhibitor of suppressive actions of phorbol-12-myristate-13-acetate on polyphosphoinositide metabolism and cytosolic Ca2+ mobilization in thrombin-stimulated human platelets

In the presence of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C in vitro, phorbol-12-myristate-13-acetate (PMA) did not suppress the thrombin-induced increase of cytosolic Ca2+ concentration in human platelets. The H-7 reversal of the inhibitory action of PMA was also observed in thrombin-induced polyphosphoinositide breakdown by phospholipase C. These results provide additional support to the developing theory that the inhibition of PMA on Ca2+ mobilization and phosphoinositide turnover may be mediated by protein kinase C activation.     

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/ phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/ phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40°C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca²⁺, the additional component was removed by an electrostatic interaction between Ca²⁺ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40°C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.     

Expression profile of the α subunit of the heterotrimeric G protein in rice

Previous studies on the activity of the rice Gα promoter using a β-Glucuronidase (GUS) reporter construct indicated that Gα expression was highest in developing organs and changed in a developmental stage-dependent manner. In this paper, GUS activity derived from the rice Gα promoter was analyzed in seeds and developing leaves. In seeds, GUS activity was detected in the aleurone layer, embryo, endosperm and scutellar epithelium. In developing leaves, the activity was detected in the mesophyll tissues, phloem and xylem of the leaf sheath and in the mesophyll tissue of the leaf blade. The activity in the aleurone layer and scutellar epithelium suggests that the Gα subunit may be involved in gibberellin signaling. The activity in the mesophyll tissues of the leaf blade suggests that the Gα subunit may be related to the intensity of disease resistance. The pattern of the activity in the developing leaf also indicates that the expression of Gα follows a developmental profile at the tissue level.Key words: expression pattern, Gα subunit, GUS staining pattern, heterotrimeric G protein, riceThe rice mutant d1 is deficient in the heterotrimeric G protein α subunit (Gα). Recently it was found that the dwarfism phenotype of d1 is due to a reduction in cell numbers.¹ This discovery has led to new questions regarding how rice Gα regulates cell number, and which other signaling molecules are involved in this process in various tissues and at different development stages. Studies of d1 suggest that rice Gα participates in both gibberellin signaling^2–4 and brassinosteroid signaling.^5–8 Promoter studies using the β-Glucuronidase (GUS) reporter indicate that Gα expression is highest in developing organs.¹ In this paper, we report on the expression pattern of a Gα promoter::GUS construct in seeds and developing leaves of rice.     

Al(3+) interaction sites of calmodulin and the Al(3+) effect on target binding of calmodulin

The interaction between calmodulin (CaM) and Al(3+) was studied by spectroscopic methods. Heteronuclear two-dimensional NMR data indicated that peaks related to the both lobes and middle of the central helix of CaM are largely affected by Al(3+). But chemical shift perturbation suggested that overall conformation of Ca(2+)-loaded CaM is not changed by Al(3+) binding. It is thought that Al(3+) interaction to the middle of the central helix is a key for the property of CaM's target recognition. If the structure and/or flexibility of the central helix are/is changed by Al(3+), target affinity to CaM must be influenced by Al(3+). Thus, we performed surface plasmon resonance experiments to observe the effect of Al(3+) on the target recognition by CaM. The data clearly indicated that target affinity to CaM is reduced by addition of Al(3+). All the results presented here support a hypothesis that Al(3+) may affect on the Ca(2+) signaling pathway in cells.     

Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin

Troponin and tropomyosin on actin filaments constitute a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle through a series of conformational changes within the actin-based thin filament. Troponin consists of three subunits: an inhibitory subunit (TnI), a Ca2+-binding subunit (TnC), and a tropomyosin-binding subunit (TnT). Ca2+-binding to TnC is believed to weaken interactions between troponin and actin, and triggers a large conformational change of the troponin complex. However, the atomic details of the actin-binding sites of troponin have not been determined. Ternary troponin complexes have been reconstituted from recombinant chicken skeletal TnI, TnC, and TnT2 (the C-terminal region of TnT), among which only TnI was uniformly labelled with 15N and/or 13C. By applying NMR spectroscopy, the solution structures of a "mobile" actin-binding domain (approximately 6.1 kDa) in the troponin ternary complex (approximately 52 kDa) were determined. The mobile domain appears to tumble independently of the core domain of troponin. Ca2+-induced changes in the chemical shift and line shape suggested that its tumbling was more restricted at high Ca2+ concentrations. The atomic details of interactions between actin and the mobile domain of troponin were defined by docking the mobile domain into the cryo-electron microscopy (cryo-EM) density map of thin filament at low [Ca2+]. This allowed the determination of the 3D position of residue 133 of TnI, which has been an important landmark to incorporate the available information. This enabled unique docking of the entire globular head region of troponin into the thin filament cryo-EM map at a low Ca2+ concentration. The resultant atomic model suggests that troponin interacted electrostatically with actin and caused the shift of tropomyosin to achieve muscle relaxation. An important feature is that the coiled-coil region of troponin pushed tropomyosin at a low Ca2+ concentration. Moreover, the relationship between myosin and the mobile domain on actin filaments suggests that the latter works as a fail-safe latch.     

The levels of main urinary metabolite of prostaglandin F1α and F2α in human subjects measured by radioimmunoassay

Radioimmunoassay technique for measuring 5α,7α-dihydroxy-11-keto-tetranorprosta-1,16-dioic acid, the main urinary metabolite of PGF₁α and PGF₂α (PGF₂α-MUM), was further improved.It was postulated based on some experimental data that the PGF₂α-MUM exists in the urine mostly as dioic acid form, not as δ-lactone formThe daily excretion of PGF₂α-MUM in men ranged from 14.43 μg to 36.14 μg and in women from 5.21 μg to 14.25 μg.     

In vitro reconstitution of the end replication problem

The end replication problem hypothesis proposes that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis. Although the idea has been widely accepted for explaining telomere attrition during cell proliferation, it has never been directly demonstrated. In order to take a biochemical approach to understand how linear DNA ends are replicated, we have established a novel in vitro linear simian virus 40 DNA replication system. In this system, terminally biotin-labeled linear DNAs are conjugated to avidin-coated beads and subjected to replication reactions. Linear DNA was efficiently replicated under optimized conditions, and replication products that had replicated using the original DNA templates were specifically analyzed by purifying bead-bound replication products. By exploiting this system, we showed that while the leading strand is completely synthesized to the end, lagging strand synthesis is gradually halted in the terminal approximately 500-bp region, leaving 3' overhangs. This result is consistent with observations in telomerase-negative mammalian cells and formally demonstrates the end replication problem. This study provides a basis for studying the details of telomere replication.     

A complete deletion mutant of the Escherichia coli dnaKdnaJ operon

Southern hydridization analyses of genomic DNAs from various dnaJ mutants of Escherichia coli showed that mutant K7052, which has well characterized dnaK706 and dnaJ705 double mutantions, is a deletion mutant. The deletion is about 8.0 kb long and encompasses the whole of the dnaKdnaJ operon.     

A 3-Mb Sequence-Ready Contig Map Encompassing the Multiple Disease Gene Cluster on Chromosome 11q13.1-q13.3

Despite the presence of several human disease genes on chromosome11q13, few of them have been molecularly cloned. Here, we reportthe construction of a contig map encompassing 11q13.1–q13.3using bacteriophage P1 (P1), bacterial artificial chromosome(BAC), and P1-derived artificial chromosome (PAC). The contigmap comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and1 YAC clone and spans a 3-Mb region from D11S480 to D11S913.The map encompasses all the candidate loci of Bardet-Biedlesyndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5),one-third of the distal region for hereditary paraganglioma2 (PGL2), and one-third of the central region for insulin-dependentdiabetes mellitus 4 (IDDM4). In the process of map construction,61 new sequence-tagged site (STS) markers were developed fromthe Not I linking clones and the termini of clone inserts. Wehave also mapped 30 ESTs on this map. This contig map will facilitatethe isolation of polymorphic markers for a more re.ned analysisof the disease gene region and identi.cation of candidate genesby direct cDNA selection, as well as prediction of gene functionfrom sequence information of these bacterial clones.