Chronic colonization of the lungs by opportunist bacteria such as Pseudomonas aeruginosa and members of the Bcc (Burkholderia cepacia complex) is the major cause of morbidity and mortality among CF (cystic fibrosis) patients. PA-IIL (lecB gene), a soluble lectin from Ps. aeruginosa, has been the subject of much interest because of its very strong affinity for fucose. Orthologues have been identified in the opportunist bacteria Ralstonia solanacearum, Chromobacterium violaceum and Burkholderia of Bcc. The genome of the J2315 strain of B. cenocepacia, responsible for epidemia in CF centres, contains three genes that code for proteins with PA-IIL domains. The shortest gene was cloned in Escherichia coli and pure recombinant protein, BclA (B. cenocepacia lectin A), was obtained. The presence of native BclA in B. cenocepacia extracts was checked using a proteomic approach. The specificity of recombinant BclA was characterized using surface plasmon resonance showing a preference for mannosides and supported with glycan array experiments demonstrating a strict specificity for oligomannose-type N-glycan structures. The interaction thermodynamics of BclA with methyl alpha-D-mannoside demonstrates a dissociation constant (K(d)) of 2.75 x 10(-6) M. The X-ray crystal structure of the complex with methyl alpha-D-mannoside was determined at 1.7 A (1 A=0.1 nm) resolution. The lectin forms homodimers with one binding site per monomer, acting co-operatively with the second dimer site. Each monomer contains two Ca2+ ions and one sugar ligand. Despite strong sequence similarity, the differences between BclA and PA-IIL in their specificity, binding site and oligomerization mode indicate that the proteins should have different roles in the bacteria.

1. Erythrocyte ghosts from human blood were produced by gentle water hemolysis. The ghost-containing hemolysate (about 20 mN) was added to media of different composition (KCl, NaCl, glucose, sucrose, etc.) and varying concentration ranging from 8 to 840 mN. The volume changes of the ghost cells were followed by a light absorption method. The potassium and sodium concentrations were also analyzed in some representative cases. 2. The ghosts shrank, or swelled, in two stages. An initial phase with a momentary expulsion, or uptake, of water leading to an osmotic equilibrium, was followed by a second phase in which a slow swelling or shrinking proceeded toward a final constant volume. 3. The ghosts were semipermeable in the sense that water always passed rapidly in either direction so as to maintain isotonicity with the external medium. The relation between ghost cell volumes (V) and the total concentration (C(e)) of the suspension medium can be expressed by a modified van't Hoff-Mariotte law: (C(e) + a)(V - b) = constant. Here a is a term correcting for an internal pressure and b is the non-solvent volume of the ghost cells. This means that the ghosts behave as perfect osmometers. 4. On the other hand appreciable concentration differences of the K and Na ions could be maintained across the intact ghost cell membranes for long periods. Whether this phenomenon is due simply to very low cation permeability or to active transport processes cannot be decided, although the first assumption appears more probable. 5. When the ghosts were treated with small concentrations of a lytic substance like Na oleate, the alkali ion transfer was greatly increased. This seems to be a simple exchange diffusion process with simultaneous, continued maintenance of osmotic equilibrium (= the second phase). A simplified theory is also given for the kinetics of the volume variations and ion exchange during the second phase (cf. the Appendix). 6. Miscellaneous observations on the effects of pH, and of some other substances are discussed. Some shape transformations of the ghost cells are also described.

Traditionally, interocular suppression is believed to disrupt high-level (i.e., semantic or conceptual) processing of the suppressed visual input. The development of a new experimental paradigm, breaking continuous flash suppression (b-CFS), has caused a resurgence of studies demonstrating high-level processing of visual information in the absence of visual awareness. In this method the time it takes for interocularly suppressed stimuli to breach the threshold of visibility, is regarded as a measure of access to awareness. The aim of the current review is twofold. First, we provide an overview of the literature using this b-CFS method, while making a distinction between two types of studies: those in which suppression durations are compared between different stimulus classes (such as upright faces versus inverted faces), and those in which suppression durations are compared for stimuli that either match or mismatch concurrently available information (such as a colored target that either matches or mismatches a color retained in working memory). Second, we aim at dissociating high-level processing from low-level (i.e., crude visual) processing of the suppressed stimuli. For this purpose, we include a thorough review of the control conditions that are used in these experiments. Additionally, we provide recommendations for proper control conditions that we deem crucial for disentangling high-level from low-level effects. Based on this review, we argue that crude visual processing suffices for explaining differences in breakthrough times reported using b-CFS. As such, we conclude that there is as yet no reason to assume that interocularly suppressed stimuli receive full semantic analysis.

The repeated administration of the hypolipidemic agent Su-13 437 (nafenopin) to neonatal rats roughly doubled the number of peroxisomes in the liver tissue and caused a sixfold volumetric expansion of the peroxisomal compartment. During the proliferative response, the size-distribution of the peroxisomes was reversibly altered, enlarged particles appearing in numbers varying according to the dose given. By means of a new method for quantitative autoradiography, it was shown that (a) the concentration of silver grains over peroxisomes was comparable to that found over the endoplasmic reticulum; (b) the peak incorporation of [3H]arginine into the peroxisomes was dealyed in comparison with that into the endoplasmic reticulum; (c) the label, once incorporated into the expanding peroxisomal compartment, displayed the same shift to large particles as did the whole population. These results are compatible with the biosynthetic pathway for peroxisomal catalase proposed earlier (cf. reference 12), and with the notion that the drug-induced size-shift might have resulted from progressive growth of a particular class of peroxisomes formed in the presence of the agent. Evidence is presented to show that during the recovery period the larger peroxisomes are removed preferentially.

Chronic fatigue syndrome (CFS) is a medically unexplained disorder, characterized by profound fatigue, infectious, rheumatological and neuropsychiatric symptoms. There is, however, some evidence that CFS is accompanied by signs of increased oxidative stress and inflammation in the peripheral blood. This paper examines the role of the inducible enzymes cyclo-oxygenase (COX-2) and inducible NO synthase (iNOS) in the pathophysiology of CFS. Toward this end we examined the production of COX-2 and iNOS by peripheral blood lymphocytes (PBMC) in 18 CFS patients and 18 normal volunteers and examined the relationships between those inflammatory markers and the severity of illness as measured by means of the FibroFatigue scale and the production of the transcription factor nuclear factor kappa beta (NFkappabeta). We found that the production of COX-2 and iNOS was significantly higher in CFS patients than in normal controls. There were significant and positive intercorrelations between COX-2, iNOS and NFkappabeta and between COX-2 and iNOS, on the one hand, and the severity of illness, on the other. The production of COX-2 and iNOS by PBMCs was significantly related to aches and pain, muscular tension, fatigue, concentration difficulties, failing memory, sadness and a subjective experience of infection. The results suggest that a) an intracellular inflammatory response in the white blood cells plays an important role in the pathophysiology of CFS; b) the inflammatory response in CFS is driven by the transcription factor NFkappabeta; c) symptoms, such as fatigue, pain, cognitive defects and the subjective feeling of infection, indicates the presence of a genuine inflammatory response in CFS patients; and d) CFS patients may be treated with substances that inhibit the production of COX-2 and iNOS.

A family of cancer cell cytotoxic cyclodepsipeptides, veraguamides A-C (1-3) and H-L (4-8), were isolated from a collection of cf. Oscillatoria margaritifera obtained from the Coiba National Park, Panama, as part of the Panama International Cooperative Biodiversity Group program. The planar structure of veraguamide A (1) was deduced by 2D NMR spectroscopy and mass spectrometry, whereas the structures of 2-8 were mainly determined by a combination of 1H NMR and MS2/MS3 techniques. These new compounds are analogous to the mollusk-derived kulomo'opunalide natural products, with two of the veraguamides (C and H) containing the same terminal alkyne moiety. However, four veraguamides, A, B, K, and L, also feature an alkynyl bromide, a functionality that has been previously observed in only one other marine natural product, jamaicamide A. Veraguamide A showed potent cytotoxicity to the H-460 human lung cancer cell line (LD50=141 nM).

The mechanisms remain uncertain by which mutations in CFTR cause lung disease in cystic fibrosis (CF). Teichgräber et al. recently reported increased ceramide in CF lungs, which was proposed to result from defective lysosomal acidification in airway epithelial cells and consequent impairment of pH-dependent ceramide-metabolizing enzymes (Teichgräber, V., Ulrich, M., Endlich, N., Reithmüller, J., Wilker, B., Conceição Ce Olivereira-Munding, C., van Heeckeren, A. M., Barr, M. L., von Kürthy, G., Schmid, K. W., Weller, M., Tümmler, B., Lang, F., Grassme, H., Döring, G., and Gulbins, E. (2008) Nat. Med. 14, 382-391). Here, we measured lysosomal pH in several CFTR-expressing and -deficient cell lines, freshly isolated airway epithelial cells from non-CF and CF mice and humans, and well-differentiated primary cultures of human non-CF and CF airway epithelial cells. Lysosomal pH was measured by ratio imaging using a fluorescent pH indicator consisting of 40-kDa dextran conjugated to Oregon Green 488 and tetramethylrhodamine. In all cell types, lysosomal pH was approximately 4.5, unaffected by the thiazolidinone CFTR inhibitor CFTR(inh)-172, and increased to approximately 6.5 following bafilomycin inhibition of the vacuolar proton pump. Lysosomal pH did not differ significantly in airway epithelial cells from non-CF versus CF humans or mice. Our results provide direct evidence against the conclusions of Teichgräber et al. that lysosomal acidification is CFTR-dependent, impaired in CF, or responsible for ceramide accumulation. As such, alternative mechanisms are needed to explain increased ceramide in CF airways. Non-CFTR mechanisms, such as ClC-type chloride channels, are likely involved in maintaining electroneutrality during organellar acidification.

Evidence is presented indicating that the induction of long-term depression (LTD) in Purkinje cells (PCs) requires a rapidly turned over protein(s) during a critical time period within 15 min after the onset of LTD-inducing stimulation and that synthesis of this protein is maintained by mRNAs supplied via transcription. LTD was induced in granule cell axon (GA)-to-PC synapses by stimulation of these synapses at 1 Hz for 5 min in conjunction with the climbing fibers (CFs) forming synapses on the same PCs and represented by a persistent reduction in the GA-induced excitatory postsynaptic potentials (EPSPs). Not only a prolonged but also a brief (5 min) pulse application of translational inhibitors (anisomycin, puromycin, or cycloheximide) effectively blocked the LTD induction. Pulses applied during the period from 30 min before to 10 min after the onset of conjunctive stimulation blocked the LTD induction, but those applied 15 min after were ineffective. The three translational inhibitors blocked the LTD induction similarly, suggesting that the effect is due to their common action of inhibiting protein synthesis. Infusion of a mRNA cap analogue (7-methyl GTP) into PCs also blocked LTD induction, ensuring that the postsynaptic protein synthesis within PCs is required for LTD induction. Transcriptional inhibitors, actinomycin D and 5,6-dichloro-l-beta-D-ribofuranosyl-benzimidazole, also blocked the LTD induction, but this effect was apparent when 5-min pulses of the transcriptional inhibitors preceded the conjunctive stimulation by 30 min or more. This time lag of 30 min is presumed to be required for depletion of the protein(s) required for LTD induction. The presently observed effects of translational and transcriptional inhibitors on the LTD induction are of temporal characteristics corresponding to their depressant effects on the type-1 metabotropic glutamate-receptor (mGluR1)-mediated slow EPSPs in PCs as we have reported recently. An antagonist of mGluR1s [(RS)-1-aminoindan-1,5-dicarboxylic acid], however, did not block LTD induction when it was applied during the 10-min period following conjunctive stimulation, where translational inhibitors effectively blocked LTD induction. This discrepancy in time course suggests that the rapidly turned over protein(s) required for LTD induction is involved in a process occurring downstream of the activation of mGluR1s.

The postsynaptic muscle-specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody-positive myasthenia gravis patients (MuSK IgG; 45 mg day(1)i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51-73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR beta-subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK-rapsyn system of AChR clustering.

A biochemical and structural analysis is presented of fractions that were obtained by a quick and mild solubilization of thylakoid membranes from spinach with the non-ionic detergent n-dodecyl-alpha,D-maltoside, followed by a partial purification using gel filtration chromatography. The largest fractions consisted of paired, appressed membrane fragments with an average diameter of about 360 nm and contain Photosystem II (PS II) and its associated light-harvesting antenna (LHC II), but virtually no Photosystem I, ATP synthase and cytochrome b (6) f complex. Some of the membranes show a semi-regular ordering of PS II in rows at an average distance of about 26.3 nm, and from a partially disrupted grana membrane fragment we show that the supercomplexes of PS II and LHC II represent the basic structural unit of PS II in the grana membranes. The numbers of free LHC II and PS II core complexes were very high and very low, respectively. The other macromolecular complexes of the thylakoid membrane occurred almost exclusively in dispersed forms. Photosystem I was observed in monomeric or multimeric PS I-200 complexes and there are no indications for free LHC I complexes. An extensive analysis by electron microscopy and image analysis of the CF(0)F(1) ATP synthase complex suggests locations of the delta (on top of the F(1) headpiece) and in subunits (in the central stalk) and reveals that in a substantial part of the complexes the F(1) headpiece is bended considerably from the central stalk. This kinking is very likely not an artefact of the isolation procedure and may represent the complex in its inactive, oxidized form.

The first discernible intermediate when fully reduced cytochrome c oxidase reacts with O2 is a dioxygen adduct (compound A) of the binuclear heme iron-copper center. The subsequent decay of compound A is associated with transfer of an electron from the low-spin heme a to this center. This reaction eventually produces the ferryl state (F) of this center, but whether an intermediate state may be observed between A and F has been the subject of some controversy. Here we show, using both optical and EPR spectroscopy, that such an intermediate (P(R)) indeed exists and that it exhibits spectroscopic properties quite distinct from F. The optical spectrum of P(R) is similar or identical to the spectrum of the P(M) intermediate that is formed after compound A when two-electron-reduced enzyme reacts with O2. An unusual EPR spectrum with features of a CuB(II) ion that interacts magnetically with a nearby paramagnet [cf. Hansson, O., Karlsson, B., Aasa, R., Vänngård, T., and Malmström, B.G (1982) EMBO J. 1, 1295-1297; Blair, D. F., Witt, S. N., and Chan, S. I. (1985) J. Am. Chem. Soc. 107, 7389-7399] can be uniquely assigned to the P(R) intermediate, not being found in either the P(M) or F intermediate. The binuclear center in the P(R) state may be assigned as having an Fe(a3)(IV)=O CuB(II) structure, as in both the P(M) and F states. The spectroscopic differences between these three intermediates are evaluated. The P(R) state has a key role as an initiator of proton translocation by the enzyme, and the thermodynamic and electrostatic bases for this are discussed.

The literature is reviewed and data are presented that relate to a model we have developed to account for the perpetuation of the perplexing disorder currently termed chronic fatigue syndrome (CFS). In patients with CFS there is chronic lymphocyte overactivation with cytokine abnormalities that include perturbations in plasma levels of proinflammatory cytokines and decrease in the ratio of Type 1 to Type 2 cytokines produced by lymphocytes in vitro following mitogen stimulation. The initiation of the syndrome is frequently sudden and often follows an acute viral illness. Our model for the subsequent chronicity of this disorder holds that the interaction of psychological factors (distress associated with either CFS-related symptoms or other stressful life events) and the immunologic dysfunction contribute to (a) CFS-related physical symptoms (e.g., perception of fatigue and cognitive difficulties, fever, muscle and joint pain) and increases in illness burden and (b) impaired immune surveillance associated with cytotoxic lymphocytes with resulting activation of latent herpes viruses.

Pseudomonas aeruginosa is a non-capsulate and non-sporing gram-negative bacillus that most commonly affects the lower respiratory system in humans. Burkholderia (previously Pseudomonas) cepacia has emerged as an important respiratory pathogen in patients with cystic fibrosis (CF). The ability of P. aeruginosa to persist and multiply in moist environments and equipment, such as humidifiers in hospital wards, bathrooms, sinks and kitchens, maybe of importance in cross-infection. P. aeruginosa infections of the lower respiratory tract can range in severity from colonisation (without an immunological response) to a severe necrotising bronchopneumonia. Infection is seen in patients with CF and other chronic lung diseases such as non-CF bronchiectasis. In patients with CF, once P. aeruginosa is established in the airways it is almost impossible to eradicate, but prior to this, aggressive treatment can delay the development of chronic infection. 30 to 40% of the present paediatric population with CF will have chronic pseudomonal infection. B. cepacia has a particular predisposition to infect patients with CF and may be distinguished from P. aeruginosa by accelerated lung disease in about one- third of patients. Overwhelming septicaemia and necrotising pneumonia are well described (cepacia syndrome); events that are rare with P. aeruginosa. With the propensity for social cross-infection, segregation policies have been accepted as means of controlling outbreaks. A number of antipseudomonal agents are available. The most commonly used are the extended-spectrum penicillins, aminoglycosides, cephalosporins, fluoroquinolones, polymixins and the monobactams. An aminoglycoside with a beta-lactam penicillin is usually considered to be the first line treatment. No trial has shown any significant clinical advantage of any particular combination regimen over another. The emergence of resistance continues to be a concern. Pipericillin, piperacillin/tazobactam and meropenem have good but equivalent antibacterial activity against P. aeruginosa. However, B. cepacia is characterised by in vitro resistance to colistin (colomycin), aminoglycosides and ciprofloxacin but better susceptibility to ceftazidime. Nebulised delivery of antipseudomonal antibiotics is thought to prevent recurrent exacerbations, reduce antibiotic usage and maintain lung function, particularly in patients with CF. Colistin, tobramycin and gentamicin are currently the most commonly prescribed nebulised antibiotics. Much effort is directed at treating chronic P. aeruginosa infection but as chronic infection is seldom if ever eradicated when first established, prevention is preferable. Early intensive treatment for P. aeruginosa infection is advocated in order to maintain pulmonary function and postpone the onset of chronic P. aeruginosa infection.

Multi-resistant strains from three UK centres, previously identified as Burkholderia (formerly Pseudomonas) cepacia, and associated with morbidity, mortality and transmission among patients with cystic fibrosis have been further characterised. Biochemical tests and fatty acid analyses indicate these strains to possess some characteristics atypical of B. cepacia but bearing close resemblance to Burkholderia gladioli, an organism previously regarded solely as a plant pathogen and a hindrance to the identification of B. cepacia. In contrast to the majority of reference strains, all multi-resistant clinical isolates possessed rough lipopolysaccharide which may be a major factor responsible for their increased antibiotic resistance and virulence. In view of the potential clinical and social problems in CF patients posed by these multi-resistant strains, it would seem prudent to consider the isolation of either B. cepacia or B. gladioli as of equal significance.

Lung disease is the major cause of morbidity and mortality of cystic fibrosis (CF), an autosomal recessive disease caused by mutations in CF transmembrane-conductance regulator (CFTR) gene. In CF, elevated levels of interleukin-8 (IL-8) signaling mediated by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) result in chronic infection, neutrophilic inflammation, and progressive airway destruction. The most frequent mutation in the CFTR gene is the deletion of phenylalanine 508 (DeltaF508), which results in its endoplasmic reticulum associated degradation (ERAD) by the ubiquitin-proteasome system. The inability of DeltaF508-CFTR to reach cell surface leads to inherently high levels of NF-kappaB. Severity of CF lung disease depends on the levels of functional CFTR on cell surface that control its chloride transport and NF-kappaB mediated innate immune response functions. NF-kappaB mediated chronic inflammation is a prominent feature of CF lung disease and the mechanism(s) by which CFTR regulates these inflammatory signaling pathways is becoming apparent. Recent data suggest that CFTR localization to lipid-rafts is critical for regulating NF-kappaB mediated innate immune response and chronic CF lung disease. We anticipate that targeting the pathways, which facilitates CFTR's rescue to the cell surface and lipid-rafts, will not only restore CFTR channel function but also control NF-kappaB mediated chronic inflammation, although the level of correction may be a critical factor for therapeutic efficacy. We discuss here the mechanisms of NF-kappaB induction in CF, pathogenesis of CF lung disease, and novel therapeutic strategies that may help in reversing the chronic CF lung disease.

Cystic fibrosis (CF) patients demonstrate a spectrum of pancreatic beta-cell abnormalities. Those with no exocrine insufficiency (NEXO) have normal insulin secretion. Exocrine-insufficient CF patients with overt diabetes (EXO-IT) have impaired insulin secretion and fasting hyperglycemia. Exocrine-insufficient patients without diabetes (EXO) have impaired insulin secretion but maintain normoglycemia. We postulated that EXO individuals compensate for insulin deficiency by increasing insulin sensitivity and investigated glucose utilization in CF. To examine hepatic and peripheral insulin sensitivity, euglycemic-hyperinsulinemic clamp studies were performed by using the hot GINF isotope dilution technique. Insulin was sequentially infused at 0.25, 1.0, and 10.0 mU.kg-1.min-1. Glucose-mediated glucose uptake (GMGU) was assessed on another day with hyperglycemic clamp studies, during which insulin and somatostatin were infused to hold insulin-mediated glucose uptake constant between the two clamp studies. Skeletal muscle GLUT4 levels were assessed in EXO and control patients with Western blotting. Three patterns of peripheral and hepatic insulin sensitivity were seen that were related to the degree of pancreatic beta-cell dysfunction. NEXO individuals had normal peripheral and hepatic insulin sensitivity. EXO individuals had enhanced peripheral insulin sensitivity that was not associated with a change in skeletal muscle glucose transporter abundance compared with control patients; paradoxically, EXO subjects demonstrated hepatic insulin resistance. EXO-IT had peripheral and hepatic insulin resistance. GMGU was diminished in both EXO and EXO-IT subjects. The unique combination of increased hepatic glucose production and increased peripheral glucose utilization seen in EXO may be a metabolic adaptation to increased peripheral energy needs.(ABSTRACT TRUNCATED AT 250 WORDS)

CFTR, the cystic fibrosis (CF) gene, encodes for the CFTR protein that plays an essential role in anion regulation and tissue homeostasis of various epithelia. In the gastrointestinal (GI) tract CFTR promotes chloride and bicarbonate secretion, playing an essential role in ion and acid-base homeostasis. Cftr has been identified as a candidate driver gene for colorectal cancer (CRC) in several Sleeping Beauty DNA transposon-based forward genetic screens in mice. Further, recent epidemiological and clinical studies indicate that CF patients are at high risk for developing tumors in the colon. To investigate the effects of CFTR dysregulation on GI cancer, we generated Apc(Min) mice that carried an intestinal-specific knockout of Cftr. Our results indicate that Cftr is a tumor suppressor gene in the intestinal tract as Cftr mutant mice developed significantly more tumors in the colon and the entire small intestine. In Apc(+/+) mice aged to ~1 year, Cftr deficiency alone caused the development of intestinal tumors in >60% of mice. Colon organoid formation was significantly increased in organoids created from Cftr mutant mice compared with wild-type controls, suggesting a potential role of Cftr in regulating the intestinal stem cell compartment. Microarray data from the Cftr-deficient colon and the small intestine identified dysregulated genes that belong to groups of immune response, ion channel, intestinal stem cell and other growth signaling regulators. These associated clusters of genes were confirmed by pathway analysis using Ingenuity Pathway Analysis and gene set enrichment analysis (GSEA). We also conducted RNA Seq analysis of tumors from Apc(+/+) Cftr knockout mice and identified sets of genes dysregulated in tumors including altered Wnt β-catenin target genes. Finally we analyzed expression of CFTR in early stage human CRC patients stratified by risk of recurrence and found that loss of expression of CFTR was significantly associated with poor disease-free survival.

The genus Burkholderia consists of diverse species which includes both "friends" and "foes." Some of the "friendly" Burkholderia spp. are extensively used in the biotechnological and agricultural industry for bioremediation and biocontrol. However, several members of the genus including B. pseudomallei, B. mallei, and B. cepacia, are known to cause fatal disease in both humans and animals. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively, while B. cepacia infection is lethal to cystic fibrosis (CF) patients. Due to the high rate of infectivity and intrinsic resistance to many commonly used antibiotics, together with high mortality rate, B. mallei and B. pseudomallei are considered to be potential biological warfare agents. Treatments of the infections caused by these bacteria are often unsuccessful with frequent relapse of the infection. Thus, we are at a crucial stage of the need for Burkholderia vaccines. Although the search for a prophylactic therapy candidate continues, to date development of vaccines has not advanced beyond research to human clinical trials. In this article, we review the current research on development of safe vaccines with high efficacy against B. pseudomallei, B. mallei, and B. cepacia. It can be concluded that further research will enable elucidation of the potential benefits and risks of Burkholderia vaccines.

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex, a group of genetically similar species that inhabit a number of environmental niches, including the lungs of patients with cystic fibrosis (CF). To colonize the lung, this bacterium requires a source of iron to satisfy its nutritional requirements for this important metal. Because of the high potential for damage in lung tissue resulting from oxygen-iron interactions, this metal is sequestered by a number of mechanisms that render it potentially unavailable to invading micro-organisms. Such mechanisms include the intracellular and extracellular presence of the iron-binding protein ferritin. Ferritin has a highly stable macromolecular structure and may contain up to 4500 iron atoms per molecule. To date, there has been no known report of a pathogenic bacterial species that directly utilizes iron sequestered by this macromolecule. To examine the ability of ferritin to support growth of B. cenocepacia J2315, iron-deficient media were supplemented with different concentrations of ferritin and the growth kinetics characterized over a 40 h period. The results indicated that B. cenocepacia J2315 utilizes iron bound by ferritin. Further studies examining the mechanisms of iron uptake from ferritin indicated that iron utilization results from a proteolytic degradation of this otherwise stable macromolecular structure. Since it is known that the ferritin concentration is significantly higher in the CF lung than in healthy lungs, this novel iron-acquisition mechanism may contribute to infection by B. cenocepacia in people with CF.

Probiotics have been shown to have a preventative role in colorectal carcinogenesis but research concerning their prophylactic potential in the later stages of colorectal cancer, specifically metastasis is limited. This study explored the potential of cell-free supernatants (CFS) from 2 probiotic Lactobacillus sp., Lactobacillus casei and Lactobacillus rhamnosus GG, to inhibit colon cancer cell invasion by influencing matrix metalloproteinase-9 (MMP-9) activity and levels of the tight junction protein zona occludens-1 (ZO-1) in cultured metastatic human colorectal carcinoma cells. HCT-116 cells were treated with CFS from L. casei, L. rhamnosus, or Bacteroides thetaiotaomicron (a gut commensal); or with uninoculated bacterial growth media. Treatment with CFS from both Lactobacillus sp. decreased colorectal cell invasion but treatment with CFS from B. thetaiotaomicron did not. CFS from both Lactobacillus sp. decreased MMP-9 and increased ZO-1 protein levels. L. rhamnosus CFS also lowered MMP-9 activity. To begin elucidating the secreted bacterial factor conveying these responses, Lactobacillus sp. CFS were fractionated into defined molecular weight ranges and cell invasion assessed. Fractionation revealed that the inhibitory activity was contained primarily in the >100 kDa and 50-100 kDa fractions, suggesting the inhibitory compound may be a macromolecule such as a protein, nucleic acid, or a polysaccharide.

BACKGROUND

Burkholderia cepacia complex bacteria are opportunistic pathogens, which can cause severe respiratory tract infections in patients with cystic fibrosis (CF). As treatment of infected CF patients is problematic, multiple preventive measures are taken to reduce the infection risk. Besides a stringent segregation policy to prevent patient-to-patient transmission, clinicians also advise patients to clean and disinfect their respiratory equipment on a regular basis. However, problems regarding the efficacy of several disinfection procedures for the removal and/or killing of B. cepacia complex bacteria have been reported. In order to unravel the molecular mechanisms involved in the resistance of biofilm-grown Burkholderia cenocepacia cells against high concentrations of reactive oxygen species (ROS), the present study focussed on the transcriptional response in sessile B. cenocepacia J2315 cells following exposure to high levels of H2O2 or NaOCl.

RESULTS

The exposure to H2O2 and NaOCl resulted in an upregulation of the transcription of 315 (4.4%) and 386 (5.4%) genes, respectively. Transcription of 185 (2.6%) and 331 (4.6%) genes was decreased in response to the respective treatments. Many of the upregulated genes in the NaOCl- and H2O2-treated biofilms are involved in oxidative stress as well as general stress response, emphasizing the importance of the efficient neutralization and scavenging of ROS. In addition, multiple upregulated genes encode proteins that are necessary to repair ROS-induced cellular damage. Unexpectedly, a prolonged treatment with H2O2 also resulted in an increased transcription of multiple phage-related genes. A closer inspection of hybridisation signals obtained with probes targeting intergenic regions led to the identification of a putative 6S RNA.

CONCLUSIONS

Our results reveal that the transcription of a large fraction of B. cenocepacia J2315 genes is altered upon exposure of sessile cells to ROS. These observations have highlighted that B. cenocepacia may alter several pathways in response to exposure to ROS and they have led to the identification of many genes not previously implicated in the stress response of this pathogen.

Chronic lymphocytic leukemia (CLL) cells express the receptor for Epstein-<em>B</em>arr virus (E<em>B</em>V) and can be infected in vitro. Infected cells do not express the growth-promoting set of E<em>B</em>V-encoded genes and therefore they do not yield LCLs, in most experiments. With exceptional clones, lines were obtained however. We describe a new line, HG3, established by in vitro E<em>B</em>V-infection from an IGHV1-2 unmutated CLL patient clone. All cells expressed E<em>B</em>NA-2 and LMP-1, the E<em>B</em>V-encoded genes pivotal for transformation. The karyotype, FISH cytogenetics and SNP-array profile of the line and the patient's ex vivo clone showed biallelic 13q14 deletions with genomic loss of DLEU7, miR15a/miR16-1, the two micro-RNAs that are deleted in 50% of CLL cases. Further features of CLL cells were: expression of CD5/CD20/CD27/CD43 and release of IgM natural antibodies reacting with oxLDL-like epitopes on apoptotic cells (<em>cf</em>. stereotyped subset-1). Comparison with two LCLs established from normal <em>B</em> cells showed 32 genes expressed at higher levels >> 2-fold). Among these were LHX2 and LILRA. These genes may play a role in the development of the disease. LHX2 expression was shown in self-renewing multipotent hematopoietic stem cells, and LILRA4 codes for a receptor for bone marrow stromal cell antigen-2 that contributes to <em>B</em> cell development. Twenty-four genes were expressed at lower levels, among these PARD3 that is essential for asymmetric cell division. These genes may contribute to establish precursors of CLL clones by regulation of cellular phenotype in the hematopoietic compartment. Expression of CD5/CD20/CD27/CD43 and spontaneous production of natural antibodies may identify the CLL cell as a self-renewing <em>B</em>1 lymphocyte.

The experiments described in this report reconcile some of the apparent differences in isoform-specific kinetics of the Na,K-ATPase reported in earlier studies. Thus, tissue-specific differences in Na+ and K+ activation kinetics of Na,K-ATPase activity of the same species (rat) were observed when the same isoform was assayed in different tissues or cells. In the case of alpha1, alpha1-transfected HeLa cell, rat kidney, and axolemma membranes were compared. For alpha3, the ouabain-insensitive alpha3*-transfected HeLa cell (<em>cf</em>. Jewell, E. A., and Lingrel, J. <em>B</em>. (1991) J. <em>B</em>iol. Chem. 266, 16925-16930), pineal gland, and axolemma (mainly alpha3) membranes were compared. The order of apparent affinities for Na+ of alpha1 pumps was axolemma approximately rat alpha1-transfected HeLa>> kidney, and for K+, kidney approximately alpha1-transfected HeLa>> axolemma. For alpha3, the order of apparent affinities for Na+ was pineal gland approximately axolemma>> alpha3*-transfected HeLa, and for K+, alpha3*-transfected HeLa>> axolemma approximately pineal gland. In addition, the differences in apparent affinities for Na+ of either kidney alpha1 or HeLa alpha3* as compared to the same isoform in other tissues were even greater when the K+ concentration was increased. A kinetic analysis of the apparent affinities for Na+ as a function of K+ concentration indicates that isoform-specific as well as tissue-specific differences are related to the apparent affinities for both Na+ and K+, the latter acting as a competitive inhibitor at cytoplasmic Na+ activation sites. Although the nature of the tissue-specific modulation of K+/Na+ antagonism remains unknown, an analysis of the nature of the <em>beta</em> isoform associated with alpha1 or alpha3 using isoform-specific immunoprecipitation indicates that the presence of distinct <em>beta</em> subunits does not account for differences of alpha1 of kidney, axolemma, and HeLa, and of alpha3 of axolemma and HeLa; in both instances <em>beta</em>1 is the predominant <em>beta</em> isoform present or associated with either alpha1 or alpha3. However, a kinetic difference in K+/Na+ antagonism due to distinct <em>betas</em> may apply to alpha3 of axolemma (alpha3<em>beta</em>1) and pineal gland ( alpha3<em>beta</em>2).

Impaired glucose tolerance and overt diabetes mellitus are becoming increasingly common complications of cystic fibrosis (CF), most probably merely as a result of increased life expectancy. In order to understand the pathophysiology of cystic fibrosis-related diabetes (CFRD), knowledge on the possible expression and cell distribution of the cystic fibrosis transmembrane conductance regulator (CFTR) protein within the endocrine pancreas is required. In this report, we establish the first evidence for expression of CFTR protein in rat pancreatic islets by using independent techniques. First reverse transcriptase-polymerase chain reaction (RT-PCR) amplification showed that CFTR mRNA is present in isolated islets of Langerhans. Furthermore, the analysis of flow cytometry-separated islet cells indicated that the level of CFTR transcripts is significantly higher in the non-beta than in beta-cell populations. The expression of CFTR protein in rat islet cells was also demonstrated by Western blotting and the level of expression was also found significantly higher in the non-beta than in beta-cell populations. Last, in situ immunocytochemistry studies with two monoclonal antibodies recognizing different CFTR epitopes indicated that CFTR expression occurs mainly in glucagon-secreting alpha-cells.