Identification and separation of Thy-1 positive mouse spleen cells active in natural cytotoxicity and antibody-dependent cell-mediated cytotoxicity.

The expression of the Thy-1 antigen on mouse spleen cells responsible for NK activity and ADCC was investigated by using a monoclonal IgM anti-Thy-1.2 antibody. Both C-mediated cytotoxicity and the fluorescence-activated cell sorter were used to fractionate cells. The effector cells were found to be heterogeneous in their expression of Thy-1. Effector cells from nude BALB/c mice were predominantly Thy-1 positive; some of the NK cells in CBA spleens appeared to be Thy-1 positive, but at least one-third of the lytic activity was due to Thy-1 negative cells. The effects of treatments on NK cytotoxicity and ADCC were very similar, supporting the hypothesis that the same cells mediate both activities.     

Do sturgeon limit burrowing shrimp populations in Pacific Northwest Estuaries?

Green sturgeon, Acipenser medirostris, and white sturgeon, Acipenser transmontanus, are frequent inhabitants of coastal estuaries from northern California, USA to British Columbia, Canada. An analysis of stomach contents from 95 green sturgeon and six white sturgeon commercially landed in Willapa Bay, Grays Harbor, and the Columbia River estuary during 2000–2005 revealed that 17–97% had empty stomachs, but those fish with items in their guts fed predominantly on benthic prey items and fish. Burrowing thalassinid shrimp (mostly Neotrypaea californiensis) were important food items for both white and especially for green sturgeon taken in Willapa Bay, Washington during summer 2003, where they represented 51% of the biomass ingested (84.9% IRI). Small pits observed in intertidal areas dominated by these shrimp, are likely made by these sturgeon and we present evidence from exclusion studies and field observation that the predator making the pits can have a significant cumulative negative effect on burrowing shrimp density. These burrowing shrimp present a threat to the aquaculture industry in Washington State due to their ability to de-stabilize the substrate on which shellfish are grown. Despite an active burrowing shrimp control program in these estuaries, it seems unlikely that current burrowing shrimp abundance and availability as food is a limiting factor for threatened green sturgeon stocks. However, these large predators may have performed an important top down control function on shrimp populations in the past when they were more abundant.     

Accommodation of GDP-linked sugars in the active site of GDP-perosamine synthase

Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 A resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K m for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k cat was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.     

Mg-activated double-stranded DNA cleavage by [(bpy)2(OH2)Ru---O---Ru(H2O) (bpy)2]

The reaction of the title complex with DNA has been examined. Addition of [(bpy)₂(OH₂)RuORu(OH₂) (bpy)₂]⁴⁺ to DNA leads to the reduction of the complex to Ru(bpy)₂(OH₂)₂²⁺, as indicated by absorption spectroscopy and cyclic voltammetry. The reaction is accelerated by Mg²⁺. The combined evidence points to a mechanism where the oxo-bridged dimer is hydrolyzed to a monomeric Ru(III) complex that is capable of oxidizing DNA to effect strand scission. Gel electrophoresis demonstrates nicking of supercoiled /gfX174 DNA by [(bpy)₂(OH₂)RuORu(OH₂) (bpy)₂]⁴⁺, and double-stranded cleavage is observed in the presence of Mg²⁺. Linearization of the plasmid prior to treatment with the complex does not lead to further fragmentation, suggesting that supercoiling is required to realize double-stranded cleavage.     

Quorum-sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other Gram-negative bacteria

In cell-free Pseudomonas aeruginosa culture supernatants, we identified two compounds capable of activating an N-acylhomoserine lactone (AHL) biosensor. Mass spectrometry and NMR spectroscopy revealed that these compounds were not AHLs but the diketopiperazines (DKPs), cyclo(DeltaAla-L-Val) and cyclo(L-Pro-L-Tyr) respectively. These compounds were also found in cell-free supernatants from Proteus mirabilis, Citrobacter freundii and Enterobacter agglomerans [cyclo(DeltaAla-L-Val) only]. Although both DKPs were absent from Pseudomonas fluorescens and Pseudomonas alcaligenes, we isolated, from both pseudomonads, a third DKP, which was chemically characterized as cyclo(L-Phe-L-Pro). Dose-response curves using a LuxR-based AHL biosensor indicated that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) activate the biosensor in a concentration-dependent manner, albeit at much higher concentrations than the natural activator N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL). Competition studies showed that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) antagonize the 3-oxo-C6-HSL-mediated induction of bioluminescence, suggesting that these DKPs may compete for the same LuxR-binding site. Similarly, DKPs were found to be capable of activating or antagonizing other LuxR-based quorum-sensing systems, such as the N-butanoylhomoserine lactone-dependent swarming motility of Serratia liquefaciens. Although the physiological role of these DKPs has yet to be established, their activity suggests the existence of cross talk among bacterial signalling systems.     

The Virtual Ventricular Wall: A Tool for Exploring Cardiac Propagation and Arrhythmogenesis

Methods for the experimental and clinical investigation of cardiac arrhythmias are limited to inferring propagation within the myocardium, from surface measurements, or from electrodes at a few sites within the cardiac wall. Biophysically and anatomically detailed computational models of cardiac tissues offer a powerful way for studying the electrical propagation processes and arrhythmias within the virtual heart. We use virtual tissues to study and visualise the effects of patho- and physiological conditions, and pharmacological interventions on transmural propagation in the virtual ventricular walls. Class III drug actions are quantitatively explained by changes induced in the transmural dispersion of action potential duration. We illustrate the automated construction of a virtual anisotropic ventricle from Diffusion Tensor MRI for individual hearts, and use it to explore mechanisms leading to ventricular fibrillation. The virtual ventricular wall provides an effective tool for exploring, evaluating and visualising processes during the initiation and maintenance of ventricular arrhythmias.     

Insertional mutagenesis of pathogenic fungi

Screening insertional mutants for loss of virulence is an effective method for investigating the molecular genetic basis of bacterial pathogenesis, but has only recently been applied to fungal pathogens. For many pathogenic fungi transformation with heterologous plasmid DNA results in complex integration events. This problem can now be circumvented for some species using restriction enzyme mediated integration. Insertional mutagenesis of Fusarium oxysporum using the naturally occurring fungal transposon impala has been described, but transposon tagging for other fungi has yet to be developed. Although insertional mutagenesis has recently identified important virulence determinants of fungal phytopathogens, the lack of suitable screening strategies has so far limited its applicability for fungal pathogens of humans.     

Toluene diffusion and reaction in unsaturated Pseudomonas putida biofilms

Biofilms are frequently studied in the context of submerged or aquatic systems. However, much less is known about biofilms in unsaturated systems, despite their importance to such processes as food spoilage, terrestrial nutrient cycling, and biodegradation of environmental pollutants in soils. Using modeling and experimentation, we have described the biodegradation of toluene in unsaturated media by bacterial biofilms as a function of matric water potential, a dominant variable in unsaturated systems. We experimentally determined diffusion and kinetic parameters for Pseudomonas putida biofilms, then predicted biodegradation rates over a range of matric water potentials. For validation, we measured the rate of toluene depletion by intact biofilms and found the results to reasonably follow the model predictions. The diffusion coefficient for toluene through unsaturated P. putida biofilm averaged 1.3 x 10(7) cm(2)/s, which is approximately two orders of magnitude lower than toluene diffusivity in water. Our studies show that, at the scale of the microbial biofilm, the diffusion of toluene to biodegrading bacteria can limit the overall rate of biological toluene depletion in unsaturated systems. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 656-670, 1997.     

Apparently unstable normal FMR1 alleles in nine developmentally delayed patients: implications for molecular diagnosis of the fragile X syndrome

The Fragile X syndrome is a common form of X-linked mental retardation, affecting approximately 1 in 4,000 males. Since the discovery of the FMR1 gene responsible for the syndrome, molecular, rather than cytogenetic, diagnosis of Fragile X syndrome has become the gold standard. Numerous molecular diagnostic centers worldwide use PCR and Southern blotting to characterize the size of the CGG repeats within the gene, expansion of which has been shown to be associated with the vast majority of cases of Fragile X syndrome. Instability of this repeat through successive generations has been demonstrated in many patients and has been associated with numerous factors, including repeat length and molecular structure of the repeat. Nine males with normal-size alleles that exhibit repeat length instability by the presence of a second normal length distinct band by repeated PCR analysis from peripheral lymphocytes are reported. Many hypotheses addressing the reason for this apparent instability were tested without elucidating the underlying molecular causes, including cytogenetic analysis, sequence analysis of the repeat locus, and analysis of flanking dinucleotide repeat loci. All patients exhibited a normal complement of sex chromosomes by cytogenetic and molecular analysis. These results from the widely used PCR analysis illustrate an interesting molecular phenomenon and raise many questions relating to the factors and mechanisms involved in trinucleotide instability as well as having implications for the diagnostic testing of the Fragile X syndrome.     

The response of excitable membrane models to a cyclic input

The response of a space-clamped patch of Hodgkin-Huxley membrane to an applied current density ofA cos(2ft)+BA/cm² is computed for frequencies from 5 to 250 Hz. The train of action potentials generated is phase-locked to the driving cycle,N action potentials occurring at fixed phases inM cycles. For frequencies whereN/M is a simple ratio a describing function for the membrane is computed. The phase-locked behaviour and describing functions are similar to those obtained for a simple leaky integrator neurone model.