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Background: The prognostic importance of renal function in patients undergoing surgery for valvular heart disease is poorly defined. The current study addresses this issue., Methods: Baseline demographic and clinical variables, including the European system for cardiac operative risk evaluation (EuroSCORE), were recorded prospectively from 514 consecutive patients undergoing heart valve surgery between April 2000 and March 2004. Patients with active infective endocarditis and/or requiring emergency surgery were excluded. The glomerular filtration rate was estimated (eGFR) using the Modification of Diet in Renal Disease equation. The primary outcome was all-cause mortality., Results: During a median follow-up of 2 years, 87 patients died. In univariable analysis, both eGFR (hazard ratio [HR] 0.69 per 10 mL/min per 1.73 m2, P<.001) and creatinine (HR 1.04 per 10 micromol/L, P<.001) predicted mortality. Estimated GFR was a stronger predictor and was used in subsequent multivariable models. It remained a powerful independent predictor of death in a multivariable model including all study variables (HR 0.70 per 10 mL/min per 1.73 m2 increase, P<.001) and in a model including EuroSCORE (HR 0.64 per 10 mL/min per 1.73 m2 increase, P<.001). After correction for preoperative EuroSCORE, an eGFR of <60 mL/min per 1.73 m2 was associated with an excess hazard of death of 2.31 (P=.001)., Conclusion: Renal function, particularly the eGFR, is a powerful predictor of outcome in patients undergoing heart valve surgery. This prognostic utility is independent of other recognized risk factors and the EuroSCORE.

Myxococcus xanthus is a gliding bacterium with a complex life cycle that includes swarming, predation and fruiting body formation. Directed movements in M. xanthus are regulated by the Frz chemosensory system, which controls cell reversals. The Frz pathway requires the activity of FrzCD, a cytoplasmic methyl-accepting chemotaxis protein, and FrzF, a methyltransferase (CheR) containing an additional domain with three tetra trico-peptide repeats (TPRs). To investigate the role of the TPRs in FrzCD methylation, we used full-length FrzF and FrzF lacking its TPRs (FrzF(CheR)) to methylate FrzCD in vitro. FrzF methylated FrzCD on a single residue, E182, while FrzF(CheR) methylated FrzCD on three residues, E168, E175 and E182, indicating that the TPRs regulate site-specific methylation. E168 and E182 were predicted consensus methylation sites, but E175 is methylated on an HE pair. To determine the roles of these sites in vivo, we substituted each methylatable glutamate with either an aspartate or an alanine residue and determined the impact of the point mutants on single cell reversals, swarming and fruiting body formation. Single, double and triple methylation site mutants revealed that each site played a unique role in M. xanthus behaviour and that the pattern of receptor methylation determined receptor activity. This work also shows that methylation can both activate and inactivate the receptor.

The complex life cycle of Myxococcus xanthus includes predation, swarming, fruiting-body formation and sporulation. The genome of M. xanthus is large and comprises an estimated 7,400 open reading frames, of which approximately 605 code for regulatory genes. These include eight clusters of chemotaxis-like genes that define eight chemosensory pathways, most of which have dedicated functions. Although many of these chemosensory pathways have a role in controlling motility, at least two of these pathways control gene expression during development.

The characteristics that allow one Campylobacter jejuni genotype to succeed over another under the influence of bacteriophage predation have been examined in experimental broiler chickens following the observation that this succession appeared to occur in naturally colonized broiler chicken flocks. Examination of three C. jejuni strains from a single flock indicated that horizontal transfer of at least 112 kb of genomic DNA from strain F2C10 (bacteriophage sensitive) to strain F2E1 (bacteriophage insensitive) had created strain F2E3. Transfer of this DNA was associated with acquisition of sensitivity to 6 of 25 lytic bacteriophage isolated from the same flock. All strains tested were capable of colonizing broiler chickens but cocolonization revealed that the bacteriophage sensitive strains F2E3 and F2C10 had a competitive advantage over the bacteriophage insensitive strain F2E1. With the addition of lytic bacteriophage the situation was completely reversed, with F2E1 dominating. The inability to replicate bacteriophage is associated with a significant fitness cost that renders the insensitive strain competitive only in the presence of bacteriophage. We demonstrate that interstrain recombination in vivo can generate genome diversity in C. jejuni and that bacteriophage predation is a strong selective pressure that influences the relative success of emergent strains in broiler chickens.

Campylobacter jejuni is a leading cause of food-borne illness. Although a natural reservoir of the pathogen is domestic poultry, the degree of genomic diversity exhibited by the species limits the application of epidemiological methods to trace specific infection sources. Bacteriophage predation is a common burden placed upon C. jejuni populations in the avian gut, and we show that amongst C. jejuni that survive bacteriophage predation in broiler chickens are bacteriophage-resistant types that display clear evidence of genomic rearrangements. These rearrangements were identified as intra-genomic inversions between Mu-like prophage DNA sequences to invert genomic segments up to 590 kb in size, the equivalent of one-third of the genome. The resulting strains exhibit three clear phenotypes: resistance to infection by virulent bacteriophage, inefficient colonisation of the broiler chicken intestine, and the production of infectious bacteriophage CampMu. These genotypes were recovered from chickens in the presence of virulent bacteriophage but not in vitro. Reintroduction of these strains into chickens in the absence of bacteriophage results in further genomic rearrangements at the same locations, leading to reversion to bacteriophage sensitivity and colonisation proficiency. These findings indicate a previously unsuspected method by which C. jejuni can generate genomic diversity associated with selective phenotypes. Genomic instability of C. jejuni in the avian gut has been adopted as a mechanism to temporarily survive bacteriophage predation and subsequent competition for resources, and would suggest that C. jejuni exists in vivo as families of related meta-genomes generated to survive local environmental pressures.

Covalent attachment of polyethylene glycol, PEGylation, has been shown to prolong the half-life and enhance the pharmacodynamics of therapeutic proteins. Current methods for PEGylation, which rely on chemical conjugation through reactive groups on amino acids, often generate isoforms in which PEG is attached at sites that interfere with bioactivity. Here, we present a novel strategy for site-directed PEGylation using glycosyltransferases to attach PEG to O-glycans. The process involves enzymatic GalNAc glycosylation at specific serine and threonine residues in proteins expressed without glycosylation in Escherichia coli, followed by enzymatic transfer of sialic acid conjugated with PEG to the introduced GalNAc residues. The strategy was applied to three therapeutic polypeptides, granulocyte colony stimulating factor (G-CSF), interferon-alpha2b (IFN-alpha2b), and granulocyte/macrophage colony stimulating factor (GM-CSF), which are currently in clinical use.

This study investigated the morphology of the mesiobuccal root of the maxillary first molar. One hundred ten extracted maxillary first molars were accessed. The first and second mesiobuccal canals were exposed, soaked in black India ink, decalcified, dehydrated, and made transparent. The configurations of the root canals in the mesiobuccal root were studied under magnification (2.6x) and classified according to Weine's four types of root canal configurations. Under the conditions of this study, a high percentage of second mesiobuccal canals were revealed.

The design, synthesis and biological evaluation of novel seco-iso-cyclopropylfurano[2,3-e]indoline (seco-iso-CFI) and the seco-cyclopropyltetrahydrofurano[2,3-f]quinoline (seco-CFQ) analogues of the duocarmycins are described. These novel analogues (4-7) were designed on the premise that the lone pair of electrons on the furano-oxygen atom could enter into conjugation with the isocyclopropylfurano[e]indolone (iso-CFI) alkylating moiety, formed from the loss of HCl in compounds 4-7. The seco-iso-CFI DNA alkylating pharmacophore was synthesized through a well precedented approach of 5-exo-trig aryl radical cyclization with a vinyl chloride. In our studies, in addition to the formation of the seco-iso-CFI product, an equal amount of an unexpected seco-CFQ product was also generated during the radical cyclization reaction. Like CC-1065 and adozelesin, using Taq DNA polymerase stop and thermal cleavage assays, the seco-iso-CFI compounds (4 and 6) and the seco-CFQ compounds (5 and 7) were shown to preferentially alkylate the adenine-N3 position within the minor groove of long stretches of A residues. A MM2 energy optimized molecular model of a 1:1 complex of compound 6 with DNA reveals that the iso-CFI compound fits snugly within the minor groove. Using a MTT based experiment, the cytotoxicity of compounds 4-7 were determined against the growth of murine leukemia (L1210), mastocytoma (P815) and melanoma (B16) cell lines. The concentrations of compounds required to inhibit the growth of these tumor cells by 50% is in the range of 10(-8)M. These compounds were also tested against a panel of human cancer cells by the National Cancer Institute, demonstrating that the compounds exhibited a high level of activity against selected solid tumors. At a concentration of 0.0084 microM (based on the IC(50) of compound 17 (seco-CBI-TMI) against the growth L1210 cells), while compounds 4 and 17 were toxic against murine bone marrow cells as judged by a colony forming study of freshly isolated murine progenitor hematopoeitic cells, compound 5, a seco-CFQ compound, was significantly less toxic. Flow cytometric analysis of P815 cells that had been incubated for 24h with compounds 4 and 5 at their cytotoxic IC(50) concentrations indicated the induction of apoptosis in a large percentage of cells, thereby suggesting that this might be the mechanism by which the iso-CFI compounds kill cells.

The ability of chemical or pharmaceutical agents to induce allergic contact dermatitis (ACD) is of major health and regulatory concern. As such, tests to identify their sensitizing capacity, such as the guinea pig maximization test and the more recently developed local lymph node assay, are broadly used. Ideally, for risk assessment it is useful to translate results from animal data into establishing safe or no-effect levels for occupational or environmental agents. This, of course, would require consideration of the quantitative relationships between sensitizing and challenge doses as well as other exposure conditions. In the present studies, we modeled two sensitizers, 2,4-dinitrochlorobenzene and squaric acid dibutyl ester, over a large range of concentrations using the LLNA and more traditional tests that measure both sensitization and elicitation responses. Both the sensitization and challenge phases provided similar dose-response curves, demonstrating a threshold followed by a shallow linear increase and eventual plateau at increasing doses. Extending earlier studies by P. S. Friedmann (1994, Immunotoxicology and Immunopharmacology, pp. 589-616, Raven Press, New York) in humans, we observed that the minimum dose required to elicit sensitization or challenge was not static, but rather reflected a "sliding-scale." That is, as the sensitization dose was increased, the concentration required to elicit a challenge response was decreased. Correspondingly, as the challenge dose was increased, the dose required for sensitization was lessened. Taken together, these findings indicate that there is a need to consider dose-response relationships for sensitization and challenge in establishing minimum exposure levels for chemicals that cause ACD.

Dandruff is a major problem, yet little is known about the underlying mechanism and subsequent biochemical changes occurring in the scalp skin that lead to its manifestation. The characteristic flaking and scaling of the scalp experienced by dandruff sufferers suggests, similar to the changes classically seen in xerosis, that the desquamation process is impaired. We initiated studies to quantify the biochemical nature of the stratum corneum in the scalp of healthy individuals and dandruff sufferers. Total amounts and relative ratios of stratum corneum lipids species were analysed in scalp stratum corneum samples collected during studies conducted in the UK and Thailand in order to examine ethnic differences. In both populations, dandruff was associated with a dramatic decrease in free lipid levels, with significant decreases in ceramides, fatty acids, and cholesterol. Detailed sub-analysis of the major ceramide species within the total ceramide fraction revealed a decrease in ceramide 1 and increased proportions of ceramide 6i and 6ii. In a separate study, we demonstrated that dandruff sufferers show both an elevated blood flow and an increased reported incidence of itch in response to histamine topically applied to the scalp compared with no-dandruff controls. Taken together these two studies indicate that the quality and resilience of the epidermal water barrier is impaired in the scalp of dandruff sufferers. We propose that the perturbed barrier leaves dandruff sufferers more prone to the adverse effects of microbial and fungal toxins, and environmental pollutants, thus perpetuating their impaired barrier.

Clinical and laboratory studies have provided evidence of oncostatic activity by the pineal neurohormone melatonin. However, these studies have not elucidated its mechanism of action. The following series of MCF-7 breast tumor cell studies conducted in the absence of exogenous steroid hormones provide evidence for a novel mechanism of oncostatic activity by this endogenous hormone. We observed a 40--60% loss of MCF-7 cells after 20-h treatment with 100 nM melatonin, which confirmed and extended previous reports of its oncostatic potency. Interestingly, there were no observed changes in tritiated thymidine uptake, suggesting a lack of effect on cell cycle/nascent DNA synthesis. Further evidence of a cytocidal effect came from morphologic observations of acute cell death and autophagocytosis accompanied by degenerative changes in mitochondria. Studies of mitochondrial function via standard polarography revealed a significant increase in oxygen consumption in melatonin-treated MCF-7 cells. Enzyme-substrate studies of electron transport chain (complex IV) activity in detergent permeabilized cells demonstrated a concomitant 53% increase (p < 0.01) in cytochrome c oxidase activity. Additional studies of succinate dehydrogenase activity (complex II) as determined by reduction of (3-4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide demonstrated a significant increase (p < 0.05) in melatonin-treated cells and further confirmed the accelerated ET activity. Finally, there was a 64% decrease (p < 0.05) in cellular ATP levels in melatonin-treated cells. The G-protein-coupled melatonin receptor antagonist luzindole abrogated the cytotoxic and mitochondrial effects. These studies suggest a receptor-modulated pathway of cytotoxicity in melatonin-treated MCF-7 tumor cells with apparent uncoupling of oxidative phosphorylation., (Copyright 2001 Academic Press.)

Chemosensory dysfunction is most often secondary to one of only a few causes: nasal/sinus disease, viral infection, toxic chemical exposure, head trauma, as well as medication-related and idiopathic conditions. Medication-related disorders are corrected by discontinuance of the causative medicine. Our experiences have also shown that only dysfunctions of smell caused by disorders of the nose and/or sinuses are amendable to therapy.

Chemosensory problems can have major consequences for those patients who develop them. Although more than 200 conditions and 40 medications have been linked to taste and smell disorders, for most patients the cause will fall into one of the following categories: nasal/sinus disease, idiopathic, postviral URI, and head trauma. Careful attention to clinical characteristics will aid immensely in the diagnosis. Parosmias, dysgeusias and the burning mouth syndrome are symptoms that deserve special consideration.