Serine Protease EspP from Enterohemorrhagic Escherichia Coli Is Sufficient to Induce Shiga Toxin Macropinocytosis in Intestinal Epithelium

Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC) O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells’ basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.     

Testosterone regulates 25-hydroxycholesterol production in testicular macrophages

Recently, we found that testicular macrophages produce 25-hydroxycholesterol (25-HC) and express 25-hydroxylase, the enzyme that converts cholesterol to 25-HC. In addition, 25-HC may be an important paracrine factor mediating the known interactions between macrophages and neighboring Leydig cells, because it is efficiently converted to testosterone by Leydig cells. The purpose of the present study was to determine if testosterone can regulate the production of 25-HC in rat testicular macrophages, representing a potential negative-feedback loop from Leydig cells. We found that expression of 25-hydroxylase mRNA and production of 25-HC by cultured testicular macrophages were significantly inhibited by testosterone at 10 micro g/ml. This dose of testosterone did not have an effect on cell viability and did not change the rate of mRNA degradation in the presence of actinomycin D. These studies indicate that production of 25-HC is negatively regulated by testosterone, which may be representative of a paracrine negative-feedback loop.     

Comparative evaluation of pectolytic and proteolytic enzyme production by free and immobilized cells of some strains of the phytopathogenic Erwinia chrysanthemi

Whole cells of the phytopathogenic Erwinia chrysanthemi strains were immobilized in k-carrageenan and grown in high-calcium Xanthomonas campestris medium containing sodium polypectate as carbon source. All the strains used survived immobilization into k-carrageenan beads. Immobilized E. chrysanthemi strains displayed higher pectolytic and proteolytic enzyme activities than free cells in liquid suspension. Carrageenan immobilization techniques could provide a system to mimic the conditions of E. chrysanthemi cells in the infected plant tissue. This could prompt a thorough study of the factors governing the biosynthesis of virulence factors by this bacterium. Journal of Industrial Microbiology & Biotechnology (2001) 27, 215–219. Received 04 April 2001/ Accepted in revised form 12 June 2001     

Analysis of molecular genetic study on the polymorphic C-159T locus of the <Emphasis Type="Italic">CD14</Emphasis> gene in children with increased risk of recurrent episodes of acute obstructive bronchitis

A molecular genetic study of the polymorphic C-159T locus of the CD14 gene has been carried out in 31 children with recurrent episodes of acute obstructive bronchitis as well as in a cohort of 50 randomly sampled subjects forming the control group. As a result of the study, no higher statistically significant frequency of the CC, CT, and TT genotypes has been recorded at the polymorphic C-159T locus of the CD14 gene among children of the main group compared with the control. However, there is a trend towards a more frequent observation of the TT genotype among children with recurrent episodes of acute obstructive bronchitis compared with the control group (32.25 vs. 22.0%).     

Mitochondria in cardiomyocyte Ca signaling

Ca²⁺ signaling is of vital importance to cardiac cell function and plays an important role in heart failure. It is based on sarcolemmal, sarcoplasmic reticulum and mitochondrial Ca²⁺ cycling. While the first two are well characterized, the latter remains unclear, controversial and technically challenging.In mammalian cardiac myocytes, Ca²⁺ influx through L-type calcium channels in the sarcolemmal membrane triggers Ca²⁺ release from the nearby junctional sarcoplasmic reticulum to produce Ca²⁺ sparks. When this triggering is synchronized by the cardiac action potential, a global [Ca²⁺]_i transient arises from coordinated Ca²⁺ release events. The ends of intermyofibrillar mitochondria are located within 20 nm of the junctional sarcoplasmic reticulum and thereby experience a high local [Ca²⁺] during the Ca²⁺ release process. Both local and global Ca²⁺ signals may thus influence calcium signaling in mitochondria and, reciprocally, mitochondria may contribute to the local control of calcium signaling. In addition to the intermyofibrillar mitochondria, morphologically distinct mitochondria are also located in the perinuclear and subsarcolemmal regions of the cardiomyocyte and thus experience a different local [Ca²⁺].Here we review the literature in regard to several issues of broad interest: (1) the ultrastructural basis for mitochondrion – sarcoplasmic reticulum cross-signaling; (2) mechanisms of sarcoplasmic reticulum signaling; (3) mitochondrial calcium signaling; and (4) the possible interplay of calcium signaling between the sarcoplasmic reticulum and adjacent mitochondria.Finally, this review discusses experimental findings and mathematical models of cardiac calcium signaling between the sarcoplasmic reticulum and mitochondria, identifies weaknesses in these models, and suggests strategies and approaches for future investigations.     

A bioluminescence method for direct measurement of phosphodiesterase activity

We have adapted bioluminescence methods to be able to measure phosphodiesterase (PDE) activity in a one-step technique. The method employs a four-enzyme system (PDE, adenylate kinase (AK) using excess CTP instead of ATP as substrate, pyruvate kinase (PK), and firefly luciferase) to generate ATP, with measurement of the concomitant luciferase-light emission. Since AK, PK, and luciferase reactions are coupled to recur in a cyclic manner, AMP recycling maintains a constant rate of ATP formation, proportional to the steady-state AMP concentration. The cycle can be initiated by the PDE reaction that yields AMP. As long as the PDE reaction is rate limiting, the system is effectively at steady state and the bioluminescence kinetics progresses at a constant rate proportional to the PDE activity. In the absence of cAMP and PDE, low concentrations of AMP trigger the AMP cycling, which allows standardizing the system. The sensitivity of the method enables detection of <1 μU (pmol/min) of PDE activity in cell extracts containing 0.25–10 μg protein. Assays utilizing pure enzyme showed that 0.2 mM IBMX completely inhibited PDE activity. This single-step enzyme- and substrate-coupled cyclic-reaction system yields a simplified, sensitive, reproducible, and accurate method for quantifying PDE activities in small biological samples.     

Light intensity regulates growth and reproduction of a snail grazer (Gyraulus chinensis) through changes in the quality and biomass of stream periphyton

1. The light : nutrient hypothesis (LNH) proposes that herbivore growth rates are maximised at intermediate light‐to‐nutrient ratios. A reduction to light intensity (i.e. decreased light‐to‐nutrient ratio) should lead to reduced food availability for herbivores while excessive light intensity in oligotrophic environments (i.e. increased light‐to‐nutrient ratios) should increase the C : N and C : P ratios of producers. However, this hypothesis has not yet been supported by studies on stream ecosystems. 2. We tested the LNH by experimental application of controlled natural gradients in light intensity to oligotrophic laboratory channels that included periphyton and the freshwater snail Gyraulus chinensis. 3. The results in this oligotrophic environment indicate that light regulated the flow of matter between trophic levels and grazer reproduction by controlling C : P ratios of the producers.     

The ecology and agronomy of Miscanthus sinensis, a species important to bioenergy crop development, in its native range in Japan: a review

Among several candidate perennial taxa, Miscanthus × giganteus has been evaluated and promoted as a promising bioenergy crop. Owing to several limitations, however, of the sterile hybrid, both at the taxon and agronomic production levels, other options need to be explored to not only improve M . × giganteus , which was originally collected in Japan, but to also consider the development of other members of its genus, including Miscanthus sinensis , as bioenergy crops. Indeed, there is likely much to be learned and applied to Miscanthus as a bioenergy crop from the long history of intensive interaction between humans and M. sinensis in Japan, which in some regions of the country spans several thousand years. Combined with its high amount of genetic variation, stress tolerance, biotic interactions with fauna, and function as a keystone species in diverse grasslands and other ecosystems within its native range, the unique and extensive management of M. sinensis in Japan as a forage grass and building material provides agronomists, agroecologists, and plant breeders with the capability of better understanding this species in terms of potential contribution to bioenergy crop development. Moreover, the studies described in this review may serve as a platform for future research of Miscanthus as a bioenergy crop in other parts of the world.     

Aboveground plant biomass,carbon, and nitrogen dynamics before and after burning in a seminatural grassland of Miscanthus sinensis in Kumamoto,Japan

Although fire has been used for several thousand years to maintain Miscanthus sinensis grasslands in Japan, there is little information about the nutrient dynamics in these ecosystems immediately after burning. We investigated the loss of aboveground biomass; carbon (C) and nitrogen (N) dynamics; surface soil C change before and after burning; and carbon dioxide (CO₂), methane (CH₄), and nitrous oxide (N₂O) fluxes 2 h after burning in a M. sinensis grassland in Kumamoto, Japan. We calculated average C and N accumulation rates within the soil profile over the past 7300 years, which were 58.0 kg C ha^?1 yr^?1 and 2.60 kg N ha^?1 yr^?1, respectively. After burning, 98% of aboveground biomass and litter were consumed. Carbon remaining on the field, however, was 102 kg C ha^?1. We found at least 43% of C was possibly lost due to decomposition. However, remaining C, which contained ash and charcoal, appeared to contribute to C accumulation in soil. There was no difference in the amount of 0–5 cm surface soil C before and after burning. The amount of remaining litter on the soil surface indicated burning appeared not to have caused a reduction in soil C nor did it negatively impact the sub‐surface vegetative crown of M. sinensis. Also, nearly 50 kg N ha^?1 of total aboveground biomass and litter N was lost due to burning. Compared with before the burning event, postburning CO₂ and CH₄ fluxes from soil appeared not to be directly affected by burning. However, it appears the short time span of measurements of N₂O flux after burning sufficiently characterized the pattern of increasing N₂O fluxes immediately after burning. These findings indicate burning did not cause significant reductions in soil C nor did it result in elevated CO₂ and CH₄ emissions from the soil relative to before the burning event.     

25-hydroxycholesterol is produced by testicular macrophages during the early postnatal period and influences differentiation of Leydig cells in vitro

Leydig cells develop inappropriately in animals lacking testicular macrophages. We have recently found that macrophages from adult animals produce 25-hydroxycholesterol, an oxysterol involved in the differentiation of hepatocytes and keratinocytes. Therefore, we hypothesized that testicular macrophages also produce 25-hydroxycholesterol during the early postnatal period and that this oxysterol plays a role in the differentiation of Leydig cells. We assessed the production of 25-hydroxycholesterol and 25-hydroxylase mRNA by cultured testicular macrophages from rats at 10, 20, and 40 days of age. We also tested the long-term effects of 25-hydroxycholesterol on basal and LH-stimulated testosterone production, and 3beta-hydroxysteroid dehydrogenase activity as end points of Leydig cell differentiation in vitro. We found that testicular macrophages from animals at all ages produced both 25-hydroxycholesterol and 25-hydroxylase mRNA, with macrophages from 10-day-old animals having the highest steady-state levels of message. We also found that chronic exposure of Leydig cells to 25-hydroxycholesterol increased basal production of testosterone but decreased LH-stimulated steroidogenesis at all ages. Finally, 25-hydroxycholesterol increased 3beta-hydroxysteroid dehydrogenase activity in both progenitor and immature Leydig cells. These findings support the hypothesis that testicular macrophages play an important role in the differentiation of Leydig cells through the secretion of 25-hydroxycholesterol.