A study of the interaction between Helicobacter pylori and components of the human fibrinolytic system

The interaction of plasminogen, tissue plasminogen activator (t-PA) and urokinase with a clinical strain of Helicobacter pylori was studied. Plasminogen bound to the surface of H. pylori cells in a concentration-dependent manner and could be activated to the enzymatic form, plasmin, by t-PA. Affinity chromatography assays revealed a plasminogen-binding protein of 58.9 kDa in water extracts of surface proteins. Surface-associated plasmin activity, detected with the chromogenic substrate CBS 00.65, was observed only when plasminogen and an exogenous activator were added to the cell suspension. The two physiologic plasminogen activators, t-PA and urokinase, were also shown to bind to and remain active on the surface of bacterial cells. epsilon-Aminocaproic acid caused partial inhibition of t-PA binding, suggesting that the kringle 2 structure of this activator is involved in the interaction with surface receptors. The activation of plasminogen by t-PA, but not urokinase, strongly depended on the presence of cells and a 25-fold enhancer effect on the initial velocity of activation by t-PA compared to urokinase was established. Furthermore, a relationship between cell concentration and the initial velocity of activation was demonstrated. These findings support the concept that plasminogen activation by t-PA on the bacterial surface is a surface-dependent reaction which offers catalytic advantages

Modulation of staphylokinase-dependent plasminogen activation by mono- and divalent ions

The effect of several ions (Cl-, Na+, K+, Ca2+) on the rate of plasminogen (Pg) activation by recombinant staphylokinase (rSTA) is reported. Both monovalent and divalent ions affect the rate at which Pg is activated by rSTA, in a concentration-dependent manner (range 0-100 mM). In almost all cases, a decrease of the initial velocity of activation was observed. Cl- showed the most striking inhibitory effect at low concentrations (64 percent at 10 mM). However, in the presence of a fibrin surface, this inhibition was attenuated to 38 percent. Surprisingly, 10 mM Ca2+ enhanced the Pg activation rate 21 percent when a polymerized fibrin matrix was present. These data support the idea that ions can modulate the rate of Pg activation through a mechanism that may be associated with changes in the molecular conformation of the zymogen. This effect is strongly dependent on the presence of a fibrin clot

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmatic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0 percent ammonium sulfate, when a linear grandient was applied. Theree major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 muM and kcat = 0.82 s(-1), when assayed with a chromogen-coupled subtrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing.

Variaciones farmacocinéticas de anticonvulsivantes en mujeres epilépticas mexicanas durante las diferentes etapas del postparto / Pharmakocinetic variation of anticonvulsants in Mexican epileptic women during different parts of puerperium

Durante el embarazo existen una serie de cambios fisiológicos que influyen en la cinética de los medicamentos administrativos durante esta etapa: Estos cambios fisiológicos no se restauran inmediatamente en el parto, de tal manera que su concentración en los fluidos biológicos es diferente cuando se administra inmediatamente después del parto que varias semanas posterior al mismo. El presente trabajo tiene como propósito identificar los cambios en las constantes farmacocinéticas de fenitoína y carbamacepina, en pacientes epilépticas cuando se mantiene una misma dosis durante diferentes etapas del postparto. En 20 mujeres mexicanas epilépticas se determinó la concentración en plasma y leche de fenitoína durante 60 días postparto y en 14 se determinó carbamacepina. A todas las pacientes se les realizó la farmacocinética de los anticonvulsivantes en cada periodo de estudio (5, 15, 30, 45 y 60 días posparto) y se determinó el índice de excreción en leche materna. Las concentraciones plasmáticas de fenitoína no presentaron variaciones, sin embargo la carbanacepina fue más alta en el periodo tardió, también se observaron diferencias en las áreas bajo la curva y vida media de eliminación y en los índices de excreción

Chlorate metabolism by whole cells and membrane vesicles of Escherichia coli K-2.

Se siguio la reduccion del clorato por Escherichia coli K-12 mediante la utilizacion de clorato marcado 36 C1-, preparado por oxidacion anodica del ion cloruro. Las celulas redujeron el nitrato y el clorato a velocidades similares. Las vesiculas de membrana y las celulas no acumularon clorato ni sus productos de reduccion (C102-, C10-, C1). El bajo nivel de radioactividad enlazado a celulas o vesiculas puede ser atribuido principalmente a absorcion inespecifica. Estos resultados son discutidos en relacion con la localizacion del sitio enlazante del clorato (y el nitrato) sobre la nitrato reductasa membranal de E. coli