Use of a microbial sensor: inhibition effect of azo-reactive dyes on activated sludge

This paper summarizes the methodology utilized for measuring the toxic and inhibitory effects of azo-reactive dyes on the activity of activated sludge. The microbial sensor employed in this study consisted of a small-fluidized bed reactor in which the microbial mass was immobilized on spherical (diameter =1-2 mm) reticulated sinter glass carriers. To sustain a highly dense population of aerobic microbes, pure oxygen was supplied via a cylindrical chamber, which comprised part of the sample re-circulation system. The mean hydraulic retention time in the microbial sensor ranged between 30 and 40 min, while temperature was maintained at 30 degrees C and pH at 6.4. Inhibition of microbial activity (toxicity) was determined as the mean percent reduction in carbon dioxide production from microorganisms' respiration. Several azo-reactive dyes demonstrated toxicity when applied at a high concentration (2 g/l), however, a portion of the microbes showed tolerance to the dyes. Moreover, textile wastewater demonstrated very efficient biodegradation.     

Soluble T-lymphocyte antigen-specific immunoproteins: a progress report

T-cell-derived proteins that bind nominal (non-MHC-associated) antigen specifically (TABM) express V and C region epitopes of the T-cell receptor (TCR) for antigen and have a significant similarity in amino acid sequence to TCR alpha-chain V and C region. The presence of these immunoproteins in human serum and a specific increase in serum TABM in infectious disease, chemical sensitivity, and food intolerance suggest that TABM may impact on pathogenesis through the modulation of cell-mediated immunity, the antigen-specific concentration and delivery of immunoregulatory cytokines such as TGF-beta and elastase, and the induction of the release of substance P by sensory neurons. In this Minireview update, we describe advances in the detection and quantitation of human TABM by monoclonal antibodies, and the association of increased human serum TABM titers in infectious disease, chemical sensitivity, and food intolerance. We suggest that the immunomodulatory mode of action of these immunoproteins may be the antigen-specific focusing of cytokines associated with TABM.     

Ascorbic acid might play a role in the sclerotial differentiation of Sclerotium rolfsii

Certain phytopathogenic fungi differentiate by forming sclerotia by an unclear biochemical mechanism. We have proposed that sclerotial differentiation might be regulated by fungal antioxidant defense. Part of this defense might be ascorbic acid, which in its reduced form is a well-known antioxidant. This natural antioxidant was studied in Sclerotium rolfsii in relation to oxidative-growth conditions, developmental stages and strain-differentiating ability. The transition of a sclerotial strain from the undifferentiated to the differentiated stage was accompanied by a sharp shift in the ratio of reduced/oxidized ascorbate toward the oxidized form. Ascorbate profiles and lipid peroxidation levels were different between the sclerotial strain grown under high- and low-oxidative stress conditions, as well as between a nonsclerotial S. rolfsii strain grown under high-oxidative stress conditions. In addition, the ratio of reduced/oxidized ascorbate in the nonsclerotial strain remained unchanged throughout growth. Lipid peroxidation under high-oxidative stress conditions in sclerotial S. rolfsii colonies one day before differentiation was 3.6-fold higher than in same-day colonies of this strain grown under low-oxidative stress conditions and 2.5-fold higher than in similar-day colonies of the nonsclerotial strain grown under high-oxidative stress conditions. Exogenous ascorbate caused a concentration-dependent reduction of lipid peroxidation and a proportional inhibition of the degree of sclerotial differentiation in the sclerotial strain grown under high-oxidative stress conditions by lowering its lipid peroxidation before differentiation to levels similar to the strain grown under low-oxidative stress conditions and to the nonsclerotial strain. Ascorbic acid might be produced by the sclerotial strain to reduce oxidative stress, although less efficiently than the nondifferenting strain. The data of this study support our theory that oxidative stress might be the triggering factor of sclerotial differentiation in phytopathogenic fungi.     

SOAP,a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development

An essential, but poorly understood part of malaria transmission by mosquitoes is the development of the ookinetes into the sporozoite-producing oocysts on the mosquito midgut wall. For successful oocyst formation newly formed ookinetes in the midgut lumen must enter, traverse, and exit the midgut epithelium to reach the midgut basal lamina, processes collectively known as midgut invasion. After invasion ookinete-to-oocyst transition must occur, a process believed to require ookinete interactions with basal lamina components. Here, we report on a novel extracellular malaria protein expressed in ookinetes and young oocysts, named secreted ookinete adhesive protein (SOAP). The SOAP gene is highly conserved amongst Plasmodium species and appears to be unique to this genus. It encodes a predicted secreted and soluble protein with a modular structure composed of two unique cysteine-rich domains. Using the rodent malaria parasite Plasmodium berghei we show that SOAP is targeted to the micronemes and forms high molecular mass complexes via disulphide bonds. Moreover, SOAP interacts strongly with mosquito laminin in yeast-two-hybrid assays. Targeted disruption of the SOAP gene gives rise to ookinetes that are markedly impaired in their ability to invade the mosquito midgut and form oocysts. These results identify SOAP as a key molecule for ookinete-to-oocyst differentiation in mosquitoes.     

Laminin alpha subunits and their role in C. elegans development

Laminins are heterotrimeric (alpha/beta/gamma) glycoproteins that form a major polymer within basement membranes. Different alpha, beta and gamma subunits can assemble into various laminin isoforms that have different, but often overlapping, distributions and functions. In this study, we examine the contributions of the laminin alpha subunits to the development of C. elegans. There are two alpha, one beta and one gamma laminin subunit, suggesting two laminin isoforms that differ by their alpha subunit assemble in C. elegans. We find that near the end of gastrulation and before other basement membrane components are detected, the alpha subunits are secreted between primary tissue layers and become distributed in different patterns to the surfaces of cells. Mutations in either alpha subunit gene cause missing or disrupted extracellular matrix where the protein normally localizes. Cell-cell adhesions are abnormal: in some cases essential cell-cell adhesions are lacking, while in other cases, cells inappropriately adhere to and invade neighboring tissues. Using electron microscopy, we observe adhesion complexes at improper cell surfaces and disoriented cytoskeletal filaments. Cells throughout the animal show defective differentiation, proliferation or migration, suggesting a general disruption of cell-cell signaling. The results suggest a receptor-mediated process localizes each secreted laminin to exposed cell surfaces and that laminin is crucial for organizing extracellular matrix, receptor and intracellular proteins at those surfaces. We propose this supramolecular architecture regulates adhesions and signaling between adjacent tissues.     

Synthesis and angiogenetic activity in the chick chorioallantoic membrane model of thymosin beta-15

Thymosin beta-15 (Tbeta15), a 44 amino acid peptide (MW = 5173) localized in human prostate and breast cancer tissues was successfully synthesized in multigram quantities using Fmoc solid-phase peptide synthesis. The synthesized product was shown to have the right structure by ESI and MALDI mass spectral techniques and amino acid analysis. Relatively high yield was achieved, which might be due to enhanced acid stability of the p-cyanotrityl resin used. The effect of the synthesized Tbeta15 on the angiogenesis process was investigated using the chick chorioallantoic membrane (CAM) in vivo model. At concentrations above 1 microg/10 microl per disc, Tbeta15 exhibited a positive effect on angiogenesis, comparable to the effect of the intense angiogenetic factor phorbol 12-myristate 13-acetate at a standard concentration of 0.1 microg/10 microl per disc. The results of this study contribute to the further elucidation of the biological regulatory role of thymosin peptides and provide helpful information in the investigation of their possible therapeutic potential.     

Enhanced crossover SCRATCHY: construction and high-throughput screening of a combinatorial library containing multiple non-homologous crossovers

SCRATCHY is a methodology for the construction of libraries of chimeras between genes that display low sequence homology. We have developed a strategy for library creation termed enhanced crossover SCRATCHY, that significantly increases the number of clones containing multiple crossovers. Complementary chimeric gene libraries generated by incremental truncation (ITCHY) of two distinct parental sequences are created, and are then divided into arbitrarily defined sections. The respective sections are amplified by skewed sets of primers (i.e. a combination of gene A specific forward primer and gene B specific reverse primer, etc.) allowing DNA fragments containing non-homologous crossover points to be amplified. The amplified chimeric sections are then subjected to a DNA shuffling process generating an enhanced crossover SCRATCHY library. We have constructed such a library using the rat theta 2 glutathione transferase (rGSTT2) and the human theta 1 glutathione transferase (hGSTT1) genes (63% DNA sequence identity). DNA sequencing analysis of unselected library members revealed a greater diversity than that obtained by canonical family shuffling or with conventional SCRATCHY. Expression and high-throughput flow cytometric screening of the chimeric GST library identified several chimeric progeny that retained rat-like parental substrate specificity.