In pigs, protection against the toxigenic extra-cellular bacterium Actinobacillus pleuropneumoniae was correlated with an increased IgG(1):IgG(2) ratio of haemolytic toxin-specific antibodies. In all species so far studied, IgG isotype expression is controlled by Type 1 (IFN-gamma, IL-12) and Type 2 (IL-4, IL-10) cytokines which dictate immune response polarization to cell-mediated (CMI) or antibody-mediated immunity (AMI), respectively. Thus, immunoglobulin (Ig) isotypes reflect Type 1 or Type 2 immune responses. Immunoglobulin isotype production by porcine B-cells cultured in the presence of recombinant porcine (rp) cytokines varies by individual, however pigs tend to generate a high IgG(1):IgG(2) ratio in response to rp IL-10 and the inverse in response to rp IFN-gamma or rp IL-12. Differential Ig isotype production should favor an isotype with a functional advantage to control the inciting infection and disease. However, functions of porcine Ig isotypes have not been described. To compare function of porcine IgM, IgG(1) and IgG(2) of defined specificity for hen eggwhite lysozyme (HEWL), Ig isotypes were affinity purified from serum by HEWL specificity and by isotype-specific mouse monoclonal antibodies. Their ability to activate complement (C') and to opsonize was tested in vitro. Porcine IgG(2) had greater guinea pig C' activating ability than did IgG(1). Neither isotype opsonized HEWL-conjugated sheep erythrocytes in vitro. Amino acid sequence analysis of IgG isotypes revealed that all subclasses have putative C' binding sites but that IgG(2a), IgG(2b) and IgG(4) were more flexible in the middle hinge region than IgG(1) and IgG(3) and would likely activate C' more efficiently. Thus, porcine IgG isotypes associated with resistance and susceptibility to disease also differ in their actual and predicted biological functions.

S100 is a family of small, acidic, calcium binding proteins involved in the control of a multitude of intra- and extracellular processes, including many pathologies. The application of the analytical methodology based on the combination of RP HPLC and ESI-MS allowed for the characterization of S-nitrosylation and S-glutathionylation in two representative S100 proteins: S100A1 and S100B. The GSNO related S-nitrosylation of the conserved C-terminal cysteine is strongly activated by the binding of Ca(II) to S100A1 and of Ca(II) and Zn(II) to S100B. This modification results in a global alteration of protein structure, as demonstrated by a variety of techniques. The presented results provide a mechanistic basis for further studies of the function of S100 proteins in the control of redox-based and metal-based signal transduction.

Whole-cell patch clamp experiments were used to investigate the transduction mechanism of adenosine A(2A) receptors in modulating N-methyl-D-aspartate (NMDA)-induced currents in rat striatal brain slices. The A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) inhibited the NMDA, but not the (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current in a subset of striatal neurons. Lucifer yellow-filled pipettes in combination with immunostaining of A(2A) receptors were used to identify CGS 21680-sensitive cells as typical medium spiny striatal neurons. Dibutyryl cyclic AMP and the protein kinase A activator Sp-cyclic AMPs, but not the protein kinase A inhibitors Rp-cyclic AMPS or PKI(14 - 24)amide abolished the inhibitory effect of CGS 21680. The phospholipase C inhibitor U-73122, but not the inactive structural analogue U-73343 also interfered with CGS 21680. The activation of protein kinase C by phorbol 12-myristate 13-acetate or the blockade of this enzyme by staurosporine did not alter the effect of CGS 21680. Heparin, an antagonist of inositol 1, 4,5-trisphosphate (InsP(3)) and a more efficient buffering of intracellular Ca(2+) by BAPTA instead of EGTA in the pipette solution, abolished the CGS 21680-induced inhibition. The calmodulin antagonist W-7 and cytochalasin B which enhances actin depolymerization also prevented the effect of CGS 21680; the calmodulin kinase II inhibitors CaM kinase II(281 - 309) and KN-93 but not the inactive structural analogue KN-92 were also effective. The calcineurin inhibitor deltamethrin did not interfere with CGS 21680. It is suggested that the transduction mechanism of A(2A) receptors to inhibit NMDA receptor channels is the phospholipase C/InsP(3)/calmodulin and calmodulin kinase II pathway. The adenylate cyclase/protein kinase A and phospholipase C/protein kinase C pathways do not appear to be involved.

A high-performance liquid chromatographic (HPLC) assay is described for the determination of the new, investigational antitumour drug SK&F 104864-A and its lactone ring opened form (SK&F 105992). The analytical methodology reported here involves a protein precipitation step with methanol as sample pretreatment procedure. The instability of the drug necessitates that the plasma fraction is obtained within 5 min after blood sampling by centrifugation, immediately followed by protein precipitation with cold methanol (-30 degrees C). The methanolic extract can be stored at -30 degrees C for several days without deterioration of the analyses. Stability data of the drug and its lactone ring opened metabolite in plasma and after methanolic extraction are discussed. The parent drug and the metabolite are separated by reversed-phase ion-pair liquid chromatography on a LiChrosorb RP-18 column, using methanol-water eluent (pH 6.0) with sodium dioctylsulphosuccinate (DOSS) as ion-pairing agent and fluorescence detection. The proposed method has been validated and, subsequently, implemented in a phase I clinical trial for pharmacokinetic evaluation of the new cytotoxic agent.

In a previous study, a repeated sequence, RPSCandida albicans. It was 2.1 kb in length and was tandemly repeated in a limited region of almost all of the chromosomes. In this study, we examined and characterized the diversity of the repeating structure of the RPS units were of 2.1, 2.3, 2.5, and 2.9 kbp in length after digestion of the genomic DNA with SmaI and 2.1 and 2.3 kbp after digestion with PstI, with the differences being multiples of approximately 0.2 kbp. Moreover, one or two types of RPS unit were present specifically on each chromosome. We cloned 14 RPS units from the mixed DNA of chromosomes 1 and 2 and 59 RPS units from chromosome 6. These RPS units were classified into four types by their SfiI digestion profiles and chromosomal origins. Sequence comparisons revealed a tandem arrangement of internal, small repeating units of 172 bp. This unit of repetition was designated alt (C. albicans tandem repeating unit). The size of RPS units was variable, with sizes representing a series of increments of approximately 0.2 kbp that corresponded to the alt sequence. By contrast, the sequences other than the tandem repeats of alts were highly conserved, with homology of more than 98% among all cloned RPS units. These results suggested that RPS plays an important role in the organization and function of the chromosomes of C. albicans even though the actual function of RPS has not yet been clarified. Structural features of RPS that contains the repeated alt sequence are discussed in relation to human alpha-satellite DNA with its tandem repeats of about 170 bp that are similar in size to alt, the repetition of which is responsible for the variations in the size of the higher-order repeats.

Electrogenic and redox events in the reaction-centre complexes from Rhodopseudomonas viridis have been studied. In contrast to the previous points of view it is shown that all the four hemes of the tightly bound cytochrome c have different Em values (-60, +20, +310 and +380 mV). The first three hemes reveal alpha absorption maxima at 554 nm, 552 nm and 556 nm respectively. The 380-mV heme displays a split alpha band with a maximum at 559 nm and a shoulder at 552 nm. Such a splitting is due to non-degenerated Qx and Qy transitions in the iron-porphyrin ring as demonstrated by magnetic circular dichroism spectra. Fast kinetic measurements show that, at redox potentials when only high-potential hemes c-559 and c-556 are reduced, heme c-559 appears to be the electron donor to P-960+ (tau = 0.32 microsecond) whereas heme c-556 serves to rereduce c-559 (tau = 2.5 microsecond). Upon reduction of the third heme (c-552), the P-960+ reduction rate increases twofold (tau = 0.17 microsecond) and all photoinduced redox events within the cytochrome appear to be complete in less than 1 microsecond after the flash. The following sequence of the redox centers is tentatively suggested: c-554, c-556, c-552, c-559, P-960. To study electrogenesis, the reaction-centre complexes from Rps. viridis were incorporated into asolectin liposomes, and fast kinetics of laser flash-induced electric potential difference has been measured in proteoliposomes adsorbed on a phospholipid-impregnated film. The electrical difference induced by a single 15-ns flash was found to be as high as 100 mV. The photoelectric response has been found to involve four electrogenic stages associated with (I) QA reduction by P-960; (II) reduction of P-960+ by heme c-559; (III) reduction of c-559 by c-556 and (IV) protonation of Q2-B. The relative contributions of stages I, II, III and IV are found to be equal to 70%, 15%, 5% and 10%, respectively, of the overall electrogenic process. At the same time, the first three respective distances along the axis normal to the membrane plane covered by electrons, calculated from X-ray data of Deisenhofer et al. [J. Mol. Biol. 180, 385-398 (1984)], are 22%, 18.5% and 26%. This indicates that the efficiency of electrogenic phases depends first of all upon the value of the dielectric constant of the respective membrane regions rather than upon the distance between the redox groups involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzo[ a]pyrene (B[ a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically activated by three enzymatic pathways: by peroxidases (e.g., cytochrome P450 peroxidase) to yield radical cations, by P4501A1/1B1 monooxygenation and epoxide hydrolase to yield diol epoxides, and by P4501A1/1B1 monooxygenation, epoxide hydrolase, and aldo-keto reductases (AKRs) to yield o-quinones. In humans, a major exposure site for environmental and tobacco smoke PAH is the lung; however, the profile of B[ a]P metabolites formed at this site has not been well characterized. In this study, human bronchoalveolar H358 cells were exposed to B[ a]P, and metabolites generated by peroxidase (B[ a]P-1,6- and B[ a]P-3,6-diones), from cytochrome P4501A1/1B1 monooxygenation [3-hydroxy-B[ a]P, B[ a]P-7,8- and 9,10- trans-dihydrodiols, and B[ a]P- r-7, t-8, t-9, c-10-tetrahydrotetrol (B[ a]P-tetraol-1)], and from AKRs (B[ a]P-7,8-dione) were detected and quantified by RP-HPLC, with in-line photo-diode array and radiometric detection, and identified by liquid chromatography-mass spectrometry (LC-MS). Progress curves showed a lag phase in the formation of 3-hydroxy-B[ a]P, B[ a]P-7,8- trans-dihydrodiol, B[ a]P-tetraol-1, and B[ a]P-7,8-dione over 24 h. Northern blot analysis showed that B[ a]P induced P4501B1 and AKR1C isoforms in H358 cells in a time-dependent manner, providing an explanation for the lag phase. Pretreatment of H358 cells with 10 nM 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) eliminated this lag phase but did not alter the levels of the individual metabolites observed, suggesting that both B[ a]P and TCDD induction ultimately yield the same B[ a]P metabolic profile. The one exception was B[ a]P-3,6-dione which was formed without a lag phase in the absence and presence of TCDD, suggesting that the peroxidase responsible for its formation was neither P4501A1 nor 1B1. Candidate peroxidases that remain include PGH synthases and uninduced P450 isoforms. This study shows that the P4501A1/1B1 and AKR pathways are inducible in human lung cells and that the peroxidase pathway was not. It also provides evidence that each of the pathways of PAH activation yields their distinctive metabolites in H358 human lung cells and that each pathway may contribute to the carcinogenic process.

1. The role of the cGMP pathway in the modulation of the cardiac L-type Ca2+ current (ICa,L) by nitric oxide (NO) was examined in rat ventricular myocytes. 2. The NO donors DEANO, SIN-1, SNP, SNAP and GSNO had no significant effects on basal ICa,L. However, DEANO (100 microM) inhibited ICa,L after the current had been previously stimulated by either isoprenaline (Iso, 1-10 nM), a beta-adrenergic agonist, or isobutylmethyl-xanthine (IBMX, 10-80 microM), a wide spectrum phosphodiesterase (PDE) inhibitor. 3. The anti-adrenergic effect of DEANO on ICa,L was not mimicked by other NO donors (SIN-1, SNAP and SPNO). 4. The NO-sensitive guanylyl cyclase inhibitor ODQ (10 microM), antagonized the inhibitory effect of DEANO on ICa,L. Likewise, inhibitors of the cGMP-dependent protein kinase (cG-PK), Rp-8-chloro-phenylthio-cGMP (10 microM) and KT5823 (0.1 and 0.3 microM), also abolished the inhibitory effect of DEANO on Iso (1-10 nM)-stimulated ICa,L. 5. Intracellular dialysis with exogenous cAMP (10-100 microM) blunted the inhibitory effect of DEANO (10 and 100 microM) on ICa,L. SNAP and SNP also had no effect on the cAMP-stimulated ICa,L. 6. Pre-treatment of the myocytes with pertussis toxin (0.5 microg ml-1, 4-6 h at 37 degrees C) eliminated the inhibitory effect of DEANO (100 microM) on ICa,L, in the presence of either Iso (0.01 and 1 nM) or IBMX (10-80 microM). 7. These results demonstrate that DEANO produces anti-adrenergic effects in rat ventricular myocytes. This effect of DEANO occurs in a cGMP-dependent manner, and involves activation of cG-PK and regulation of a pertussis toxin-sensitive G protein.

OBJECTIVE

(18)F-Fluorocholine (FCH) and (11)C-acetate (ACE) PET are widely used for detection of recurrent prostate cancer (PC). We present the first results of a comparative, prospective PET/CT study of both tracers evaluated in the same patients presenting with recurrence and low PSA to compare the diagnostic information provided by the two tracers.

METHODS

The study group comprised 23 patients studied for a rising PSA level after radical prostatectomy (RP, 7 patients, PSA ≤ 3 ng/ml), curative radiotherapy (RT, 7 patients, PSA ≤ 5 ng/ml) or RP and salvage RT (9 patients, PSA ≤ 5 ng/ml). Both FCH and ACE PET/CT scans were performed in a random sequence a median of 4 days (range 0 to 11 days) apart. FCH PET/CT was started at injection (307 ± 16 MBq) with a 10-min dynamic acquisition of the prostate bed, followed by a whole-body PET scan and late (45 min) imaging of the pelvis. ACE PET/CT was performed as a double whole-body PET scan starting 5 and 22 min after injection (994 ± 72 MBq), and a late view (45 min) of the prostate bed. PET/CT scans were blindly reviewed by two independent pairs of two experienced nuclear medicine physicians, discordant subgroup results being discussed to reach a consensus for positive, negative end equivocal results.

RESULTS

PET results were concordant in 88 out of 92 local, regional and distant findings (Cohen's kappa 0.929). In particular, results were concordant in all patients concerning local status, bone metastases and distant findings. Lymph-node results were concordant in 19 patients and different in 4 patients. On a per-patient basis results were concordant in 22 of 23 patients (14 positive, 5 negative and 3 equivocal). In only one patient was ACE PET/CT positive for nodal metastases while FCH PET/CT was overall negative; interestingly, the ACE-positive and FCH-negative lymph nodes became positive in a second FCH PET/CT scan performed a few months later.

CONCLUSIONS

Overall, ACE and FCH PET/CT showed excellent concordance, on both a per-lesion and a per-patient basis, suggesting that both tracers perform equally for recurrent prostate cancer staging.

OBJECTIVE

To evaluate the clinical usefulness of wide-field fundus autofluorescence (FAF) imaging in patients with retinitis pigmentosa (RP).

METHODS

Cross-sectional case series.

METHODS

Seventy-five eyes of 75 patients with RP.

METHODS

We examined the eyes of the RP patients using the Optos 200Tx imaging system (Optos PLC, Dunfermline, United Kingdom) and identified abnormal FAF patterns such as ring hyperautofluorescence and patchy hypoautofluorescent areas. Patients with hyperautofluorescent rings or foveal hyperautofluorescence were compared with those without such findings. We determined the percentage area occupied by the FAF abnormalities within a defined region of the eye and examined the relationship between the percentage area of these abnormalities and the visual field area. Moreover, we categorized the patients into 3 different groups based on the presence of a patchy hypoautofluorescent lesion larger than 1 disc diameter: Group A consisted of those with patchy lesions smaller than 1 disc diameter, group B consisted of those with patchy lesions larger than 1 disc diameter but present in only 1 quadrant, and group C consisted of those with patchy lesions larger than 1 disc diameter and present in more than 1 quadrant. In addition, various clinical characteristics were compared among these 3 groups.

METHODS

Predicting the visual field size and duration of the disease in RP patients based on FAF patterns.

RESULTS

Patients without hyperautofluorescent rings or foveal hyperautofluorescence had better visual acuity or mean deviation measured with a Humphrey perimeter. The total area of the abnormal FAF image correlated with the visual field area measured with a Goldmann perimeter (R = -0.64, P<0.001). The individuals with the large patchy hypofluorescent areas (i.e., larger than 1 disc diameter) were older than those with small patchy hypofluorescent areas (group A vs. groups B and C, P = 0.002 and P<0.001, respectively) and had experienced the symptoms for longer durations (group A vs. groups B and C, P<0.05 and P<0.001, respectively).

CONCLUSIONS

We can estimate the visual field in patients with RP using the objective measurements from wide-field FAF. The presence of patchy hypofluorescent lesions can be used an indicator of the duration of RP.

BACKGROUND

The author(s) have no proprietary or commercial interest in any materials discussed in this article.

This report presents 2-year outcome data from an outpatient continuing care study in which cocaine-dependent patients (N = 132) were randomly assigned to either standard group counseling (STND) or individualized relapse prevention (RP). Data on cocaine outcomes during the 6-month treatment phase of the study were presented in an earlier report (J. R. McKay, A. I. Alterman, J. S. Cacciola, M. R. Rutherford, & C. P. O'Brien, 1997). In the present report, a continuing care condition main effect was obtained on only 1 of 8 outcome variables examined. However, patients who endorsed a goal of absolute abstinence on entering continuing care had better cocaine use outcomes in RP than in STND, whereas the opposite was the case for those with less stringent abstinence goals. In addition, patients with current cocaine or alcohol dependence on entering continuing care who received RP had better cocaine use outcomes in Months 1-6 and better alcohol use outcomes in Months 13-24 than those in STND.

PGE(2) and PGI(2) stimulate renin secretion and cAMP accumulation in juxtaglomerular granular (JG) cells. We addressed, at the single-cell level, the receptor subtypes and intracellular transduction mechanisms involved. Patch clamp was used to determine cell capacitance (C(m)), current, and membrane voltage in response to PGE(2), EP2 and EP4 receptor agonists, and an IP receptor agonist. PGE(2) (0.1 micromol/l) increased C(m) significantly, and the increase was abolished by intracellular application of the protein kinase A antagonist Rp-8-CPT-cAMPS. EP2-selective ligands butaprost (1 micromol/l), AE1-259-01 (1 nmol/l), EP4-selective agonist AE1-329 (1 nmol/l), and IP agonist iloprost (1 micromol/l) significantly increased C(m) mediated by PKA. The EP4 antagonist AE3-208 (10 nmol/l) blocked the effect of EP4 agonist but did not alter the response to PGE(2). Application of both EP4 antagonist and EP2-antagonist AH-6809 abolished the effects of PGE(2) on C(m) and current. EP2 and EP4 ligands stimulated cAMP formation in JG cells. PGE(2) rapidly stimulated renin secretion from superfused JG cells and diminished the membrane-adjacent granule pool as determined by confocal microscopy. The membrane potential hyperpolarized significantly after PGE(2), butaprost, AE1-329 and AE1-259 and outward current was augmented in a PKA-dependent fashion. PGE(2)-stimulated outward current, but not C(m) change, was abolished by the BK(Ca) channel inhibitor iberiotoxin (300 nmol/l). EP2 and EP4 mRNA was detected in sampled JG cells, and the preglomerular and glomerular vasculature was immunopositive for EP4. Thus IP, EP2, and EP4 receptors are associated with JG cells, and their activation leads to rapid PKA-mediated exocytotic fusion and release of renin granules.

OBJECTIVE

The factors preventing the translation of preclinical findings supporting the clinical development mTOR-targeted therapy in pancreatic cancer therapy remain undetermined. Stromal cell.derived factor 1α (SDF-1α)-CXCR4 signaling was examined as a representative microenvironmental factor able to promote mTOR-targeted therapy resistance in pancreatic cancer.

METHODS

Primary pancreas explant xenografts and in vitro experiments were used to perform pharmacodynamic analyses of SDF-1α-CXCR4 regulation of the mTOR pathway. Combinatorial effects of CXCR4, EGFR, and mTOR pharmacologic inhibition were evaluated in temsirolimus-resistant and -sensitive xenografts. Intratumoral gene and protein expressions of mTOR pathway effectors cyclin D1, c-Myc, and VEGF were evaluated.

RESULTS

Baseline intratumoral SDF-1α gene expression correlated with temsirolimus resistance in explant models. SDF-1α stimulation of pancreatic cells resulted in CXCR4-mediated PI3-kinase-dependent S6-RP phosphorylation (pS6-RP) on exposure to temsirolimus. Combinatorial therapy with AMD3465 (CXCR4 small-molecule inhibitor) and temsirolimus resulted in effective tumor growth inhibition to overcome temsirolimus resistance. In contrast, SDF-1α exposure induced a temsirolimus-resistant phenotype in temsirolimus-sensitive explants. AMD3465 inhibited CXCR4-mediated intratumoral S6-RP phosphorylation and cyclin D and c-myc gene expression. Next, CXCR4 promoted intratumoral EGFR expression in association with temsirolimus resistance. Treatment with AMD3465, temsirolimus- and erlotinib-mediated tumor growth inhibition to overcome temsirolimus resistance in the explant model. Lastly, SDF-1α-CXCR4 signaling increased intratumoral VEGF gene and protein expression.

CONCLUSIONS

SDF-1α-CXCR4 signaling represents a microenvironmental factor that can maintain mTOR pathway fidelity to promote resistance to mTOR-targeted therapy in pancreatic cancer by a variety of mechanisms such as recruitment of EGFR signaling and angiogenesis.

BACKGROUND

Lipid transfer proteins (LTPs) are clinically relevant plant food panallergens and have been proposed as ideal tools to study true food allergy. Pru p 3, the major peach allergen in the Mediterranean area, is among the best-characterized allergenic members of the LTP family. Its diagnostic value for Rosaceae fruit allergy has been demonstrated both in vivo and in vitro.

OBJECTIVE

We sought to locate major IgE-binding epitopes of Pru p 3.

METHODS

A serum pool and individual sera from patients with peach allergy and positive skin prick test results to Pru p 3 were used. Three-dimensional modeling was achieved by using experimentally available structures of Pru p 3 homologues as templates. Theoretical prediction of potential IgE-binding regions was performed by selecting specific residues on the molecular surface displaying prominent electrostatic potential features. Point mutants of Pru p 3 were constructed by standard polymerase chain reaction procedures with the appropriate primers. Mutants were expressed in P pastoris by means of the pPIC 9 vector and purified from the corresponding supernatants by gel-filtration chromatography followed by RP-HPLC. IgE binding by Pru p 3 mutants was tested by immunodetection and quantified by ELISA and ELISA inhibition assays. Synthetic peptides (10 mer; 5 amino acids overlapping) covering the full Pru p 3 sequence were used to detect IgE epitopes by (125)I-anti-IgE immunodetection.

RESULTS

Pru p 3 showed a 3-dimensional structure comprising 4 alpha-helixes and a nonstructured C-terminal coil (residues 73 to 91). Regions around amino acids in positions 23 to 36, 39 to 44, and 80 to 91, particularly residues R39, T40, and R44, K80 and K91, were predicted as potential antibody recognition sites according to their relevant surface and electrostatic properties. Point mutants K80A and K91A were found to have an IgE-binding capacity similar to that of recombinant Pru p 3, but the triple mutant R39A/T40A/R44A showed a substantial decrease (approximately 5 times) of IgE binding. IgE immunodetection of synthetic peptides led to the identification of Pru p 3 sequence regions 11 to 25, 31 to 45, and 71 to 80 as major IgE epitopes.

CONCLUSIONS

Main IgE-binding regions of the Pru p 3 amino acid sequence were identified. The three major ones comprised the end of an alpha-helix and some residues of the following interhelix loop. These data can help to search for Pru p 3 hypoallergenic forms.

Autosomal recessive retinitis pigmentosa (arRP) is a clinically and genetically heterogeneous retinal disease that causes blindness. Our purpose was to identify the causal gene, describe the phenotype and delineate the mutation spectrum in a consanguineous Quebec arRP family. We performed Arrayed Primer Extension (APEX) technology to exclude ∼500 arRP mutations in ∼20 genes. Homozygosity mapping [single nucleotide polymorphism (SNP) genotyping] identified 10 novel significant homozygous regions. We performed next generation sequencing and whole exome capture. Sanger sequencing provided cosegregation. We screened another 150 retinitis pigmentosa (RP) and 200 patients with Senior-Løken Syndrome (SLS). We identified a novel missense mutation in WDR19, c.2129T>C which lead to a p.Leu710Ser. We found the same mutation in a second Quebec arRP family. Interestingly, two of seven affected members of the original family developed 'sub-clinical' renal cysts. We hypothesized that more severe WDR19 mutations may lead to severe ciliopathies and found seven WDR19 mutations in five SLS families. We identified a new gene for both arRP and SLS. WDR19 is a ciliary protein associated with the intraflagellar transport machinery. We are currently investigating the full extent of the mutation spectrum. Our findings are crucial in expanding the understanding of childhood blindness and identifying new genes.

OBJECTIVE

Salt restriction has been reported to increase white adipose tissue (WAT) mass in rodents. The objective of this study was to investigate the effect of different sodium content diets on the lipogenic and lipolytic activities of WAT.

METHODS

Male Wistar rats were fed on normal-sodium (NS; 0.5% Na(+)), high-sodium (HS; 3.12% Na(+)), or low-sodium (LS; 0.06% Na(+)) diets for 3, 6, and 9 weeks after weaning. Blood pressure (BP) was measured using a computerized tail-cuff system. At the end of each period, rats were killed and blood samples were collected for leptin determinations. The WAT from abdominal and inguinal subcutaneous (SC), periepididymal (PE) and retroperitoneal (RP) depots was weighed and processed for adipocyte isolation, rate measurement of lipolysis and d-[U-(14)C]-glucose incorporation into lipids, glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme activity evaluation, and determination of G6PDH and leptin mRNA expression.

RESULTS

After 6 weeks, HS diet significantly increased BP; SC, PE, and RP WAT masses; PE adipocyte size; plasma leptin concentration; G6PDH activity in SC WAT; and PE depots and malic activity only in SC WAT. The leptin levels correlated positively with WAT masses and adipocyte size. An increase in the basal and isoproterenol-stimulated lipolysis and in the ability to incorporate glucose into lipids was observed in isolated adipocytes from HS rats.

CONCLUSIONS

HS diet induced higher adiposity characterized by high plasma leptin concentration and adipocyte hypertrophy, probably due to an increased lipogenic capacity of WAT.

The mechanisms by which oxytocin (OT) stimulates extracellular signal-regulated kinase 1/2 (ERK1/2) are only partially understood. OT receptor (OTR) signals predominantly through Galpha(q), but ERK1/2 phosphorylation (ERK1/2-P) in PHM1 myometrial cells was not eliminated by inhibition of downstream effectors such as phospholipase C or protein kinase C. Inconsistent with a Galpha(i)-coupled response, pertussis toxin inhibition of OT-induced ERK1/2-P was reversed by the protein kinase A inhibitors Rp-cAMPS and KT5720. Consistent with an inhibitory role for protein kinase A, pertussis toxin pretreatment raised cellular cAMP and 8-(4-chlorophenylthio)-cAMP inhibited OT-induced ERK1/2-P. Attenuation of the OT response by the Gbetagamma scavenger carboxyl terminus of the beta-adrenergic receptor kinase implicated a Gbetagamma-mediated pathway. In both COSM6 cells overexpressing OTR (OTR-COSM6) and in PHM1 cells, the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 markedly reduced OT-induced ERK1/2-P, whereas the platelet-derived growth factor receptor tyrosine kinase inhibitor AG1296 had no effect. Furthermore, OT increased EGFR tyrosine phosphorylation in OTR-COSM6 cells, which was inhibited by AG1478 or EGTA plus thapsigargin pretreatment. AG1478 did not affect inositol 1,4,5-triphosphate production by OT or protein kinase C-stimulated ERK1/2-P but completely blocked ionomycin-induced ERK1/2-P and EGFR tyrosine phosphorylation. In both OTR-COSM6 and PHM1 cells, EGTA reduced OT-stimulated ERK1/2-P; no ERK1/2-P was observed when intracellular calcium increases were blocked by pretreatment with thapsigargin plus EGTA. These data are consistent with activation of a Gbetagamma-mediated pathway as a consequence of Galpha(q) activation in myometrium and OTR-COSM6 cells that results in increased ERK1/2-P. This pathway involves both EGFR activation and an influence of calcium.

Prostaglandin E2 (PGE2) is an important local regulator in bone. The present study was performed to investigate the effect of PGE2 on osteoclast-like cell formation and bone-resorbing activity of mature osteoclasts in the presence or absence of osteoblasts, PGE2 (10(-8) to 10(-6) M) significantly stimulated osteoclast-like cell formation in osteoblast-containing mouse bone cell cultures, although it did not affect osteoclast-like cell formation from hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor in osteoblast-free mouse spleen cell cultures. The conditioned medium from osteoblastic UMR-106 cells pretreated with PGE2 (10(-8) and 10(-6) M) significantly stimulated osteoclast-like cell formation from hemopoietic blast cells. PGE2 also significantly stimulated the bone-resorbing activity of mature osteoclasts in osteoblast-containing mouse bone cell cultures. In contrast, PGE2 significantly inhibited the bone-resorbing activity and osteopontin mRNA expression in isolated rabbit osteoclasts. Rp-cAMPS, a direct protein kinase (PKA) antagonist, significantly inhibited PGE2-stimulated osteoclast-like cell formation and the bone-resorbing activity of mature osteoclasts, although protein kinase C inhibitors, dantrolene (an inhibitor of calcium release from the intracellular calcium pool) and voltage-dependent calcium channel blockers did not affect PGE2-stimulated osteoclast-like cell formation. In conclusion, PGE2 stimulated osteoclast-like cell formation and bone-resorbing activity in mouse bone cell cultures presumably through osteoblasts. The activation of PKA is linked to PGE2-stimulated osteoclast-like cell formation and bone-resorbing activity.

OBJECTIVE

The Suspicion Index (SI) screening tool was developed to identify patients suspected of having Niemann-Pick disease type C (NP-C). The SI provides a risk prediction score (RPS) based on NP-C manifestations within and across domains (visceral, neurological, and psychiatric). The aim of these subanalyses was to further examine the discriminatory power of the SI by age and manifestation-associations by NP-C suspicion-level and leading manifestations.

METHODS

The original retrospectively collected data were split into three patient age groups, where NP-C-positive cases were >16 years (n = 30), 4-16 years (n = 18), and <4 years (n = 23), and patients' RPS were analyzed by logistic regression. Co-occurrence of manifestations within groups of suspicion level (low, medium, high) and leading manifestations (presence/absence of ataxia, cognitive decline, psychosis, and splenomegaly) were analyzed descriptively.

RESULTS

NP-C-positive cases versus controls showed strong discriminatory power of RPS. Area under the receiver operating characteristic curve was 0.964 (>16 years) and 0.981 (4-16 years) but weaker 0.562 for infants (<4 years). Patients with RPS <70 were characterized by a lack of psychiatric manifestations and low levels of neurological involvement, suggestive of a preneurological phase of the disease. In patients >4 years, prominent leading manifestation-associations were ataxia with dystonia, dysarthria/dysphagia, and cognitive decline. Psychosis was associated with dysarthria/dysphagia but also with cognitive decline and treatment-resistant psychiatric symptoms.

CONCLUSIONS

The SI tool maintains strong discriminatory power in patients >4 years but is not as useful for infants <4 years. The SI is also informative regarding the association and co-occurrence of manifestations in patients with NP-C.

The effects of anoxia on intracellular pH (pH(i)) were examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H(+)-sensitive fluorophore, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. During perfusion with HCO/CO(2)- or HEPES-buffered media (pH 7.35) at 37 degrees C, 5- or 10-min anoxic insults were typified by an intracellular acidification on the induction of anoxia, a subsequent rise in pH(i) in the continued absence of O(2), and a further internal alkalinization on the return to normoxia. The steady-state pH(i) changes were not consequent on changes in [Ca(2+)](i) and, examined in the presence of HCO, were not significantly affected by (DIDS). In the absence of HCO, the magnitude of the postanoxic alkalinization was attenuated when external Na(+) was reduced by substitution with N-methyl-D-glucamine (NMDG(+)), but not Li(+), suggesting that increased Na(+)/H(+) exchange activity contributes to this phase of the pH(i) response. In contrast, 100-500 microM Zn(2+), a known blocker of H(+)-conductive pathways, reduced the magnitudes of the internal alkalinizations that occurred both during and following anoxia. The effects of NMDG(+)-substituted medium and Zn(2+) to reduce the increase in pH(i) that occurred after anoxia were additive. Consistent with the steady-state pH(i) changes, rates of pH(i) recovery from internal acid loads imposed immediately after anoxia were increased, and the application of Zn(2+) and/or perfusion with NMDG(+)-substituted medium slowed pH(i) recovery. Reducing extracellular pH from 7.35 to 6.60, or reducing ambient temperature from 37 degrees C to room temperature, also attenuated the increases in steady-state pH(i) observed during and after anoxia and reduced rates of pH(i) recovery from acid loads imposed in the immediate postanoxic period. Finally, inhibition of the cAMP/protein kinase A second-messenger system reduced the magnitude of the rise in pH(i) after anoxia in a manner that was dependent on external Na(+); conversely, activation of the system with isoproterenol increased the postanoxic alkalinization, an effect that was attenuated by pretreatment with propranolol, Rp-cAMPS, or when NMDG(+) (but not Li(+)) was employed as an external Na(+) substitute. The results suggest that a Zn(2+)-sensitive acid efflux mechanism, possibly a H(+)-conductive pathway activated by membrane depolarization, contributes to the internal alkalinization observed during anoxia in adult rat CA1 neurons. The rise in pH(i) after anoxia reflects acid extrusion via the H(+)-conductive pathway and also Na(+)/H(+) exchange, activation of the latter being mediated, at least in part, through a cAMP-dependent signaling pathway.

BACKGROUND

Substantial discrepancies exist in the type of sleep studies performed to diagnose pediatric obstructive sleep apnea (OSA) in different countries. Respiratory polygraphic (RP) recordings are primarily performed in sleep laboratories in Europe, whereas polysomnography (PSG) constitutes the majority in the US and Australia. Home RP show consistent apnea-hypopnea index (AHI) underscoring, primarily because the total recording time is used as the denominator when calculating the AHI compared to total sleep time (TST). However, laboratory-based RP are less likely affected, since the presence of sleep technicians and video monitoring may enable more accurate TST estimates. We therefore examined differences in AHI in PSG and in-lab RP, and whether RP-based AHI may impact clinical decision making.

METHODS

Of all the children assessed for possible OSA who underwent PSG evaluation, 100 were identified and divided into 4 groups: (A) those with AHI < 1/h TST (n = 20), (B) 1 ≤ AHI < 5/h TST (n = 40), (C) 5 ≤ AHI < 10/h TST (n = 20), and (D) AHI ≥ 10/h TST (n = 20). Electroencephalography, electrooculography, and electromyography channels were deleted from the original unscored recordings to transform them into RP, and then rescored in random sequence. AHI-RP were compared to AHI-PSG, and therapeutic decisions based on AHI-RP and AHI-PSG were formulated and analyzed using clinical details derived from the patient's clinic letter.

RESULTS

Bland Altman analysis showed that in lab RP underestimated the AHI despite more accurate estimates of TST. This underestimation was due to missed hypopneas causing arousals without desaturation. Basing the therapeutic management decision on RP instead of PSG results changed the clinical management in 23% of all patients. The clinical management for patients in groups A and D was unaffected. However, 27.5% of patients in group B would have been given no treatment, as they would be diagnosed as having no OSA (AHI < 1/h TST) when they should have received a trial of anti-inflammatory therapy or been referred for ear, nose, and throat (ENT) review. Sixty percent of patients in group C would have received either a trial of medical treatment to treat mild OSA or no treatment, instead of referral to ENT services or commencement of continuous positive airway pressure.

CONCLUSIONS

Apnea-hypopnea index (AHI) is underestimated in respiratory polygraphy (RP), and the disparity in AHI-RP and AHI-polysomnography can significantly affect clinical management decisions, particularly in children with mild and moderate obstructive sleep apnea (1 < AHI < 10/h total sleep time).

Cardiovascular morbidity and mortality is increased in patients with chronic obstructive pulmonary disease (COPD). Reduced levels of circulating endothelial progenitor cells (EPCs) are associated with increased risk of death in patients with stable coronary artery disease (CAD). Likewise, during acute events of CAD, the number of circulating EPCs increases under the influence of vascular endothelial growth factor (VEGF) and systemic inflammation. Abnormal levels of circulating EPCs have been reported in patients with COPD. However, the response of EPCs to episodes of exacerbation of the disease (ECOPD) has not been investigated yet. We hypothesized that similar to what occurs during acute events of CAD, levels of circulating EPCs would increase during ECOPD. We compared levels of circulating EPCs (assessed by the % of CD34(+)KDR(+) cells determined by flow cytometry) in patients hospitalized because of ECOPD (n = 35; 65 +/- 9 years [mean +/- SD]; FEV(1) = 46 +/- 15% predicted), patients with stable COPD (n = 44; 68 +/- 8 years; FEV(1) = 49 +/- 17% predicted), smokers with normal lung function (n = 10; 60 +/- 9 years), and healthy never smokers (n = 10; 62 +/- 4 years). To investigate potential mechanisms of EPC regulation, we assessed both VEGF and high-sensitivity C-reactive protein (hsC-RP) in plasma. Our results show that EPC levels were higher (p < 0.05) in patients with ECOPD (1.46 +/- 1.63%) than in those with stable disease (0.68 +/- 0.83%), healthy smokers (0.65 +/- 1.11%), and healthy never smokers (1.05 +/- 1.36%). The percentage of circulating EPCs was positively related to VEGF plasma levels during ECOPD (r = 0.51, p = 0.003). In a subset of 12 patients who could be studied during both ECOPD and clinical stability, the EPCs levels increased during ECOPD. We conclude that EPC levels are increased during ECOPD, likely in relation to VEGF upregulation.

The moderately thermophilic bacterium Acidithiobacillus caldus is found in bacterial populations in many bioleaching operations throughout the world. This bacterium oxidizes elemental sulfur and other reduced inorganic sulfur compounds as the sole source of energy. The purpose of this study was to purify and characterize the tetrathionate hydrolase of A. caldus. The enzyme was purified 16.7-fold by one step chromatography using a SP Sepharose column. The purified enzyme resolved into a single band in 10% polyacrylamide gel, both under denaturing and native conditions. Its homogeneity was confirmed by N-terminal amino acid sequencing. Tetrathionate hydrolase was shown to be a homodimer with a molecular mass of 103 kDa (composed from two 52 kDa monomers). The purified enzyme had optimum activity at pH 3.0 and 40 degrees C and an isoelectric point of 9.8. The periplasmic localization of the enzyme was determined by differential fractionation of A. caldus cells. Detected products of the tetrathionate hydrolase reaction were thiosulfate and pentathionate as confirmed by RP-HPLC analysis. The activity of the purified enzyme was drastically enhanced by divalent metal ions.

Bardet-Biedl syndrome (BBS) is a multisystem disorder caused by ciliary defects. To date, mutations in 15 genes have been associated with the disease and BBS1 is most frequently affected in patients with BBS. The use of homozygosity mapping in a large consanguineous family allowed us to identify the splice donor site (SD) mutation c.479G>A in exon 5 of BBS1. Clinically affected family members show symptoms of retinitis pigmentosa (RP) but lack other primary features that would clearly support the diagnosis of BBS. In agreement with this exceptionally mild BBS1-associated phenotype, we did not detect obvious ciliary defects in patient-derived cells. SDs are bound by the U1 small nuclear RNA (U1), a process that initiates exon recognition during splicing. The mutation described herein interferes with U1 binding and induces aberrant splicing of BBS1. For a gene therapeutic approach, we have adapted the sequence of U1 to increase its complementarity to the mutated SD. Lentiviral treatment of patient-derived fibroblasts with the adapted U1 partially corrected aberrant splicing of endogenously expressed BBS1 transcripts. This therapeutic effect was dose-dependent. Our results show that the adaptation of U1 can correct pathogenic effects of splice donor site mutations and suggest a high potential for gene therapy.