In the first section of this paper, various research designs in human behavior genetics are compared. In this context, the commonly used concept of biometric genetics is critically evaluated from the point of view of science theory. It is contrasted with the Mendelian gene concept, which, in principle, leads to a much deeper theoretical understanding by offering clues for basic mechanisms. To explore this advantage fully, a research strategy is needed that first looks for genetic variability in a physiological parameter of possible importance for human behavior and then tries to explore the influence of this parameter on the function of the human brain and on behavior. If possible, this genetic parameter should be selected in a way that inferences as to the mechanism of its influence on behavior become feasible. Such genetic variability is provided by the hereditary variants of the normal EEG discovered by earlier work (cf. Vogel, 1970). In the following section, a research program on 298 adult healthy males, most of them soldiers, with various inherited EEG variants is described. Apart from controls with inconspicuous EEGs, this material comprises probands with the following EEG variants: low-voltage (N); low-voltage borderline (NG); monotonous alpha-waves (R); occipital fast alpha-variants (BO); fronto-precentral beta-groups (BG), and diffuse beta-waves (BD). In addition to an EEG examination, the probands were examined with various test methods measuring intelligence (IST; LPS; Raven); working speed and concentration (d-2; KLT); personal attitudes (MMPI; 16PF; RKS); and sensory and motor abilities (flicker fusion; tachistoscopy; reaction time to optic, acoustic and combined stimuli; two-hand dexterity; pursuit rotor; tapping). In a supplementary twin study on 52 male adult twin pairs (26 MZ, 26 DZ), heritabilities were determined for the test scores included in the main study. For most test scores, heritabilities are relatively low; the data are compared with those from the literature. We conclude that the test methods utilized in the main study (on EEG variants) are expected to demonstrate at the most a small to moderate correlation of the EEGs with psychological phenotypes as defined by test examinations, even if a major part of the genetic variability underlying these phenotypes would be due to differences in brain physiology that could be revealed by EEG variation.

Cystic fibrosis (CF) is a common autosomal recesses disease in which loss of CFTR-Cl- channel function of defective cAMP-stimulated Cl- transport transfer across airway epithelia. Recombinant adenoviruses have shown progress as vectors with which to transfer CFTR cDNA to CF airway epithelia. Here we investigated variables involved in adenovirus-mediated transfer of CFTR by measuring cAMP- stimulated Cl- transport in CF airway epithelia grown as monolayers on permeable filter supports. When we compared the effects of different promoters, we found that persistent correction of Cl- transport was obtained when the vector contained the E1a promoter, or to a lesser extent the PGK promoter. Vector containing the CMV promoter produced a greater initial cAMP-stimulated Cl- current, but the duration of correction was shorter and the infection procedure itself increased CFTR expression, suggesting that high input doses of virus stimulate expression. We compared the level of expression, measured with a beta-galactosidase reporter of CFTR mRNA, with CFTR-mediated Cl- transport. Even low levels of expression generated significant Cl- current and marked increases in expression produced only modest increments in Cl- current. Correction of the CF Cl- transport defect was also improved when the concentration of adenovirus vector was high and when the duration of contact with the epithelium was prolonged. These findings may help optimize the ability of adenovirus vectors encoding CFTR to correct the CF Cl- transport defect.

In previous studies with cystic fibrosis (CF) IBB., Ji, X.-D., Jozwik, C. J., and Jacobowitz, D. M. (2005) High abundance protein profiling of cystic fibrosis lung epithelial cells. Proteomics 5, 2210-2226). In the present work we compared the IBBCFTR. We report that gene transfer resulted in significant changes in silver stain intensity of only 20 of the 194 proteins. However, simultaneous measurement of de novo biosynthetic rates with [(35)S]methionine of all 194 proteins in both cell types resulted in the identification of an additional 31 CF-specific proteins. Of the 51 proteins identified by this hybrid approach, only six proteins changed similarly in both the mass and kinetics categories. This kinetic portion of the high abundance CF proteome, hidden from direct analysis of abundance, included proteins from transcription and signaling pathways such as NFkappaB, chaperones such as HSC70, cytoskeletal proteins, and others. Connectivity analysis indicated that approximately 30% of the 51-member hybrid high abundance CF proteome interacts with the NFkappaB signaling pathway. In conclusion, measurement of biosynthetic rates on a global scale can be used to identify disease-specific differences within the high abundance cystic fibrosis proteome. Most of these kinetically defined proteins are unaffected in expression level when using conventional silver stain analysis. We anticipate that this novel hybrid approach to discovery of the high abundance CF proteome will find general application to other proteomic problems in biology and medicine.

Labeling biomolecules with ¹⁸F is usually done through coupling with prosthetic groups, which requires several time-consuming radiosynthesis steps and therefore in low labeling yield. In this study, we designed a simple one-step ¹⁸F-labeling strategy to replace the conventional complex and the long process of multiple-step radiolabeling procedure. Both monomeric and dimeric cyclic RGD peptides were modified to contain 4-NO₂-3-CF₃ arene as precursors for direct ¹⁸F labeling. Binding of the two functionalized peptides to integrin α(v)β₃ was tested in vitro using the MDA-MB-435 human breast cell line. The most promising functionalized peptide, the dimeric cyclic RGD, was further evaluated in vivo in an orthotopic MDA-MB-435 tumor xenograft model. The use of relatively low amount of precursor (~0.5 μmol) gave reasonable yield, ranging from 7 to 23% (decay corrected, calculated from the start of synthesis) after HPLC purification. Overall reaction time was 40 min, and the specific activity of the labeled peptide was high. Modification of RGD peptides did not significantly change the biological binding affinities of the modified peptides. Small animal PET and biodistribution studies revealed integrin specific tumor uptake and favorable biokinetics. We have developed a novel one-step ¹⁸F radiolabeling strategy for peptides that contain a specific arene group, which shortens reaction time and labor significantly, requires low amount of precursor, and results in specific activity of 79 ± 13 GBq/μmol. Successful introduction of 4-fluoro-3-trifluoromethylbenzamide into RGD peptides may be a general strategy applicable to other biologically active peptides and proteins.

Cystic fibrosis (CF) airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that actin plays a role in the regulation of a cloned epithelial Na+ channel (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR). We found that immunopurified bovine tracheal CFTR coreconstituted into a planar lipid bilayer with alpha,beta,gamma-rat ENaC (rENaC) decreased single-channel open probability (Po) of rENaC in the presence of actin by over 60%, a significantly greater effect than was observed in the absence of actin (approximately 20%). In the presence of actin, protein kinase A plus ATP activated both CFTR and rENaC, but CFTR was activated in a sustained manner, whereas the activation of rENaC was transitory. ATP alone could also activate ENaC transiently in the presence ofactin but had no effect on CFTR. Stabilizing short actin filaments at a fixed length with gelsolin (at a ratio to actin of 2:1) produced a sustained activation of alpha,beta,gamma-rENaC in both the presence or absence of CFTR. Gelsolin alone (i.e., in the absence of actin) had no effect on the conductance or Po of either CFTR or rENaC. We have also found that short actin filaments produced their modulatory action on alpha-rENaC independent of the presence of the beta- or gamma-rENaC subunits. In contrast, CFTR did not affect any properties of the channel formed by alpha-rENaC alone, i.e., in the absence of beta- or gamma-rENaC. These results indicate that CFTR can directly downregulate single Na+ channel activity, which may account for the observed differences between Na+ transport in normal and CF-affected airway epithelia. Moreover, the presence of actin confers an enhanced modulatory ability of CFTR on Na+ channels.

High rates of colonization and the challenge of managing Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF) have necessitated a search for safe and effective antibiotics. Currently, therapy with an aminoglycoside in combination with a beta-lactam or a quinolone antibiotic is the standard. Unfortunately, it is difficult to deliver high doses of these antibiotics via the IV route without significant systemic adverse events (AEs) (eg, ototoxicity and nephrotoxicity). Recently, a reformulation of the aminoglycoside antibiotic tobramycin has become available in a preservative-free, pH-adjusted solution for inhalation by jet nebulizer. A 96-week series of clinical studies including 520 patients, aged>> or = 6 years, with moderate-to-severe CF has evaluated the long-term safety and effectiveness of this formulation. Patients received tobramycin solution for inhalation (TSI) or placebo, which was administered in alternating cycles of 28-days-on and 28-days-off therapy, plus their usual CF care for 6 months with open-label follow-up extended to 2 years. Most AEs declined in frequency with increasing TSI exposure. Patients receiving TSI spent 25 to 33% fewer days in the hospital. Following the initiation of TSI treatment, patients experienced significant increases in FEV(1). FEV(1) values were maintained above baseline for the duration of the study series. Antibiotic susceptibility of the bacterial isolates did not predict clinical response. TSI was safe, well-tolerated, and effective for long-term treatment (96 weeks) of P aeruginosa colonization and infection in CF patients.

BACKGROUND

The disease progression of cystic fibrosis (CF) is marked by an increase in clinical conditions and therapeutic interventions, which have the potential to affect health-related quality of life (HRQoL). This cross-sectional study explored associations between clinical variables and HRQoL.

METHODS

HRQoL was measured using the Cystic Fibrosis Quality of Life (CFQoL) questionnaire, which consists of nine domains: physical, social, treatment, chest symptoms, emotional functioning, concerns for the future, relationships, body image, and career concerns. The CFQoL was completed by 223 adults with CF. Clinical and demographic data collected were: age, gender, FEV1% predicted, BMI, Burkholderia cepacia status, lung transplant status, diabetic status, level of nutritional intervention, and presence of an intravenous access device. Multiple regression using forward selection was used to construct models relating these variables to each HRQoL domain.

RESULTS

Despite many of the variables being inter-related, some variables were associated with CFQoL domains even in the presence of other important clinical factors. FEV1% predicted was weakly positively associated with all nine domains. Strong evidence emerged that patients who had received a lung transplant reported a higher HRQoL in physical and social functioning, chest symptoms, and treatment issues. Females tended to report a lower quality of life for chest symptoms and career issues, but higher values for body image. Patients with an access device expressed more career concerns. There was no evidence of an association between B. cepacia and any of the nine CFQoL domains. The model for the body image domain explained a high percentage of the variance (R2=30%): negative body image was associated with lower BMI, having an access device, diabetes, and enteral feeding.

CONCLUSIONS

While important associations were identified, much of the variance in HRQoL remains unexplained. Other clinical and psychosocial variables merit investigation. A longitudinal study is required to investigate how the disease trajectory and associated treatments affect an individual's quality of life.

Potassium (trifluoromethyl)trimethoxyborate is introduced as a new source of CF(3) nucleophiles in copper-catalyzed trifluoromethylation reactions. The crystalline salt is stable on storage, easy to handle, and can be obtained in near-quantitative yields simply by mixing B(OMe)(3), CF(3)SiMe(3), and KF. The trifluoromethylation reagent allows the conversion of various aryl iodides into the corresponding benzotrifluorides in high yields under mild, base-free conditions in the presence of catalytic quantities of a Cu(I)/1,10-phenanthroline complex.

BACKGROUND

We tested the activity of BAL30072, a novel siderophore monobactam, against multiresistant clinical isolates of Pseudomonas aeruginosa, Burkholderia cepacia group and Acinetobacter spp. and against laboratory P. aeruginosa strains with defined resistance mechanisms.

METHODS

MICs were determined on Mueller-Hinton agar supplemented with 2,2' bipyridyl to induce iron transport; comparators were aztreonam, imipenem, meropenem and piperacillin/tazobactam.

RESULTS

BAL30072 was strikingly active against Acinetobacter baumannii, with 73% of 200 carbapenemase-producing isolates, most of them belonging to the UK-dominant OXA-23 clone 1 and SE clone lineages, susceptible at 1 mg/L and 89% at 8 mg/L. Resistance nevertheless was seen in a few representatives of these clones and appeared commoner among isolates representing other A. baumannii clones. Sixty-eight per cent of 50 B. cepacia complex isolates from cystic fibrosis (CF) were susceptible to BAL30072 at 1 mg/L and 78% at 8 mg/L, compared with only 22% susceptible to aztreonam at 8 mg/L. Activity against P. aeruginosa was good, though less dramatic, with 36% of 50 (mostly multiresistant) CF isolates susceptible at 8 mg/L, compared with 12% susceptible to aztreonam at 8 mg/L. BAL30072 was active against 11/19 metallo-beta-lactamase-producing P. aeruginosa at 8 mg/L compared with 3/19 for aztreonam (12/19 versus 8/19 at 16 mg/L). Studies on P. aeruginosa mutants, isolates and transconjugants showed that BAL30072 was affected by efflux, AmpC and by a few uncommon acquired beta-lactamases, including some extended-spectrum OXA types and PER-1.

CONCLUSIONS

BAL30072 displayed impressive activity against many carbapenemase-producing A. baumannii, particularly against the two clones most prevalent in the UK, and also against B. cepacia complex isolates from CF; it was more active than aztreonam against P. aeruginosa.

Cachexia and anorexia commonly occur in patients with cystic fibrosis (CF), particularly those with severe pulmonary compromise and heavy tracheobronchial colonization with Pseudomonas aeruginosa. Current understanding of the pathophysiology of cachexia attributes much of the anorexia and weight loss to the effects of the cytokine tumor necrosis factor (TNF), which is secreted by endotoxin-stimulated macrophages. It has further been suggested that TNF may play a role in the pathobiochemistry of CF cachexia, secondary to the localized inflammatory response in the lung or wider systemic activation of cells of the monocyte-macrophage series in response to endotoxin. This study investigates TNF production and gene expression by peripheral blood monocyte-derived macrophages from CF patients, compared with normals (NL). The results indicate that although both cell populations responded dose-dependently to lipopolysaccharide (LPS); CF macrophages, upon stimulation with LPS at concentrations of 1 to 1,000 ng/ml, consistently produced substantially higher amounts of TNF than NL macrophages. At the molecular level, Northern blot analysis also revealed that both macrophage populations expressed TNF mRNA in response to LPS in a dose-dependent manner. However, at the same LPS concentrations, CF macrophage TNF mRNA expression was 2- to 4-fold greater than that of NL macrophages. LPS had no effect in either macrophage population on mRNA for CHO-B, a constitutive probe. To investigate differences between NL and CF macrophage TNF regulation, nuclear run-on/half-life studies as well as studies addressing potential differences in LPS membrane interactions and signal transduction were performed.(ABSTRACT TRUNCATED AT 250 WORDS)

The hemoprotein ligninase of Phanerochaete chrysosporium Burds. catalyzes the oxidative cleavage of lignin model dimers between C alpha and C beta of their propyl side chains. The model dimers hitherto used give multiple products and complex stoichiometries upon enzymatic oxidation. Here we present experiments with a new model dimer, 1-(3,4-dimethoxyphenyl)-2-phenylethanediol (dimethoxyhydrobenzoin, DMHB) which is quantitatively cleaved by ligninase in air to give benzaldehyde and veratraldehyde according to the stoichiometry: 2DMHB + O2----2PhCHO + 2Ph(OMe)2CHO. Catalytic amounts of H2O2 are required for this aerobic reaction. Under anaerobic conditions, ligninase uses H2O2 as the oxidant for cleavage: DMHB + H2O2----PhCHO + Ph(OMe)2CHO. Electron spin resonance experiments done in the presence of spin traps, 2-methyl-2-nitrosopropane or 5,5-dimethyl-1-pyrroline-N-oxide, show that C alpha-C beta cleavage yields alpha-hydroxybenzyl radicals as intermediate products. Under anaerobic conditions, these radicals react further to give the final aldehyde products. In air, O2 adds to the carbon-centered radicals, probably giving alpha-hydroxybenzylperoxyl radicals which fragment to yield superoxide, benzaldehyde, and veratraldehyde. These results lead us to propose a mechanism for C alpha-C beta cleavage in which attack by ligninase and H2O2 on the methoxylated ring of DMHB yields a cation radical, which then cleaves to give either benzaldehyde and an alpha-hydroxy(dimethoxybenzyl) radical or veratraldehyde and an alpha-hydroxybenzyl radical (cf. Kersten, P. J., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612; Snook, M. E., and Hamilton, G. A. (1974) J. Am. Chem. Soc. 96, 860-869). Similar mechanisms probably apply to the enzymatic C alpha-C beta cleavage of natural lignin.

BACKGROUND

The dysfunction of the mucosal interface of the upper respiratory tract in cystic fibrosis (CF) patients is clinically visible by the development of nasal polyps (NP) at a young age. Innate defence markers and inflammatory mediators in NP from patients with CF were compared with non-cystic fibrosis nasal polyps (non-CF-NP) to determine a possible different immunological background in macroscopically similar tissue.

METHODS

Surgical samples were obtained from patients with non-CF-NP, cystic fibrosis patients with nasal polyps (CF-NP) and control patients (CO). With real time PCR, the mRNA expression of human beta defensins (HBD) 2 and 3, toll-like receptors (TLR) 2 and 4 and the macrophage mannose receptor (MMR) were measured. On homogenates of the surgical samples eotaxin, myeloperoxidase (MPO), IL-5 and IL-8 protein content was measured using commercial ELISA kits; IgE and eosinophilic cationic protein (ECP) were measured by the Unicap system.

RESULTS

In CF-NP we found a statistically significant higher mRNA expression of HBD 2 compared with non-CF-NP and CO and of TLR 2 compared with non-CF-NP. In the non-CF-NP group, MMR mRNA expression was significantly elevated compared with CO and CF-NP. For TLR 4 mRNA expression no statistically significant differences were found between groups. IL-5 was below detection level in all CO and CF-NP, but was measurable in 80% of the non-CF-NP. MPO and IL-8 concentrations were significantly higher in CF-NP compared with CO and non-CF-NP, whereas ECP, eotaxin and IgE were significantly higher in the non-CF-NP group.

CONCLUSIONS

We here demonstrate that CF-NP and non-CF-NP not only differ in terms of inflammatory mediator profile, but also in terms of innate markers.

The surface charge of cultured neurons was investigated with the electron microscope markers anionized ferritin (AF) and cationized ferritin (CF). To determine which membrane components could react with the markers, model reactions were used. Both protein-coated Sepharose beads and lipid vesicles were reacted at physiological pH. Results with these model reactions indicate that the following groups may contribute to the surface charge: acidic groups--the sialic acid of both glycoproteins and gangliosides, the carboxyl group of proteins, and the phosphates of phospholipids; basic groups--the amines of proteins. The effect of chemical fixation on the surface charge was investigated. Glutaraldehyde fixation was shown to increase the charge of neutral proteins but not by a mechanism involving unbound aldehydes. Glutaraldehyde fixation of phospholipid vesicles in the presence of CF showed that amine-containing phospholipids were cross-linked to CF. This cross-linkage was seen with the electron microscope as the clumping of CF and the burying of CF in the membrane. Paraformaldehyde fixation had a lesser effect on the charge of proteins but did react with phospholipids as did glutaraldehyde. It is concluded that at physiological pH: (a) most of the charged proteins and lipids on cell surface can contribute to the membrane surface charge, and (b) the membrane surface charge of cells can be greatly changed by chemical fixation.

1. The equations derived by Heath (1968) were applied to data from experiments on rats in four metabolic states: fed, post-absorptive, starved and 2hr. after an eventually lethal injury. The data used were: (a) The fractions of label injected as C1-, C2- and C3-pyruvate (where the prefix indicates the position of labelling) that are incorporated into carbon dioxide and glucose in post-absorptive and injured rats (yields). Yields could be corrected to yields on label taken up by the liver. (b) The (C5-label in glutamate)/(total label in glutamate) ratio in the liver after C2-pyruvate in rats in all four states. (c) The distribution of label within glutamate after C2-pyruvate or C2-alanine in the livers of fed, post-absorptive and starved rats. (d) The distribution of label within glucose after C2-lactate or C2-pyruvate in starved rats. (e) The relative specific radioactivities of pyruvate, aspartate, glutamate and (in two states only) of glucose 6-phosphate after injection of [U-(14)C]glucose into rats in all four states. These data were previously published, except those after (e) and some after (b) above, which are given in this paper. 2. In addition the concentrations of pyruvate, citrate, glutamate and aspartate in the livers of post-absorptive and injured rats were found. Injury decreased glutamate and citrate concentrations and to a smaller extent aspartate and pyruvate concentrations. 3. Non-steady-state theory showed that most of the data could be used without serious error in steady-state theory. Steady-state theory correlated all but one observation (the relative yields of (14)CO(2) from C2- and C3-pyruvate) listed after (a)-(e) above within the experimental errors, and gave rough estimates of the rates of pyruvate carboxylation, conversion of pyruvate and fat into acetyl-CoA and utilization of glutamate. The main conclusions were: (a) symmetrization of label in oxaloacetate both in the mitochondrion and in the cytoplasm was far from complete, because oxaloacetate did not equilibrate with fumarate in either. From this and other findings it was deduced: (b) that malate or fumarate or both left the mitochondrion, and not oxaloacetate; (c) that there was a loss from the mitochondrion of a fraction of the malate or fumarate or both formed from succinate, and (d) the resulting deficiency of oxaloacetate for the perpetuation of the tricarboxylic acid cycle was made up from pyruvate in fed and post-absorptive rats, but (e) in the starved rat could only be made up by utilization of glutamate. (f) In the fed rat the tricarboxylic acid cycle ran mostly on pyruvate, but in the post-absorptive and starved rat mostly on fat. (g) In the injured rat the tricarboxylic acid cycle was slowed, label in oxaloacetate was completely symmetrized (cf. conclusion a), and the tricarboxylic acid cycle utilized glutamate. (h) The conclusions were not invalidated by isotopic exchange, i.e. flux of label without net flux of compound, nor by interaction with lipogenic processes. (i) In the kidneys interaction between the tricarboxylic acid cycle and gluconeogenesis was different from in the liver, and was much less. The effects on the theory were roughly assessed, and were small. 4. The experiments and optimum experimental conditions required to check the theory are listed, and several predictions, open to experimental confirmation, are made.

BACKGROUND

Peroxisome proliferator activated receptor gamma (PPARgamma) is a ligand-activated transcription factor known to be central to both adipose tissue development and insulin action. Growth of adipose tissue requires differentiation of preadipocytes with acquisition of specific cellular functions including insulin sensitivity, leptin secretion and the capacity to store triglyceride. Dietary fatty acids and members of the thiazolidinedione class of compounds have been reported to influence adipogenesis at the transcriptional level. Here, we compare the effects of a dietary fatty acid, linoleic acid, and a thiazolidinedione, rosiglitazone, on biochemical and functional aspects of human preadipocyte differentiation in vitro.

METHODS

Human omental and subcutaneous preadipocytes were subcultured 2-3 times and subsequently differentiated for 21 days in the presence of either linoleic acid or rosiglitazone. Differentiation was assessed using a number of biochemical and functional parameters.

RESULTS

Omental and subcutaneous preadipocytes differentiated in the presence of linoleic acid showed marked cytoplasmic triacylglycerol accumulation however, no biochemical markers of differentiation (LPL expression, G3PDH gene expression and enzyme activity and leptin expression or secretion) were detected. In contrast, treatment of these cells with rosiglitazone induced full biochemical differentiation as judged by all markers assessed, despite comparatively little lipid accumulation. The rosiglitazone effects were subcutaneous depot-specific. Cells treated with linoleic acid showed decreased glucose uptake cf rosiglitazone-treated cells. A luciferase reporter assay demonstrated that rosiglitazone potently activates h-peroxisome proliferator activated receptor gamma while linoleic acid had no effect.

CONCLUSIONS

These studies demonstrate that (a) human preadipocytes have the potential to accumulate triacylglycerol irrespective of their stage of biochemical differentiation; (b) while omental preadipocytes are refractory to biochemical differentiation in vitro, they are able to accumulate triacylglycerol; and (c) rosiglitazone and linoleic acid may exert their effects via different biochemical pathways.

There have been conflicting reports in the literature concerning the polypeptide composition of the vacuolar H(+)-translocating inorganic pyrophosphatase (tonoplast H(+)-PPase) of plant cells. The major subunit(s) of the enzyme have been attributed to polypeptides of relative molecular weight (M(r)) 64,500 (Beta vulgaris), 67,000 (Beta vulgaris), 73,000 (Vigna radiata), and 37,000 to 45,000 (Zea mays). Here, we reconcile these differences to show, through the combined application of independent purification, affinity-labeling, sequencing, and immunological procedures, that the major polypeptide associated with the H(+)-PPase from all of these organisms, and Arabidopsis thaliana, corresponds to the same moiety. The principal polypeptide components of the H(+)-PPase purified from Beta and Vigna by independent procedures have similar apparent subunit masses when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under identical conditions (M(r(Beta)) = 64,500; M(r(Vigna)) = 66,000) and exhibit identical kinetics of irreversible inhibition and ligand-modified labeling by [(14)C]-N-ethylmaleimide. Similarly, the M(r) 64,500 and 67,000 polypeptides isolated from Beta by independent methods (cf. C.J. Britten, J.C. Turner, P.A. Rea [1989] FEBS Lett 256: 200-206 versus V. Sarafian and R.J. Poole [1989] Plant Physiol 91: 34-38) are indistinguishable: the two polypeptides comigrate when electrophoresed under the same conditions and yield tryptic fragments with identical overlapping sequences. Because both the N-terminal sequence of the M(r) 66,000 subunit of the H(+)-PPase isolated from Vigna and the direct sequence data from Beta align precisely with the deduced amino acid sequence of cDNAs encoding the H(+)-PPase of Arabidopsis, all three enzymes are inferred to be highly conserved structurally. Accordingly, immunoblots of membranes prepared from Arabidopsis, Beta, Vigna, and Zea, probed with antibody affinity purified against the magnesium inorganic pyrophosphate-binding, M(r) 66,000 polypeptide of Vigna, reveal a single immunoreactive band at M(r) 64,500 to 67,000 in all four preparations. The M(r) 66,000 polypeptide of Zea membranes is, however, prone to proteolysis during membrane fractionation and selective aggregation during sample denaturation for SDS-PAGE. The anomalous M(r) 37,000 to 45,000 subunit pattern previously ascribed to the H(+)-PPase from Zea (A. Chanson and P.E. Pilet [1989] Plant Physiol 90: 934-938) is attributed to loss of the M(r) 66,000 subunit and the appearance of polypeptide fragments of M(r) 44,700 and 39,000 through the combined effects of sample aggregation before SDS-PAGE and proteolysis, respectively. It is, therefore, concluded that the substrate-binding subunit of the tonoplast H(+)-PPase has a common identity in all four organisms.

Bdellovibrio bacteriovorus is a predator bacterial species found in the environment and within the human gut, able to attack Gram-negative prey. Cystic fibrosis (CF) is a genetic disease which usually presents lung colonization by Pseudomonas aeruginosa or Staphylococcus aureus biofilms. Here, we investigated the predatory behavior of B. bacteriovorus against these two pathogenic species with: (1) broth culture; (2) "static" biofilms; (3) field emission scanning electron microscope (FESEM); (4) "flow" biofilms; (5) zymographic technique. We had the first evidence of B. bacteriovorus survival with a Gram-positive prey, revealing a direct cell-to-cell contact with S. aureus and a new "epibiotic" foraging strategy imaged with FESEM. Mean attaching time of HD100 to S. aureus cells was 185 s, while "static" and "flow" S. aureus biofilms were reduced by 74 (at 24 h) and 46% (at 20 h), respectively. Furthermore, zymograms showed a differential bacteriolytic activity exerted by the B. bacteriovorus lysates on P. aeruginosa and S. aureus. The dual foraging system against Gram-negative (periplasmic) and Gram-positive (epibiotic) prey could suggest the use of B. bacteriovorus as a "living antibiotic" in CF, even if further studies are required to simulate its in vivo predatory behavior.

Between January 1991 and January 1993, 265 patients who fulfilled the CDC criteria of the working case definition of Chronic Fatigue Syndrome (CFS) have been observed at our Institution and submitted for clinical and laboratory evaluation. One hundred and sixty-three patients were females and 102 males, the median age was 35 years (range 4-55 years); all patients reported profound and prolonged fatigue, lasting for a median of 3 years (range 6 months-10 years), preceded or accompanied at appearance by fever in 185 cases, and neuropsychologic problems including inability to concentrate, difficulty in thinking, confusion, irritability, forgetfulness, and depression. The fatigue was so severe that it required 102 patients to stop their working activities for a period of time ranging from 3 months to 2 years (range 7 months). In 40 consecutive patients a comprehensive immunologic testing by single and two-colour flow cytometry was performed and results compared with a group of 35 healthy, age- and sex-matched controls. Whilst no significant differences were found in the absolute numbers of circulating total T cells (CD3+) and of total helper/inducer (CD4+) or suppressor/cytotoxic (CD8+) T cells, an evident reduction in CD3-/CD16+ and CD57+/CD56+ NK lymphocytes along with an expansion of the CD8+/CD56+ and CD16-/CD56+ NK subsets, were found in the CFS group. In addition, CD56+ NK cells from CFS subjects were found to express an increased amount of cell adhesion molecules (CD11b, CD11c, CD54) and activation antigens (CD38). Both the percentage and absolute numbers of CD4+ T cells bearing the CD45RA antigen appeared significantly reduced in CFS patients, and CD4+ T lymphocytes from CFS subjects displayed an increased expression of the intercellular adhesion molecule-1 (ICAM-1/CD54). Finally, the total numbers of circulating (CD19+) B lymphocytes, were significantly higher in CFS cases than in controls, and in 11 out of 30 CFS patients the increase in circulating B cells was sustained by the expansion of the CD5+/CD19+ subset of B lymphocytes. We conclude that CFS is a syndrome not previously described in Italy, with already known clinical characteristics and appears to be associated with several immunologic abnormalities, including those reported previously in cohort of patients from different countries. We also show for the first time that CD56- NK cell subsets from CFS patients display an abnormally increased expression of cell adhesion molecules and activation markers.

BACKGROUND

The cause of chronic fatigue syndrome (CFS) is unknown but is thought to be associated with immune abnormalities or infection. Because cancer can arise from similar conditions, associations between CFS and cancer were examined in a population-based case-control study among the US elderly.

METHODS

Using linked Surveillance, Epidemiology, and End Results (SEER)-Medicare registry data, approximately 1.2 million cancer cases and 100,000 controls (age range, 66-99 years; 1992-2005) were evaluated. CFS was identified in the period more than 1 year prior to selection, using linked Medicare claims. Unconditional logistic regression was used to estimate the odds ratios (ORs) comparing the CFS prevalence in cases and controls, adjusting for age, sex, and selection year. All statistical tests were 2-sided.

RESULTS

CFS was present in 0.5% of cancer cases overall and 0.5% of controls. CFS was associated with an increased risk of non-Hodgkin lymphoma (NHL) (OR = 1.29, 95% confidence interval [CI] = 1.16-1.43, P = 1.7 × 10(-6) ). Among NHL subtypes, CFS was associated with diffuse large B cell lymphoma (OR = 1.34, 95% CI = 1.12-1.61), marginal zone lymphoma (OR = 1.88, 95% CI = 1.38-2.57), and B cell NHL not otherwise specified (OR = 1.51, 95% CI = 1.03-2.23). CFS associations with NHL overall and NHL subtypes remained elevated after excluding patients with medical conditions related to CFS or NHL, such as autoimmune conditions. CFS was also associated, although not after multiple comparison adjustment, with cancers of the pancreas (OR = 1.25, 95% CI = 1.07-1.47), kidney (OR = 1.27, 95% CI = 1.07-1.49), breast (OR = 0.85, 95% CI = 0.74-0.98), and oral cavity and pharynx (OR = 0.70, 95% CI = 0.49-1.00).

CONCLUSIONS

Chronic immune activation or an infection associated with CFS may play a role in explaining the increased risk of NHL.

The in vivo activity and source of beta-lactamase in sputum samples from 43 patients with cystic fibrosis (CF) during a 2-week antipseudomonal treatment were studied. A colorimetric method, based on the conversion of nitrocefin, was used for quantitation of the sputum beta-lactamase activity. beta-Lactamases in sputum were characterized by isoelectric focusing and inhibition profile and were compared with the beta-lactamases extracted from Pseudomonas aeruginosa isolated from the paired sputum samples. We found that the beta-lactamase activity increased to high levels in sputum from patients with CF during the course of piperacillin, ceftazidime, cefsulodin, or imipenem therapy. Aztreonam therapy lead to opposite results because the beta-lactamase activity decreased and aztreonam was able to mask beta-lactamase activity by acting as an inhibitor. All sputum beta-lactamases displayed characteristics indicative of a class I enzyme, identical to the beta-lactamases extracted from P. aeruginosa. The presence of beta-lactamase at such levels could lead to in vivo inactivation of beta-lactam antibiotics. This study supports the hypothesis that beta-lactamase production is an important in vivo resistance mechanism in P. aeruginosa-infected patients with CF.

Among a group of 70 individuals who met the criteria established by the Centers for Disease Control and Prevention (Atlanta) for chronic fatigue syndrome (CFS), 12%-28% had serum levels exceeding 95% of control values for tumor necrosis factor (TNF) alpha, TNF-beta, interleukin (IL) 1 alpha, IL-2, soluble IL-2 receptor (sIL-2R), or neopterin; overall, 60% of patients had elevated levels of one or more of the nine soluble immune mediators tested. Nevertheless, only the distributions for circulating levels of TNF-alpha and TNF-beta differed significantly in the two populations. In patients with CFS--but not in controls--serum levels of TNF-alpha, IL-1 alpha, IL-4, and sIL-2R correlated significantly with one another and (in the 10 cases analyzed) with relative amounts (as compared to beta-globin or beta-actin) of the only mRNAs detectable by reverse transcriptase-coupled polymerase chain reaction in peripheral-blood mononuclear cells: TNF-beta, unspliced and spliced; IL-1 beta, lymphocyte fraction; and IL-6 (in order of appearance). These findings point to polycellular activation and may be relevant to the etiology and nosology of CFS.

Chronic fatigue syndrome (CFS) is a newly-recognized clinical entity characterized by chronic, debilitating fatigue lasting longer than six months. Common associated findings are chronic and recurrent fever, pharyngitis, myalgias, adenopathy, arthralgias, difficulties in cognition and disorders of mood. In the majority of patients, the illness starts suddenly with an acute, 'flu-like' illness. The following abnormalities are seen with some frequency although none are seen in all patients: lymphocytosis, atypical lymphocytosis, monocytosis, elevation of hepatocellular enzymes, low levels of antinuclear antibodies, low levels of immune complexes. Clinical and serologic studies suggest an association of CFS with all of the human herpesviruses, particularly Epstein-Barr virus (EBV) and the recently-discovered human B-lymphotropic virus (HBLV) or human herpesvirus-6; neither EBV nor HBLV has yet been shown to play a causal role in the illness.

<A<em>b</em>stractText>It has <em>b</em>een reported that the triglyceride-glucose (TyG) index may serve as a simple and credi<em>b</em>le surrogate marker of insulin resistance (IR). However, its association with macrovascular and microvascular damage is unclear. Accordingly, the o<em>b</em>jective of the present study is to investigate the association of macrovascular and microvascular damage with the TyG index.</A<em>b</em>stractText><A<em>b</em>stractText>A total of 2830 elderly participants from the Northern Shanghai Study (NSS) were enrolled. The TyG index was calculated as ln[fasting triglycerides (mg/dL) × fasting glucose (mg/dL)/2]. Parameters of vascular damage, including carotid-femoral pulse wave velocity (<em>cf</em>-PWV), <em>b</em>rachial-ankle pulse wave velocity (<em>b</em>a-PWV), ankle-<em>b</em>rachial index (ABI), carotid intima-media thickness (CMT), carotid plaque, estimated glomerular filtration rate (eGFR) and the urine al<em>b</em>umin-to-creatinine ratio (UACR), were measured and calculated.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>In univariate logistic regression, an increased TyG index was associated with a higher risk of <em>cf</em>-PWV > 10 m/s, <em>b</em>a-PWV > 1800 cm/s, ABI < 0.9, microal<em>b</em>uminuria (MAU) and chronic kidney disease (CKD). In multivaria<em>b</em>le logistic regression, there was a significant increase in the risk of <em>cf</em>-PWV > 10 m/s (OR = 1.86, 95% confidence interval [95% CI] 1.37-2.53, P<su<em>b</em>)for trend</su<em>b</em>) < 0.001), <em>b</em>a-PWV > 1800 cm/s (OR = 1.39, [95% CI] 1.05-1.84, P<su<em>b</em>)for trend</su<em>b</em>)= 0.02), MAU (OR = 1.61, [95% CI] 1.22-2.13, P<su<em>b</em>)for trend</su<em>b</em>) < 0.001) and CKD (OR = 1.67, [95% CI] 1.10-1.50, P<su<em>b</em>)for trend</su<em>b</em>)= 0.02) after adjustment for age, sex, BMI, waist circumference, smoking ha<em>b</em>it, hypertension, family history of premature CVD, dia<em>b</em>etes, HDL-C, LDL-C, insulin therapy and statin therapy. However, no significant relationship was o<em>b</em>served <em>b</em>etween the TyG index and lower extremity atherosclerosis, carotid hypertrophy or carotid plaque.</p><A<em>b</em>stractText>An elevated TyG index was significantly associated with a higher risk of arterial stiffness and nephric microvascular damage. This conclusion lends support to the clinical significance of the TyG index for the assessment of vascular damage.</A<em>b</em>stractText>

BACKGROUND

A clinical study to investigate the leukotriene B(4) (LTB(4))-receptor antagonist BIIL 284 in cystic fibrosis (CF) patients was prematurely terminated due to a significantly increased risk of adverse pulmonary events. We aimed to establish the effect of BIIL284 in models of Pseudomonas aeruginosa lung infection, thereby contributing to a better understanding of what could have led to adverse pulmonary events in CF patients.

METHODS

P. aeruginosa DNA in the blood of CF patients during and after acute pulmonary exacerbations and in stable patients with non-CF bronchiectasis (NCFB) and healthy individuals was assessed by PCR. The effect of BIIL 284 treatment was tested in an agar bead murine model of P. aeruginosa lung infection. Bacterial count and inflammation were evaluated in lung and other organs.

RESULTS

Most CF patients (98%) and all patients with NCFB and healthy individuals had negative P. aeruginosa DNA in their blood. Similarly, the P. aeruginosa-infected mice showed bacterial counts in the lung but not in the blood or spleen. BIIL 284 treatment decreased pulmonary neutrophils and increased P. aeruginosa numbers in mouse lungs leading to significantly higher bacteremia rates and lung inflammation compared to placebo treated animals.

CONCLUSIONS

Decreased airway neutrophils induced lung proliferation and severe bacteremia in a murine model of P. aeruginosa lung infection. These data suggest that caution should be taken when administering anti-inflammatory compounds to patients with bacterial infections.