Ail Proteins of Yersinia pestis and Y. pseudotuberculosis Have Different Cell Binding and Invasion Activities

The Yersinia pestis adhesin Ail mediates host cell binding and facilitates delivery of cytotoxic Yop proteins. Ail from Y. pestis and Y. pseudotuberculosis is identical except for one or two amino acids at positions 43 and 126 depending on the Y. pseudotuberculosis strain. Ail from Y. pseudotuberculosis strain YPIII has been reported to lack host cell binding ability, thus we sought to determine which amino acid difference(s) are responsible for the difference in cell adhesion. Y. pseudotuberculosis YPIII Ail expressed in Escherichia coli bound host cells, albeit at ∼50% the capacity of Y. pestis Ail. Y. pestis Ail single mutants, Ail-E43D and Ail-F126V, both have decreased adhesion and invasion in E. coli when compared to wild-type Y. pestis Ail. Y. pseudotuberculosis YPIII Ail also had decreased binding to the Ail substrate fibronectin, relative to Y. pestis Ail in E. coli. When expressed in Y. pestis, there was a 30–50% decrease in adhesion and invasion depending on the substitution. Ail-mediated Yop delivery by both Y. pestis Ail and Y. pseudotuberculosis Ail were similar when expressed in Y. pestis, with only Ail-F126V giving a statistically significant reduction in Yop delivery of 25%. In contrast to results in E. coli and Y. pestis, expression of Ail in Y. pseudotuberculosis led to no measurable adhesion or invasion, suggesting the longer LPS of Y. pseudotuberculosis interferes with Ail cell-binding activity. Thus, host context affects the binding activities of Ail and both Y. pestis and Y. pseudotuberculosis Ail can mediate cell binding, cell invasion and facilitate Yop delivery.     

In vitro stabilization of 6-methylsalicylic acid synthetase from Penicillium urticae

In continuing studies of patulin biosynthesis, the first enzyme of the pathway, 6-methylsalicylic acid synthetase, was found to be far more labile than were the later enzymes of the pathway. Attempts were made to stabilize 6-methylsalicylic acid synthetase in vitro. The combined addition of the cofactor NADPH, the substrates acetyl-CoA and malonyl-CoA, the reducing agent dithiothreitol, and the proteinase inhibitor phenylmethylsulfonyl fluoride to cell-free extracts was found to prolong the half-life of the enzyme as much as 12-fold. This suggested that proteolysis and the conformational integrity of the enzyme may play an important role in controlling the duration of antibiotic biosynthesis in vivo. This was in agreement with the finding that the intracellular proteinase content of antibiotic-producing cells of Penicillium urticae rapidly increased just before the loss of 6-methylsalicylic acid synthetase content. These in vitro stabilization studies have provided some insight into the metabolic conditions that may stabilize these enzymes in vivo, and into possible ways of extending the life of these catalysts.     

Photooxidation of dinitrophenylhistidine-200 human carbonic anhydrase B.

Partial inactivation of tau-dinitrophenylhistidine-200 human carbonic anhydrase B, induced by visible light, followed first order kinetics (k(app) = 6.05 times 10-2 min-1). After 50 min the tau-dinitrophenylhistidine (tau-DNP-histidine) content decreased to a negligible level, but the illuminated enzyme retained, at pH 7.6, approximately 9.2 percent of the esterase activity of the native enzyme. The following lines of evidence suggest that the loss of activity results from the destruction of tau-DNP-histidine-200. (1) No significant loss of amino acid other than tau-DNP-histidine was detected after illumination. (2) The rate of loss of activity correlated well with the loss of tau-DNP-histidine. (3) In the photooxidized enzyme the DNP moiety was retained but had lost the characteristic sensitivity of tau-DNP-histidine to nucleophilic attack. Titration of the illuminated enzyme with acetazolamide indicated that the residual activity is an intrinsic property of the modified enzyme. The chromatographically purified photooxidized enzyme migrated as a single band on isoelectrofocusing in polyacylamide gel, and at pH 7.6 possessed 7.5 percent esterase activity relative to the native enzyme. By establishing effective destruction of histidine-200, it can be concluded that neither the pi N nor, as previously shown, the tau N of histidine-200 is critical for the catalysis.     

A yeast's eye view of mammalian reproduction: cross-species gene co-expression in meiotic prophase

Background  

Meiotic prophase is a critical stage in sexual reproduction. Aberrant chromosome recombination during this stage is a leading cause of human miscarriages and birth defects. However, due to the experimental intractability of mammalian gonads, only a very limited number of meiotic genes have been characterized. Here we aim to identify novel meiotic genes important in human reproduction through computational mining of cross-species and cross-sex time-series expression data from budding yeast, mouse postnatal testis, mouse embryonic ovary, and human fetal ovary.     

Effect of hyperthermia on growth of Ehrlich ascites tumor cells

Hyperthermic treatment at 43 degrees C suppressed the growth of Ehrlich ascites tumor (EAT) cells in vitro. Incubation of EAT cells at 43 degrees C for as little as 1.5 h totally abolished the transplantability of the tumor. At the same time, the rate of cellular glucose uptake, the density of glucose transporter on the cells as well as the extent of thymidine, uridine and leucine incorporation were significantly reduced.     

Conscious Brain-to-Brain Communication in Humans Using Non-Invasive Technologies

Human sensory and motor systems provide the natural means for the exchange of information between individuals, and, hence, the basis for human civilization. The recent development of brain-computer interfaces (BCI) has provided an important element for the creation of brain-to-brain communication systems, and precise brain stimulation techniques are now available for the realization of non-invasive computer-brain interfaces (CBI). These technologies, BCI and CBI, can be combined to realize the vision of non-invasive, computer-mediated brain-to-brain (B2B) communication between subjects (hyperinteraction). Here we demonstrate the conscious transmission of information between human brains through the intact scalp and without intervention of motor or peripheral sensory systems. Pseudo-random binary streams encoding words were transmitted between the minds of emitter and receiver subjects separated by great distances, representing the realization of the first human brain-to-brain interface. In a series of experiments, we established internet-mediated B2B communication by combining a BCI based on voluntary motor imagery-controlled electroencephalographic (EEG) changes with a CBI inducing the conscious perception of phosphenes (light flashes) through neuronavigated, robotized transcranial magnetic stimulation (TMS), with special care taken to block sensory (tactile, visual or auditory) cues. Our results provide a critical proof-of-principle demonstration for the development of conscious B2B communication technologies. More fully developed, related implementations will open new research venues in cognitive, social and clinical neuroscience and the scientific study of consciousness. We envision that hyperinteraction technologies will eventually have a profound impact on the social structure of our civilization and raise important ethical issues.     

Swabbing Often Fails to Detect Amphibian Chytridiomycosis under Conditions of Low Infection Load

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (denoted Bd), causes large-scale epizootics in naïve amphibian populations. Intervention strategies to rapidly respond to Bd incursions require sensitive and accurate diagnostic methods. Chytridiomycosis usually is assessed by quantitative polymerase chain reaction (qPCR) amplification of amphibian skin swabs. Results based on this method, however, sometimes yield inconsistent results on infection status and inaccurate scores of infection intensity. In Asia and other regions where amphibians typically bear low Bd loads, swab results are least reliable. We developed a Bd-sampling method that collects zoospores released by infected subjects into an aquatic medium. Bd DNA is extracted by filters and amplified by nested PCR. Using laboratory colonies and field populations of Bombina orientalis, we compare results with those obtained on the same subjects by qPCR of DNA extracted from swabs. Many subjects, despite being diagnosed as Bd-negative by conventional methods, released Bd zoospores into collection containers and thus must be considered infected. Infection loads determined from filtered water were at least 1000 times higher than those estimated from swabs. Subjects significantly varied in infection load, as they intermittently released zoospores, over a 5-day period. Thus, the method might be used to compare the infectivity of individuals and study the periodicity of zoospore release. Sampling methods based on water filtration can dramatically increase the capacity to accurately diagnose chytridiomycosis and contribute to a better understanding of the interactions between Bd and its hosts.     

Effect of trichome density on soybean pod feeding by adult bean leaf beetles (Coleoptera: Chrysomelidae).

The role of soybean, Glycine max (L.) Merrill, pod trichomes on feeding by adult bean leaf beetles. Cerotoma trifurcata (Forster), was evaluated under laboratory and field conditions during 1997 and 1998. Three Clark isolines and 'Corsoy 79' were used to compare the feeding preference of the beetle on pods with different trichome densities. The three Clark isolines, including densely pubescent, sparsely pubescent, and glabrous, were isogenic except for trichome density. The trichome densities on the pods of Clark densely pubescent, Clark sparsely pubescent, and Corsoy 79 were significantly different. In no-choice tests, under both laboratory and field conditions, the feeding on pods of Clark densely pubescent was significantly lower than that on the other isolines. In the choice test of Corsoy 79 conducted in the laboratory and field, when the trichomes on one of the two pods were shaven off, the feeding on the shaven pods was significantly higher than that of the intact ones. In the choice test among Clark isolines under laboratory condition, the result was significant, with the lowest feeding on the densely pubescent pods. This study demonstrates that densely pubescent soybean has the potential to resist bean leaf beetle feeding on pods.