We found that engagement of beta2 integrins on human neutrophils increased the levels of GTP-bound Rap1 and Rap2. Also, the activation of Rap1 was blocked by PP1, SU6656, LY294002, GF109203X, or BAPTA-AM, which indicates that the downstream signaling events in Rap1 activation involve Src tyrosine kinases, phosphoinositide 3-kinase, protein kinase C, and release of calcium. Surprisingly, the beta2 integrin-induced activation of Rap2 was not regulated by any of the signaling pathways mentioned above. However, we identified nitric oxide as the signaling molecule involved in beta2 integrin-induced activation of Rap1 and Rap2. This was illustrated by the fact that engagement of beta2 integrins increased the production of nitrite, a stable end-product of nitric oxide. Furthermore, pretreatment of neutrophils with Nomega-monomethyl-L-arginine, or 1400W, which are inhibitors of inducible nitric-oxide synthase, blocked beta2 integrin-induced activation of Rap1 and Rap2. Similarly, Rp-8pCPT-cGMPS, an inhibitor of cGMP-dependent serine/threonine kinases, also blunted the beta2 integrin-induced activation of Rap GTPases. Also nitric oxide production and its downstream activation of cGMP-dependent serine/threonine kinases were essential for proper neutrophil adhesion by beta2 integrins. Thus, we made the novel findings that beta2 integrin engagement on human neutrophils triggers production of nitric oxide and its downstream signaling is essential for activation of Rap GTPases and neutrophil adhesion.

We have previously reported that human eosinophil granule major basic protein and synthetic cationic proteins such as poly-L-arginine and poly-L-lysine, can increase airway responsiveness in vivo. In the present study, we have investigated whether activation of sensory C-fibers is important in this phenomenon. Dose-response curves to methacholine were constructed before and 1 h after intratracheal instillation of poly-L-lysine in anaesthetized spontaneously breathing rats, and the concentration of methacholine required to induce a doubling in total lung resistance was calculated. Poly-L-lysine induced a fivefold increase in airway responsiveness, which was inhibited by neonatal capsaicin treatment and potentiated by phosphoramidon (100 micrograms/ml). Furthermore, pretreatment with either CP, 96-345, or RP-67580 two selective NK-1 receptor antagonists inhibited poly-L-lysine-induced airway hyperresponsiveness and plasma protein extravasation. In vitro, cationic proteins stimulated the release of calcitonin gene-related peptide-like immunoreactivity from perfused slices of the main bronchi. Our results demonstrate that cationic proteins can activate sensory C-fibers in the airways, an effect which is important in the subsequent development of airway hyperresponsiveness and plasma protein extravasation. Cationic proteins may therefore function as a link between inflammatory cell accumulation and sensory nerve activation.

Chemical immobilization, an in situ remediation method where inexpensive chemicals are used to reduce contaminant solubility in contaminated soil, has gained attention. We investigated the effectiveness of lime-stabilized biosolid (LSB), N-Viro Soil (NV), rock phosphate (RP), and anaerobic biosolid (AB) to reduce extractability and plant and gastrointestinal (GI) bioavailability in three Cd-, Pb-, and Zn-contaminated soils from smelter sites. Treated (100 g kg(-1) soil) and control soils were incubated at 27 degrees C and -0.033 MPa (0.33 bar) water content for 90 d. The effect of soil treatment on metal extractability was evaluated by sequential extraction, on phytoavailability by a lettuce bioassay (Lactuca sativa L.), on human GI availability of Pb from soil ingestion by the Physiologically Based Extraction Test. The largest reductions in metal extractability and phytoavailability were from alkaline organic treatments (LSB and NV). Phytotoxic Zn [1188 mg Zn kg(-1) extracted with 0.5 M Ca(NO3)2] in Blackwell soil (disturbed soil) was reduced by LSB, NV, and RP to 166, 25, and 784 mg Zn kg(-1), respectively. Rock phosphate was the only treatment that reduced GI-available Pb in both gastric and intestinal solutions, 23 and 92%, respectively. Alkaline organic treatments (LSB, NV) decreases Cd transmission through the food chain pathway, whereas rock phosphate decreases risk from exposure to Pb via the soil ingestion pathway. Alkaline organic treatments can reduce human exposure to Cd and Pb by reducing Zn phytotoxicity and revegetation of contaminated sites.

Head-to-tail cyclic peptides of 4-10 residues consisting of alternating hydrophilic (Lys) and hydrophobic (Leu and Phe) amino acids were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Xanthomonas vesicatoria and Pseudomonas syringae. The antibacterial activity, evaluated as the minimal inhibitory concentration (MIC), the cytotoxicity against human red blood cells and stability towards protease degradation were determined. The influence of cyclization, ring size, and replacement of l-Phe with d-Phe on antibacterial and hemolytic activities was studied and correlated with the degree of structuring and hydrophobicity. Our results showed that linear peptides were inactive against the three bacteria tested. Cyclic peptides were active only toward X. vesicatoria and P. syringae, being c(KLKLKFKLKQ) (BPC10L) the most active peptide with MIC values of 6.25 and 12.5 microM, respectively. The improved antibacterial activity of cyclic peptides compared to their linear counterparts was associated to an increase of the hydrophobicity, represented as RP-HPLC retention time (t(R)), and secondary structure content which are related to an enhanced amphipathicity. A decrease of antibacterial and hemolytic activities was observed when a d-Phe was introduced into the cyclic sequences, which was attributed to their low amphipathicity as shown by their low secondary structure content and low t(R). The small size, simple structure, bactericidal effect, and stability to protease degradation of the best peptides make them potential candidates for the development of effective antibacterial agents for use in plant protection.

Postprandial chylomicron remnant clearance was studied in six patients with familial combined hyperlipidemia (FCH) and seven control subjects by using an oral retinyl palmitate (RP) fat-loading test. The chylomicron remnant clearance (Sf < 1,000 fraction), expressed as the area under the RP curve (AUC-RP), was delayed in FCH subjects (65.05 +/- 12.84 hours x [mg/L]) compared with control subjects (25.1 +/- 5.4 hours x [mg/L]; p = 0.01). Postprandial lipoprotein particle size and composition in the Sf>> 1,000 fraction were different between FCH and control subjects as analyzed by molecular-sieve chromatography. Fasting high density lipoprotein cholesterol was lower in FCH patients (0.54 +/- 0.09 mmol/L) than in control subjects (0.89 +/- 0.05 mmol/L; p < 0.01). Mean plasma postheparin lipoprotein lipase and hepatic lipase activities were similar between FCH patients (94 +/- 25 and 427 +/- 57 milliunits/mL, respectively) and control subjects (126 +/- 16 and 362 +/- 33 milliunits/mL, respectively). In FCH, a 54% reduction (p < 0.05) of plasma triglycerides to 2.63 +/- 0.41 mmol/L by drug treatment resulted in an enhanced, but not normalized, clearance of chylomicron remnants (39.4 +/- 6.0 hours x [mg/L]). Univariate regression analysis revealed that in FCH subjects the changes in fasting plasma apolipoprotein C-III concentrations after therapy were significantly associated with the changes in chylomicron remnant AUC-RP (r = 0.87; p = 0.02). Delayed elimination of atherogenic chylomicron remnants may contribute to the increased risk of premature atherosclerosis in FCH.

To examine whether adrenomedullin (AM), a novel vasodilator peptide, acts as a growth modulator in the vasculature, the effects of AM on protein tyrosine phosphorylation, mitogen-activated protein kinase (MAPK) activation, protooncogene expression, DNA synthesis, and cell proliferation were studied in cultured rat vascular smooth muscle cells (VSMC). AM and calcitonin gene-related peptide (CGRP), although weaker than AM, stimulated DNA synthesis and cell proliferation of quiescent VSMC, whose effects were inhibited by a CGRP receptor antagonist, CGRP-(8-37). AM induced a rapid increase in MAPK activity, followed by the expression of the immediate early protooncogene c-fos. AM-induced MAPK activation and cell proliferation were completely blocked by protein tyrosine kinase inhibitors (genistein and ST638). Moreover, AM rapidly induced tyrosine phosphorylation of several proteins (approximately 120, approximately 90, and approximately 50 kDa) and transiently increased association of a tyrosine-phosphorylated protein (approximately 120 kDa) and Shc with the glutathione-S-transferase-Grb2 fusion protein. A MAPK kinase inhibitor (PD98059) also reduced the AM-induced MAPK activation, c-fos messenger RNA expression, and cell proliferation. Although AM has been shown to induce vasodilation through cAMP production in VSMC, a cAMP antagonist (Rp-cAMP-thionate) and a protein kinase A inhibitor (KT5720) failed to block AM-induced MAPK activation and DNA synthesis. Moreover, 8-bromo-cAMP and forskolin did not affect the MAPK activity. AM had no effect on either the intracellular Ca2+ concentration or inositol 1,4,5-trisphosphate formation. In addition, a protein kinase C inhibitor (GF109203X) did not inhibit the AM-induced MAPK activation. These data suggest that in addition to its vasodilatory effect through the cAMP-dependent pathway, AM exerts its mitogenic activity via protein tyrosine kinase-mediated MAPK activation in quiescent rat VSMC.

Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca2+ release and Ca2+ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca2+ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP3), cyclic AMP, and the intracellular Ca2+ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP3 production, the Ca2+ release induced by PA-CAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP3 channels. The potencies of the peptides to cause Ca2+ release in the presence of cinnarizine were similar. The Ca2+ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca2+ release was unaffected by Rp-adenosine 3',5'-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2+ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca2+ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca2+ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca2+ in adrenal chromaffin cells.

BACKGROUND

The genetic features that facilitate Campylobacter jejuni's adaptation to a wide range of environments are not completely defined. However, whole genome expression studies showed that respiratory proteins (RPs) were differentially expressed under varying conditions and stresses, suggesting further unidentified roles for RPs in C. jejuni's adaptation. Therefore, our objectives were to characterize the contributions of selected RPs to C. jejuni's i- key survival phenotypes under different temperature (37°C vs. 42°C) and oxygen (microaerobic, ambient, and oxygen-limited/anaerobic) conditions and ii- its interactions with intestinal epithelial cells from disparate hosts (human vs. chickens).

RESULTS

C. jejuni mutant strains with individual deletions that targeted five RPs; nitrate reductase (ΔnapA), nitrite reductase (ΔnrfA), formate dehydrogenase (ΔfdhA), hydrogenase (ΔhydB), and methylmenaquinol:fumarate reductase (ΔmfrA) were used in this study. We show that only the ΔfdhA exhibited a decrease in motility; however, incubation at 42°C significantly reduced the deficiency in the ΔfdhA's motility as compared to 37°C. Under all tested conditions, the ΔmfrA showed a decreased susceptibility to hydrogen peroxide (H(2)O(2)), while the ΔnapA and the ΔfdhA showed significantly increased susceptibility to the oxidant as compared to the wildtype. Further, the susceptibility of the ΔnapA to H(2)O(2) was significantly more pronounced at 37°C. The biofilm formation capability of individual RP mutants varied as compared to the wildtype. However, the impact of the deletion of certain RPs affected biofilm formation in a manner that was dependent on temperature and/or oxygen concentration. For example, the ΔmfrA displayed significantly deficient and increased biofilm formation under microaerobic conditions at 37°C and 42°C, respectively. However, under anaerobic conditions, the ΔmfrA was only significantly impaired in biofilm formation at 42°C. Additionally, the RPs mutants showed differential ability for infecting and surviving in human intestinal cell lines (INT-407) and primary chicken intestinal epithelial cells, respectively. Notably, the ΔfdhA and the ΔhydB were deficient in interacting with both cell types, while the ΔmfrA displayed impairments only in adherence to and invasion of INT-407. Scanning electron microscopy showed that the ΔhydB and the ΔfdhA exhibited filamentous and bulging (almost spherical) cell shapes, respectively, which might be indicative of defects in cell division.

CONCLUSIONS

We conclude that the RPs contribute to C. jejuni's motility, H(2)O(2) resistance, biofilm formation, and in vitro interactions with hosts' intestinal cells. Further, the impact of certain RPs varied in response to incubation temperature and/or oxygen concentration. Therefore, RPs may facilitate the prevalence of C. jejuni in a variety of niches, contributing to the pathogen's remarkable potential for adaptation.

The pathogenic fungus Cryptococcus neoformans must overcome multiple stressors to cause disease in its human host. In this study, we report that C. neoformans rapidly and transiently repressed ribosomal protein (RP) transcripts during a transition from 30°C to host temperature. This repression was accompanied by accelerated mRNA degradation mediated by the major deadenylase, Ccr4, and influenced by the dissociable RNA polymerase II subunit, Rpb4. Destabilization and deadenylation of RP transcripts were impaired in an rpb4Δ mutant, suggesting that Rpb4 may be involved in host temperature-induced Ccr4-mediated decay. Accelerated decay of ER stress transcripts 1 h following a shift to host temperature was also impaired in the rpb4Δ mutant. In response to host temperature, Rpb4 moved from the nucleus to the cytoplasm, supporting a role for Rpb4 in coupling transcription and degradation. The PKH signalling pathway was implicated as a regulator of accelerated degradation of the RP transcripts, but not of the ER stress transcripts, revealing a further level of specificity. When transcription and degradation were uncoupled by deletion of Rpb4, growth at host temperature was impaired and virulence was attenuated. These data suggest that mRNA synthesis and decay are coupled in C. neoformans via Rpb4, and this tight coordination promotes host-temperature adaptation and pathogenicity.

We report the comparative proteomic characterization of the venoms of Bothrops atrox, B. barnetti and B. pictus. The venoms were subjected to RP-HPLC and the resulting fractions analyzed by SDS-PAGE. The proteins were cut from the gels, digested with trypsin and identified via peptide mass fingerprint and manual sequencing of selected peptides by MALDI-TOF/TOF mass spectrometry. Around 20-25 proteins were identified belonging to only 6-7 protein families. Metalloproteinases of the classes P-I and P-III were the most abundant proteins in all venoms (58-74% based on peak area A214 nm), followed by phospholipases-A(2) (6.4-14%), disintegrins (3.2-9%) and serine proteinases (7-11%), and some of these proteins occurred in several isoforms. In contrast cysteine-rich secretory proteins and L-amino acid oxidases appeared only as single isoforms and were found only in B. atrox and B. barnetti. C-type lectins were also detected in all venoms but at low levels (~ 5%). Furthermore, the venoms contain variable numbers of peptides (<3 kDa) and non-protein compounds which were not considered in this work. The protein composition of the investigated Bothrops species is in agreement with their pharmacological and pathological effects.

An ion-pairing reversed-phase liquid chromatography-mass spectrometry (IP-RP-LC-MS) was developed for the determination of nucleotides, nucleosides and their transformation products in Cordyceps. Perfluorinated carboxylic acid, namely pentadecafluorooctanoic acid (PDFOA, 0.25mM), was used as volatile ion-paring agent and a reversed-phase column (Agilent ZORBAX SB-Aq column) was used for the separation of three nucleotides namely uridine-5'-monophosphate (UMP, 0.638-10.200microg/mL), adenosine-5'-monophosphate (AMP, 0.24-7.80microg/mL) and guanosine-5'-monophosphate (GMP, 0.42-13.50microg/mL), seven nucleosides including adenosine (0.55-8.85microg/mL), guanosine (0.42-6.75microg/mL), uridine (0.33-10.50micro/mL), inosine (0.21-6.60microg/mL), cytidine (0.48-15.30microg/mL), thymidine (0.20-6.30microg/mL) and cordycepin (0.09-1.50microg/mL), as well as six nucleobases, adenine (0.22-6.90microg/mL), guanine (0.26-4.20microg/mL), uracil (0.38-12.15microg/mL), hypoxanthine (0.13-4.20microg/mL), cytosine (0.39-12.45microg/mL) and thymine (0.26-8.25microg/mL) with 5-chlorocytosine arabinoside as the internal standard. The overall LODs and LOQs were between 0.01-0.16microg/mL and 0.04-0.41microg/mL for the 16 analytes, respectively. The contents of 16 investigated compounds in natural and cultured Cordyceps were also determined and compared after validation of the developed IP-RP-LC-MS method. The transformations of nucleotides and nucleosides in Cordyceps were evaluated based on the quantification of the investigated compounds in three extracts, including boiling water extraction (BWE), 24h ambient temperature water immersion (ATWE) and 56h ATWE extracts. Two transformation pathways including UMP->>uridine->>uracil and GMP->>guanosine->>guanine were proposed in both natural Cordyceps sinensis and cultured Cordyceps militaris. The pathway of AMP->>adenosine->>inosine->>hypoxanthine was proposed in natural C. sinensis, while AMP->>adenosine->>adenine in cultured C. militaris. However, the transformation of nucleotides and nucleosides was not found in commercial cultured C. sinensis.

The proteasome is the most complex protease known, with a molecular mass of approx. 3 MDa and 33 distinct subunits. Recent studies reported the discovery of four chaperones that promote the assembly of a 19-subunit subcomplex of the proteasome known as the regulatory particle, or RP. These and other findings define a new and highly unusual macromolecular assembly pathway. The RP mediates substrate selection by the proteasome and injects substrates into the CP (core particle) to be degraded. A heterohexameric ring of ATPases, the Rpt proteins, is critical for RP function. These ATPases abut the CP and their C-terminal tails help to stabilize the RP-CP interface. ATPase heterodimers bound to the chaperone proteins are early intermediates in assembly of the ATPase ring. The four chaperones have the common feature of binding the C-domains of Rpt proteins, apparently a remarkable example of convergent evolution; each chaperone binds a specific Rpt subunit. The C-domains are distinct from the C-terminal tails, but are proximal to them. Some, but probably not all, of the RP chaperones appear to compete with CP for binding of the Rpt proteins, as a result of the proximity of the tails to the C-domain. This competition may underlie the release mechanism for these chaperones. Genetic studies in yeast point to the importance of the interaction between the CP and the Rpt tails in assembly, and a recent biochemical study in mammals suggests that RP assembly takes place on pre-assembled CP. These results do not exclude a parallel CP-independent pathway of assembly. Ongoing work should soon clarify the roles of both the CP and the four chaperones in RP assembly.

The onset and duration of resistance in experimental survivors of Penaeus japonicus produced by an intramuscular injection with white spot syndrome virus (WSSV) were surveyed by re-challenge tests with the virus conducted at weeks 1-4 and months 1-3 post initial exposure (PIE) to the virus. Virus neutralising activity in the survivors' plasma was also examined. Plasma-treated WSSV was separated from the plasma by centrifugation and then injected into naïve shrimp, in parallel with each re-challenge test. Re-challenge tests of the survivors conducted at weeks 1-4 PIE revealed that the resistance commenced at week 3 (relative percent survival, RPS: 39%) and almost fully developed at week 4 (RPS: 58%), because statistically significant differences in survival rates were observed between the test (previously virus exposed) and control groups at weeks 3 and 4. Re-challenge at months 1-3 PIE resulted in RPS values of 67, 54 and 6%, respectively, indicating the resistance persisted until month 2. RPS values in neutralisation tests performed at weeks 1-4 and months 1-3 PIE were -5, 14, 36, 50, 100, 38 and 6%, respectively, which coincided with the RPS values in each re-challenge test conducted in parallel. The present results demonstrated that resistance of P. japonicus against the viral pathogen developed 3 or 4 weeks after an exposure to the virus, and it persisted for another month at 24 degrees C. The resistance was paralleled by a humoral neutralising factor(s) in the plasma of shrimp.

This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of Clostridium perfringens. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an N-acetylglucosaminidase activity. The study of acp expression showed a constant expression during growth, which suggested an important role of Acp in growth of C. perfringens. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of C. perfringens. A knockout acp mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the acp mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and acp mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall N-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.

OBJECTIVE

The inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory conditions that affect the gastrointestinal tract. In regulation of this inflammatory process, Interleukin-6 (IL-6) has a major role. Overproduction of IL-6 by immunocompetent cells contributes to development of the inflammatory condition. Elevated levels of IL-6 in saliva could be expected, because the saliva-producing cells are part of the digestive system.

METHODS

IL-6 concentrations in saliva and plasma were studied in patients with CD (n=15), UC (n=7) and reference persons (RP) (n=19) by use of an ELISA method.

RESULTS

A significant difference in saliva IL-6 concentration between CD patients (median 16.9 ng/L; p<0.05) and RP (median 6.3 ng/L) was found. A significant difference in plasma IL-6 concentration between CD (median 10.3 ng/L; p<0.001) or UC (median 7.8 ng/L; p<0.001) and RP (median 0.8 ng/L) was observed. In patients with CD, plasma IL-6 correlated significantly with C-reactive protein (CRP) as well as albumin. In patients with UC, saliva IL-6 and plasma IL-6 correlated significantly with AI (activity index) scores as well as albumin. In patients with UC, a significant correlation between the saliva and plasma IL-6 concentrations was found.

CONCLUSIONS

IL-6 was found in saliva in patients with IBD, documenting the general involvement of the gastrointestinal tract extending to the mouth cavity, and measuring IL-6 may be an additional method for evaluating and monitoring the disease activity.

Cyclosporine A (CsA) has been clinically used as immunosuppressant, and new application for airway inflammation was also proposed. However, the clinical use of CsA was limited due to severe adverse effects after systemic exposure and the poor solubility. In the present investigation, novel respirable powder (RP) of CsA was developed for pulmonary administration with use of solid dispersion of wet-milled CsA (WM/CsA), and the physicochemical and pharmacological properties of the WM/CsA and its RP formulation were characterized. CsA in the solid dispersion was found to be amorphous by X-ray powder diffraction and differential scanning calorimetry. It exhibited the improved dissolution behavior as compared to active pharmaceutical ingredients. Laser diffraction and cascade impactor analysis of newly developed WM/CsA-RP, consisting of jet-milled WM/CsA and lactose carriers, suggested high dispersion and deposition in the respiratory organs with the emitted dose and the fine particle fraction of 96 and 54%, respectively. Intratracheal administration of WM/CsA-RP (100 microg CsA) in experimental inflammatory rats led to 71 and 85% reduction of granulocyte recruitment in bronchoalveolar lavage fluids and lung tissues, respectively, with showing ca 10(2)-fold reduced AUC and C(max) values of plasma CsA as compared to the oral dosage form of CsA at toxic concentration (10 mg/kg). Upon these findings, WM/CsA-RP would be efficacious dosage form for clinical treatment of airway inflammations with minimal systemic side effects.

Columns for reversed-phase HPLC (RP-LC) can be characterized by five, retention-related parameters: H (hydrophobicity), S (steric selectivity), A (hydrogen-bond acidity), B (hydrogen-bond basicity), and C (cation-exchange behavior). In the present study, values of the latter parameters have been measured for 92 type-B (low metals content)alkyl-silica columns and compared to column properties such as ligand length,ligand concentration, pore diameter, and the presence or absence of end-capping. With the exception of five columns of unusual design, retention factors, k, for 16 representative test compounds were correlated with values of H, S, etc., within an average +/- 1.2% (1 standard deviation, SD), suggesting that all significant solute-column interactions are recognized by these five column parameters. A single-valued function F(s) is proposed to measure differences in selectivity for any two RP-LC columns whose values of H, S, etc., are known. This allows the easy selection of columns whose selectivity is desired to be either similar to or different from a starting column, for application in either routine analysis or method development.

S-adenosyl methionine (SAM) is an important methyl group donor that is formed from methionine. S-adenosyl homocysteine (SAH) is formed after demethylation of SAM and represents a potent inhibitor of many methyltransferases. We developed an improved stable-isotope dilution ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of SAM and SAH in biological samples. The method comprises a phenylboronic acid-containing solid-phase extraction procedure, serving for binding and clean-up of SAM and SAH. After extraction, samples were separated and detected using either a HPLC SymmetryShield RP(18) or an Acquity UPLC BEH C(18) column with a HPLC-MS/MS or an UPLC-MS/MS system. The best results were obtained by Acquity UPLC BEH C(18) column. In plasma samples, the estimated intraassay coefficients of variation (CVs) for SAM and SAH were 3.3% and 3.9%, respectively, the interassay CVs were 10.1% for SAM and 8.3% for SAH. Mean recovery of SAM and SAH at two different concentrations was 100.0% for SAM and 101.7% for SAH. The quantification limits were 0.5 and 0.7nmol/L for SAM and SAH, respectively. In 31 plasma samples, the mean concentrations (SD) were 85.5 (11.1)nmol/L for SAM and 13.3 (5.0)nmol/L for SAH with a SAM/SAH ratio of 7.0 (1.8). The new UPLC-MS/MS method showed very high sensitivity and selectivity for SAM and SAH, low CVs and fast sample preparation (40 samples in 60min) and analysis time (3min). This new assay can be used for large-scale clinical studies.

Gonadotropin-releasing hormone (GnRH) controls the reproductive physiology and behavior of vertebrates by stimulating synthesis and release of gonadotropin from the pituitary gland. In 2000, another hypothalamic neuropeptide, gonadotropin-inhibitory hormone (GnIH), was discovered in quail and found to be an inhibiting factor for gonadotropin release. GnIH homologs are present in the brains of vertebrates, including birds, mammals, amphibians, and fish. These peptides, categorized as RF amide-related peptides (RFRPs), possess a characteristic LPXRF-amide (X = L or Q) motif at their C-termini. GnIH/RFRP precursor mRNA encodes a polypeptide that is possibly cleaved into three mature peptides in birds and two in mammals. The names of these peptides are GnIH, GnIH-related peptide-1 (GnIH-RP-1) and GnIH-RP-2 in birds, and RFRP-1 and RFRP-3 in mammals. GnIH/RFRP is synthesized in neurons of the paraventricular nucleus of the hypothalamus in birds and the dorsomedial hypothalamic area in mammals. GnIH neurons project to the median eminence, thus providing a functional neuroanatomical infrastructure to regulate anterior pituitary function. In quail, GnIH inhibits gonadal activity by decreasing synthesis and release of gonadotropin. The widespread distribution of GnIH/RFRP immunoreactive fibers in all animals tested suggests various actions within the brain. In accordance, GnIH/RFRP receptor mRNA is also expressed widely in the brain and the pituitary. GnIH/RFRP immunoreactive axon terminals are in probable contact with GnRH neurons in birds and mammals, and we recently demonstrated expression of GnIH receptor mRNA in GnRH-I and GnRH-II neurons in European starlings. Thus, GnIH/RFRP may also inhibit gonadotropin synthesis and release by inhibiting GnRH neurons in addition to having direct actions on the pituitary gland. Intracerebroventricular administration of GnIH/RFRP further inhibits reproductive behaviors in songbirds and rodents, possibly via direct actions on the GnRH system. The expression of GnIH/RFRP is regulated by melatonin which is an internal indicator of day length in vertebrates. Stress stimuli also regulate the expression of GnIH/RFRP in songbirds and rodents. Accordingly, GnIH/RFRP may serve as a transducer of environmental information and social interactions into endogenous physiology and behavior of the animal. Recently, it was shown that GnIH/RFRP and its receptor are also expressed in the gonads of birds, rodents and primates. In sum, the existing data suggest that GnIH/RFRP is an important mediator of reproductive function acting at the level of the brain, pituitary, and the gonad in birds and mammals.

BACKGROUND

Clinicians reported an increasing trend of rapid progression (RP) (AIDS within 3 years of infection) in Cuba.

METHODS

Recently infected patients were prospectively sampled, 52 RP at AIDS diagnosis (AIDS-RP) and 21 without AIDS in the same time frame (non-AIDS). 22 patients were sampled at AIDS diagnosis (chronic-AIDS) retrospectively assessed as>> 3 years infected. Clinical, demographic, virological, epidemiological and immunological data were collected. Pol and env sequences were used for subtyping, transmission cluster analysis, and prediction of resistance, co-receptor use and evolutionary fitness. Host, immunological and viral predictors of RP were explored through data mining.

RESULTS

Subtyping revealed 26 subtype B strains, 6 C, 6 CRF18_cpx, 9 CRF19_cpx, 29 BG-recombinants and other subtypes/URFs. All patients infected with CRF19 belonged to the AIDS-RP group. Data mining identified CRF19, oral candidiasis and RANTES levels as the strongest predictors of AIDS-RP. CRF19 was more frequently predicted to use the CXCR4 co-receptor, had higher fitness scores in the protease region, and patients had higher viral load at diagnosis.

CONCLUSIONS

CRF19 is a recombinant of subtype D (C-part of Gag, PR, RT and nef), subtype A (N-part of Gag, Integrase, Env) and subtype G (Vif, Vpr, Vpu and C-part of Env). Since subtypes D and A have been associated with respectively faster and slower disease progression, our findings might indicate a fit PR driving high viral load, which in combination with co-infections may boost RANTES levels and thus CXCR4 use, potentially explaining the fast progression. We propose that CRF19 is evolutionary very fit and causing rapid progression to AIDS in many newly infected patients in Cuba.

Retinitis pigmentosa (RP) is a genetically heterogeneous retinal disorder. Despite the numerous genes associated with RP already identified, the genetic basis remains unknown in a substantial number of patients and families. In this study, we performed whole-exome sequencing to investigate the molecular basis of a syndromic RP case that cannot be solved by mutations in known disease-causing genes. After applying a series of variant filtering strategies, we identified an apparently homozygous frameshift mutation, c.31delC (p.Q11Rfs*24) in the ADIPOR1 gene. The reported phenotypes of Adipor1-null mice contain retinal dystrophy, obesity, and behavioral abnormalities, which highly mimic those in the syndromic RP patient. We further confirmed ADIPOR1 retina expression by immunohistochemistry. Our results established ADIPOR1 as a novel disease-causing gene for syndromic RP and highlight the importance of fatty acid transport in the retina.

BACKGROUND

Increasing numbers of tyrosine kinase inhibitors (TKIs) were studied and approved for therapy of malignancies and other diseases. The aim of this study was to develop and validate a specific, simple and rapid quantification method for various TKIs in human plasma.

METHODS

A simultaneous test for six TKIs (erlotinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib) was developed using liquid chromatography tandem mass spectrometry in a multiple reaction monitoring mode. After protein precipitation the specimens were applied to the HPLC system and separated using a gradient of acetonitrile containing 1% formic acid with 10 mM ammoniumformiate on an analytic RP-C18 column.

RESULTS

The calibration range was 10-1000 ng/mL for sunitinib and 50-5000 ng/mL for the other TKIs with coefficients of determination ≥0.99 for all analytes. The intra- and inter day coefficients of variation were ≤15% and the chromatographic run time was 12 min. Plasma specimens were stable for measurement for at least 1 week at 4°C. Clinical applications of the assay are exemplarily discussed.

CONCLUSIONS

This novel high-throughput method is suitable for specific simultaneous determination of different TKIs in routine clinical practice.

Bioceramic materials are used for the reconstruction or replacement of the damaged parts of the human body. In this study an improved procedure is described for producing ceramic scaffolds with controlled porosity. Bioinert alumina ceramic was used to make porous scaffolds by using indirect fused deposition modeling (FDM), a commercially available rapid prototyping (RP) technique. Porous alumina samples were coated with hydroxyapatite (HAp) to increase the biocompatibility of the scaffolds. Initial biological responses of the porous alumina scaffolds were assessed in vitro using rat pituitary tumor cells (PR1). Both porous alumina and HAp coated alumina ceramics provided favorable sites for cell attachments in a physiological solution at 37 degrees C, which suggests that these materials would promote good bonding while used as bone implants in vivo. Based on these preliminary studies, similar tests were performed with human osteosarcoma cells. Cell proliferation studies show that both the ceramic materials can potentially provide a non-toxic surface for bone bonding when implanted in vivo.

A reversed-phase ion-pair high-performance liquid chromatografic method (RP-IPC) was developed to assay some water-soluble vitamins in solution dosage forms. Vitamins of the B-group B1, B2, B3, and B6, including vitamin C were determined in Oligovit coated tablets. In Beviplex coated tablets the vitamins B1, B2, B3, B6 and p-aminobenzoic acid were analysed. Hexanesulphonic acid sodium salt and triethanolamine in water methanol were used as mobile phase with adjusting pH to 2.8 with orthophosphoric acid. Phenol was used as an internal standard. For quantitative simultaneous analysis of vitamins in pharmaceutical formulations, the method of internal standard was used. All parameters for the validation of the method are given.