Regulation of the xylan-degrading apparatus of Cellvibrio japonicus by a novel two-component system

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.     

Regulation of expression of a sheep metallothionein 1a-sheep growth hormone fusion gene in transgenic mice.

Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.     

Identification of a novel inositol bisphosphate isomer formed in chemoattractant stimulated human polymorphonuclear leukocytes

Analysis of inositol phosphate formation in chemoattractant-stimulated human polymorphonuclear leukocytes demonstrated the production of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,4-bisphosphate and another inositol bisphosphate isomer not detected in unstimulated cells. Studies in cell sonicates provided evidence that the previously unidentified inositol bisphosphate isomer is produced via the degradation of inositol 1,3,4-trisphosphate. This unidentified inositol bisphosphate peak was purified by high pressure liquid chromatography, and base hydrolyzed to form a mixture of inositol monophosphate isomers. Based on these studies, the unidentified peak was identified as inositol 3,4-bisphosphate. Identification of this isomer defines a new metabolic product derived from the initial inositol 1,4,5-trisphosphate formation, and also suggests another substrate for the inositol 1-phosphatase.     

Comparative epidemiology and control of trypanosomes

Tsetse-transmitted trypanosomiasis in man and domestic livestock is a major constraint to livestock and socio-economic development in Africa. Analysis of the limited number of databases on matching animal health and productivity available for cattle in tsetse-infested areas of Africa has provided new information about the value of current methods of controlling the disease. It was established that the trypanotolerant breeds of cattle, namely N'Dama and West African Shorthorn, were much more productive than originally believed, despite their small stature, and, as a result, could be considered for livestock development programmes in tsetse-infested regions. Moreover, in areas of low tsetse challenge, it was shown that the strategic use of therapeutic trypanocidal drugs allowed the maintenance of high levels of productivity in dairy cattle and demonstrated genotype and acquired differences in treatment requirements. On the other hand, the use of a prophylactic drug regime allowed beef cattle to be reared to an economically acceptable level of productivity in an area where tsetse challenge was high that animals rapidly succumbed if left untreated. These results show that the current methods available for the control of animal African trypanosomiasis can be effective if properly applied.     

Sugar control of the plant cell cycle: differential regulation of Arabidopsis D-type cyclin gene expression

In most plants, sucrose is the major transported carbon source. Carbon source availability in the form of sucrose is likely to be a major determinant of cell division, and mechanisms must exist for sensing sugar levels and mediating appropriate control of the cell cycle. We show that sugar availability plays a major role during the G(1) phase by controlling the expression of CycD cyclins in Arabidopsis. CycD2 mRNA levels increase within 30 min of the addition of sucrose; CycD3 is induced after 4 h. This corresponds to induction of CycD2 expression early in G(1) and CycD3 expression in late G(1) near the S-phase boundary. CycD2 and CycD3 induction is independent both of progression to a specific point in the cell cycle and of protein synthesis. Protein kinase activity of CycD2- and CycD3-containing cyclin-dependent kinases is consistent with the observed regulation of their mRNA levels. CycD2 and CycD3 therefore act as direct mediators of the presence of sugar in cell cycle commitment. CycD3, but not CycD2, expression responds to hormones, for which we show that the presence of sugars is required. Finally, protein phosphatases are shown to be involved in regulating CycD2 and CycD3 induction. We propose that control of CycD2 and CycD3 by sucrose forms part of cell cycle control in response to cellular carbohydrate status.     

Structure of an S layer on a pathogenic strain of Aeromonas hydrophila.

Negative staining revealed a tetragonal surface array (S layer) on all the members of a serogroup of Aeromonas hydrophila which possess high virulence for fish. The S layers were similar on all the strains examined, with unit cell dimensions of approximately 12 nm. A single representative strain, strain TF7, was selected for further analysis. Freeze-cleaved and etched preparations and sections for electron microscopy showed that the S layer was the outermost component of the cell envelope. This was confirmed by observation of thin sections. Computer-generated enhancements of the negatively stained micrographs showed the subunit organization to a resolution of less than 4 nm. Two structural units of identical lattice constants alternated in the array in both axes, and one of them was apparently dominant as the center of mass. The lesser unit was rotated 20 degrees from the dominant axes of symmetry and was formed by the junction of linker projections from a corner of the four components of the dominant unit. This interpretation was supported by finding that the array consists of a single polypeptide (molecular weight, 52,000). The unit cell as defined showed p4 symmetry, and a = b = 12.2 nm.     

Thiophilic paramagnetic particles as a batch separation medium for the purification of antibodies from various source materials

A preparation of thiophilic agarose-based paramagnetic particles (T-Gel) has been developed with physical characteristics (particle size and particle density) that facilitate its use as a batch separation medium suitable for the large-scale purification and isolation of immunoglobulins. The medium was used to extract immunoglobulins from a wide range of starting materials, including sera, ascites fluid, tissue culture medium, and whole blood. None of these starting materials required pretreatment such as clarification by centrifugation or filtration prior to antibody extraction. The antibody purity obtained using T-Gel compared well with that obtained using protein A agarose column chromatography. Yields were approximately 30 mg of immunoglobulins per milliliter of T-Gel, and little was required in the way of specialist equipment. The method is uncomplicated and involves a roll mix extraction overnight, followed by magnetic separation to facilitate supernatant removal and subsequent washing of the particles. Elution of bound antibodies was carried out at neutral pH to yield a concentration of immunoglobulins that was approximately 7 mg/ml. The method was found to be applicable to antibody purification from the blood serum of seven different mammalian species and for all immunoglobulin classes.