Tissue contents and plasma levels of IR-CGRP were studied following administration of capsaicin to newborn rats. A depletion of>> 50% of IR-CGRP content was seen in the cardiovascular tissues (e.g., heart and peripheral arteries), lungs, gastrointestinal tract (e.g., esophagus, stomach, and intestine), genitourinary tract (e.g., ureter, bladder, uterus, and penis), and in the nervous system (e.g., dorsal root and trigeminal ganglia, sciatic and trigeminal nerves, and dorsal spinal cord) in capsaicin-treated rats, in comparison with the control rat tissues (p < 0.01). These findings are compatible with the known involvement of capsaicin of the unmyelinated sensory C and A delta fibers and hence their distribution in the nervous system and other organs. Plasma IR-CGRP levels were also significantly lower in the capsaicin-treated rats throughout their life span (p < 0.001), suggesting that, at least in part, circulating CGRP is derived from the nervous system. RP-HPLC confirmed the identity of CGRP in both tissue and plasma extracts.

Extracellular ATP increases intracellular Ca2+ ([Ca2+]i) in HL-60 cells. When cells are stimulated with supramaximal concentrations of ATP, although the initial [Ca2+]i increase is similar over a range of 30, 100, and 300 microM ATP, the rate of the return to basal [Ca2+]i level is faster in cells treated with higher concentrations of ATP. This probably results from differences in Ca2+ influx rather than Ca2+ release, since the influx of the unidirectional Ca2+ surrogates Ba2+ and Mn2+ also exhibit similar responses. Furthermore, while 300 microM ATP had an inhibitory effect on the thapsigargin-induced capacitative Ca2+ entry, 30 microM ATP potentiated the response. However, the inhibitory action of 300 microM ATP was blocked by protein kinase C (PKC) inhibitors, such as GF 109203X and chelerythrine, and the potentiating action of 30 microM ATP was blocked by protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. The PKC inhibitors also slowed the decay rate of the Ca2+ response induced by 300 microM ATP, and the PKA inhibitors increased it when induced by 30 microM ATP. In the measurements of PKA and PKC activity, 30 microM ATP activates only PKA, while 300 microM ATP activates both kinases. Taken together, these data suggest that the changes in the ATP-induced Ca2+ response result from differential modulation of ATP-induced capacitative Ca2+ entry by PKC and PKA in HL-60 cells.

BACKGROUND

Brachypodium distachyon L. is a newly emerging model plant system for temperate cereal crop species. However, its grain protein compositions are still not clear. In the current study, we carried out a detailed proteomics and molecular genetics study on grain glutenin proteins in B. distachyon.

RESULTS

SDS-PAGE and RP-HPLC analysis of grain proteins showed that Brachypodium has few gliadins and high molecular weight glutenin subunits. In contrast the electrophoretic patterns for the albumin, globulin and low molecular weight glutenin subunit (LMW-GS) fractions of the grain protein were similar to those in wheat. In particular, the LMW-C type subunits in Brachypodium were more abundant than the equivalent proteins in common wheat. Southern blotting analysis confirmed that Brachypodium has 4-5 copies of LMW-GS genes. A total of 18 LMW-GS genes were cloned from Brachypodium by allele specific PCR. LMW-GS and 4 deduced amino acid sequences were further confirmed by using Western-blotting and MALDI-TOF-MS. Phylogenetic analysis indicated that Brachypodium was closer to Ae. markgrafii and Ae. umbellulata than to T. aestivum.

CONCLUSIONS

Brachypodium possessed a highly conserved Glu-3 locus that is closely related to Triticum and related species. The presence of LMW-GS in B. distachyon grains indicates that B. distachyon may be used as a model system for studying wheat quality attributes.

A rapid, sensitive, and reliable method for measuring anandamide amidase activity in rat brain microsomes by reversed-phase high-performance liquid chromatography (<em>RP</em>-HPL<em>C</em>) and its applications are described. Enzymatic activity was assayed by the determination of the rates of hydrolysis of anandamide or its analogs at 37 degrees <em>C</em>. The reaction products were separated using an ODS guard column eluted with aqueous phosphoric acid-acetonitrile and quantitated with uv detection at 204 nm and an external standard method. Baseline separation of the acid products from their substrates was completed in less than 2 min. The detection limits were 1.4 pmol for arachidonic acid and 0.22 pmol for anandamide at a signal to noise ratio of 4:1. The stability of anandamide in the acidic mobile phase was tested, and no significant decomposition was observed up to 1 h. The method was successfully applied to the examination of substrate specificity as well as for testing the ability of amidase inhibitors to block its hydrolysis. Kinetic constants obtained for (S)-methanandamide were an apparent Km of 8.6 +/- 1.3 microM and a Vmax of 362 +/- 16 pmol/min/mg of protein. A highly potent inhibitor, palmitylsulfonyl fluoride (PSF), was found to have an I<em>C</em>50 of 50 nM. PSF is 210 times as potent as phenylmethylsulfonyl fluoride. The method offers several advantages over existing methodology using radioisotopes or a solvent extraction procedure.

A highly transcribed region in Oenothera mitochondria codes for a reading frame (orf206) which shows high homology to the Marchantia encoded mitochondrial open reading frame orf277 and is also conserved in the mitochondrial genomes of Arabidopsis thaliana and Daucus carota. Transcripts of orf206 are modified by cytidine to uridine changes in 46 positions by RNA editing, affecting 30% of all cytidines and 15% of the total encoded amino acids. This ORF is cotranscribed with an upstream reading frame and with the downstream rps 14 gene. The orf206 deduced protein shows high similarity to polypeptides which are proposed to be part of an ABC-type heme transporter involved in cytochrome c biogenesis in Bradyrhizobium and Rhodobacter.

Natural hirudin variant 2 with a lysine residue in position 47 (rHV2-Lys47) was produced in a genetically engineered strain of Saccharomyces cerevisiae as a secreted protein of 65 amino acids and purified to greater than 99% homogeneity. Only reversed-phase high-performance liquid chromatography (RP-HPLC) using very shallow acetonitrile gradients indicated the presence of a component in the final product (approximately 1% of total protein) with a slightly increased retention time. Using successive RP-HPLC purification steps, this hydrophobic impurity was isolated and separated into two constituents defined as components A1 and A2 which differed from the parent molecule by mass reductions of 17.2 Da (A1) and 17.6 Da (A2), respectively, as determined by electrospray mass spectrometry (ESMS). Proteolytic digestion with endoprotease Glu-C from Staphylococcus aureus (V8 protease) and analysis of the peptide mixture by ESMS showed that the mass difference between rHV2-Lys47 and component A1 was due to a modification between amino acids 1 and 43, while the corresponding mass difference with component A2 was the result of a modification within the peptide fragment comprising residues 50-61. Further analyses using amino acid sequencing and ESMS in combination with collision-activated dissociation (CAD) detected modifications at residues Asn33-Gly34 in component A1 and at Asn53-Gly54 in component A2. Both of these sites were previously shown to be susceptible to spontaneous deamidation under slightly basic pH conditions. Thus, the mass reductions of approximately 17 Da and the fact that both asparagines, Asn33 in component A1 and Asn53 in component A2, proved to be resistant to Edman degradation provided strong support for them being stable succinimide intermediates of the corresponding deamidation reactions. Both intermediates were shown to have inhibition constants for human alpha-thrombin on the order of 1 pM, identical to that of rHV2-Lys47. The isoelectric point of component A2 was determined to be within 0.01 pH unit of that of the parent molecule by isoelectric focusing in an immobilized pH gradient.

Genistein, a soybean-derived isoflavone with an inhibitory effect on protein tyrosine kinases (PTKs), has been shown to suppress osteoclastic bone resorption. To clarify the mechanisms underlying this action, we investigated the effects of genistein on inward rectifier K(+) current (I(Kir)) in rat osteoclasts by using the whole-cell patch-clamp technique. Extracellularly applied genistein inhibited I(Kir) in a concentration-dependent manner. Physiologically attainable concentrations of genistein inhibited I(Kir). IC(50) values obtained 5 and 10 min after the application of genistein were 54 and 27 microM, respectively. The removal of genistein partially restored the current. Daidzein, an isoflavone without PTK-inhibiting activity, also showed a weak inhibitory effect on I(Kir), but genistin had no effect. Other PTK inhibitors, tyrphostin A25, tyrphostin B42, and tyrphostin B46, inhibited I(Kir), whereas herbimycin A and lavendustin A were without effect. The inactive tyrphostin, A1, showed a similar inhibitory effect as tyrphostin A25. The tyrosine phosphatase inhibitor, orthovanadate, did not affect the inhibitory potency of genistein on I(Kir). The inhibitory action of genistein was unaffected by changing intracellular Ca(2+) concentration ([Ca(2+)]i) or by pretreatment of the cell with GDPbetaS, Rp-cAMPS, okadaic acid, or staurosporine. Therefore the inhibition of I(Kir) by genistein does not depend on PTK inhibition, involvement of changes in [Ca(2+)]i, or secondary interaction with protein kinase A or protein kinase C. Genistein-induced inhibition of I(Kir) would cause membrane depolarization, elevation of [Ca(2+)]i, and inhibition of osteoclastic bone resorption.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates catecholamine secretion from cultured porcine adrenal medullary chromaffin cells in a dose-dependent manner with the half-maximal and maximal doses of 30 nM and 1 microM, respectively. Either removal of extracellular Ca2+ or addition of Gd3+, an inorganic Ca2+ channel blocker, very potently inhibits PACAP-induced catecholamine secretion. Both nicardipine (1 microM) and methoxyverapamil (1 microM), blockers of voltage-dependent Ca2+ channels, are also effective in inhibiting PACAP-induced catecholamine secretion. When the intracellular free Ca2+ concentration ([Ca2+]i) is measured in a fura 2-loaded single chromaffin cell, PACAP is found to cause a sustained increase in [Ca2+]i by mobilizing Ca2+ from both extra- and intracellular pools. It is also found that PACAP stimulates the production of inositol phosphates in a dose-dependent manner, which is not abolished by removal of extracellular Ca2+ unlike the case of nicotine. PACAP increases cAMP content in chromaffin cells in a dose-dependent manner. Removal of extracellular Ca2+ enhances PACAP-induced cAMP production but strongly inhibits PACAP-induced catecholamine secretion. Pretreatment of cells with adenosine-3':5'-monophosphothioate, cyclic, Rp-isomer, a cAMP antagonist, does not block PACAP-induced catecholamine secretion. The addition of forskolin or 3-isobutyl-1-methylxanthine does not enhance the PACAP-induced catecholamine secretion. These results indicate that PACAP activates voltage-dependent Ca2+ channels and phospholipase C as well as adenylate cyclase in cultured porcine adrenal medullary cells and strongly suggest that PACAP-induced catecholamine secretion is mainly mediated by activation of voltage-dependent Ca2+ channels.

Antibacterial proteins are an important part of the innate immune system for all animals. They have been extensively studied in mammals, amphibians and invertebrates, but have received only scant attention in fish. Their expression and processing, however, provide a way of monitoring defence vigour during development or with seasonal changes in physiology. The aim of the present work was to identify and characterise antibacterial proteins in rainbow trout. In vitro analyses of extracts of the peripheral blood leucocytes, head kidney leucocytes and mucus from adult unstimulated (non-immune) fish showed marked antibacterial activity against Gram positive bacteria. Fractionation by ion exchange chromatography and RP-HPLC of head kidney extracts showed the presence of two forms of lysozyme but no constitutively expressed antimicrobial proteins of < 10 kDa. By contrast, chromatographic analyses of mucus revealed at least four antibacterial proteins. Two are conventional lysozymes, a third is an unusual lysozyme-like protein with a low isoelectric point, and the fourth is a highly hydrophobic, cationic peptide of c. 3 kDa.

The electric (linear) dichroisms observed in the membrane electroporation of salt-filled lipid bilayer vesicles (diameter O = 2 alpha = 0.32 micron; inside [NaCl] = 0.2 M) in isotonic aqueous 0.284 M sucrose-0.2 mM NaCl solution indicate orientation changes of the anisotropic light scattering centers (lipid head groups) and of the optical transition moments of the membrane-inserted probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Both the turbidity dichroism and DPH absorbance dichroism show peculiar features: (1) at external electric fields E>> or = Esat the time course of the dichroism shows a maximum value (reversal): Esat = 4.0 (+/- 0.2) MV m-1, T = 293 K (20 degrees C), (2) this reversal value is independent of the field strength for E>> or = Esat, (3) the dichroism amplitudes exhibit a maximum value Emax = 3.0 (+/- 0.5) MV m-1, (4) for the pulse duration of 10 microseconds there is one dominant visible normal mode, the relaxation rate increases up to tau-1 approximately 0.6 x 10(6) s-1 at Esat and then decreases for E>> Esat. The data can be described in terms of local lipid phase transitions involving clusters Ln of n lipids in the pore edges according to the three-state scheme C<->>HO<->>HI, C being the closed bilayer state, HO the hydrophobic pore state and HI the hydrophilic or inverted pore state with rotated lipid and DPH molecules. At E>> or = Esat, further transitions HO<->>HO* and HI<->>HI* are rapidly coupled to the C<->>HO transition, which is rate-limiting. The vesicle geometry conditions a cos theta dependence of the local membrane field effects relative to the E direction and the data reflect cos theta averages. The stationary induced transmembrane voltage delta phi (theta, lambda m) = -1.5 aEf(lambda m) magnitude of cos theta does not exceed the limiting value delta phi sat = -0.53 V, corresponding to the field strength Em,sat = -delta phi sat/d = 100 MV m-1 (10(3) kV cm-1), due to increasing membrane conductivity lambda m. At E = Esat, f(lambda m) = 0.55, lambda m = 0.11 mS m-1. The lipid cluster phase transition model yields an average pore radius of rp = 0.35 (+/- 0.05) nm of the assumed cylindrical pore of thickness d = 5 nm, suggesting an average cluster size of= 12 (+/- 2) lipids per pore edge. For E>> Esat, the total number of DPH molecules in pore states approaches a saturation value; the fraction of DPH molecules in HI pores is 12 (+/- 2)% and that in HO pores is 48 (+/- 2)%. The percentage of membrane area P approximately (lambda m/lambda i) x 100% of conductive openings filled with the intravesicular medium of conductance lambda i = 2.2 S m-1 linearly increases from P approximately 0% (E = 1.8 MV m-1) to P = 0.017% (E = 8.5 MV m-1). Analogous estimations made by Kinosita et al. (1993) on the basis of fluorescence imaging data for sea urchin eggs give the same order of magnitude for P (0.02-0.2%). The increase in P with the field strength is collinear with the increase in concentration of HI and HI* states with the field strength, whereas the HO and HO* states exhibit a sigmoid field dependence. Therefore our data suggest that it is only the HI and HI* pore states which are conductive. It is noted that the various peculiar features of the dichroism data cannot be described by simple whole particle deformation.

Aortic impedance data of infants, children and adults (age range 0.8-54 yr), previously reported by others, were interpreted by means of three alternative four-element windkessel models: W4P, W4S, and IVW. The W4P and W4S are derived from the three-element windkessel (W3) by connecting an inertance (L) in parallel or in series, respectively, with the aortic characteristic resistance (Rc). In the IVW, L is connected in series with a viscoelastic windkessel (VW). The W4S and IVW (same input impedance) fit the data best. The W4S, however, suffers from the assumption that Rc is part of total peripheral resistance (Rp). The IVW model offers a new paradigm for interpretation of resistive properties in terms of viscous (Rd) properties of vessel wall motion, distinguished from Rp. Results indicated that rapid reduction of Rd/Rp during early development is functional to modulation of decay time constant (taud) of pressure in diastole, such that normalization over heart period (taud/T) is independent of body size. Estimates of total arterial compliance (C) vs. age were fitted by a bell-shaped curve with a maximum at 33 yr. With body weight (BW) factored out by normalization, the C/BW data scattered about a bell-shaped curve centered at 66 mmHg. Inertance was significantly higher in pediatric patients than in adults, in accordance with a lower cross-sectional area of the vasculature, commensurate to a lower aortic flow. Changes of arterial properties appear functional to control the ratio of pulsatile power to active power and keep arterial efficiency as high as 97% in infants and children.

This study was performed to determine whether the compound isolated from Phyllostachys nigra could attenuate oxidative stress in transformed retinal ganglion cells (RGC-5 cells) death. RGC-5 cells in culture were given two different insults such as l-buthionine-(S,R)-sulfoximine (BSO) plus glutamate for 24h or hydrogen peroxide for 24h, after which cell survival were measured. Among the four systematic fractions tested, ethyl acetate fraction showed a significantly higher inhibition which was in a concentration dependent manner. Eight compounds were isolated from ethyl acetate fraction of P. nigra using preparative RP-HPLC and Sephadex LH-20 column chromatography. Their chemical structures were elucidated by chemical and spectral analysis as isoorientin (1), orientin (2), vitexin (3), cis-coumaric acid (4), p-coumaric acid (5), luteolin 6-C-(6''-O-trans-caffeoylglucoside) (6), vittariflavone (7) and tricin (8). The luteolin 6-C-(6''-O-trans-caffeoylglucoside) (compound 6) significantly attenuated the negative effects of BSO plus glutamate or hydrogen peroxide to RGC-5 cells. Treatment of the RGC-5 cells with compound 6 reduced the reactive oxygen species (ROS) as quantified using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). This compound also replenished the reduced glutathione level. In conclusion, these results implicate that compound 6 isolated from P. nigra could be used as a leading compounds for retinal disease via anti-oxidative effects.

The mineral phosphate solubilizing (MPS) ability of a Serratia marcescens strain, namely CTM 50650, isolated from the phosphate mine of Gafsa, was characterized on a chemically defined medium (NBRIP broth). Various insoluble inorganic phosphates, including rock phosphate (RP), calcium phosphate (CaHPO(4)), tri-calcium phosphate (Ca(3)(PO(4))(2)) and hydroxyapatite were tested as sole sources of phosphate for bacterial growth. Solubilization of these phosphates by S. marcescens CTM 50650 was very efficient. Indeed, under optimal conditions, the soluble phosphorus (P) concentration it produced reached 967, 500, 595 and 326 mg/l from CaHPO(4), Ca(3)(PO(4))(2), hydroxyapatite and RP, respectively. Study of the mechanisms involved in the MPS activity of CTM 50650, showed that phosphate solubilization was concomitant with significant drop in pH. HPLC-analysis of culture supernatants revealed the secretion of gluconic acid (GA) resulting from direct oxidation pathway of glucose when the CTM 50650 cells were grown on NBRIP containing glucose as unique carbon source. This was correlated with the simultaneous detection by PCR for the first time in a S. marcescens strain producing GA, of a gene encoding glucose dehydrogenase responsible for GA production, as well as the genes pqqA, B, C and E involved in biosynthesis of its PQQ cofactor. This study is expected to lead to the development of an environmental-friendly process for fertilizer production considering the capacity of S. marcescens CTM 50650 to achieve yields of P extraction up to 75% from the Gafsa RP.

ABSTRACT We mapped and characterized quantitative trait loci (QTL) for partial resistance to Puccinia sorghi and investigated consistency across different European flint maize populations. Four independent populations, containing 280 F(3) lines (AxB(I)), 120 F(5) lines (AxB(II)), 131 F(4) lines (AxC), and 133 F(4) lines (CxD) were produced from four European elite flint inbreds (A, B, C, and D) and genotyped at 89, 151, 104, and 122 restriction fragment length polymorphism marker loci, respectively. All F(n) lines were evaluated in field trials with two replications in three or five (AxB(I)) environments. Genotypic variance was highly significant for rust ratings in all populations, and heritabilities exceeded 0.64. Between 4 and 13 QTL were detected in individual populations using composite interval mapping, explaining between 33 and 71% of the phenotypic variance. Twenty QTL were distributed over all ten chromosomes, without preference to chromosomes 3, 4, 6, and 10, which harbor qualitatively acting Rp loci. In most cases, gene action was additive or partially dominant. Four pairs of QTL displayed significant digenic epistatic interactions, and QTL-environment interactions were observed frequently. Approximately half of the QTL were consistent between AxB(I) and AxB(II) or AxC and CxD; fewer were consistent between AxB(I) and AxC or CxD. In European flint maize germ plasm, conventional selection for partial rust resistance seems to be more promising than marker-assisted selection.

Reduced port surgery (RPS), in which fewer ports are used than that in a conventional laparoscopic procedure, is becoming increasingly popular for various surgeries. However, the application of RPS to the field of gastrectomy is still underdeveloped. We started laparoscopy-assisted total gastrectomy through an umbilical port plus another 5 mm port (dual port laparoscopy-assisted total gastrectomy: DP-LATG) as an RPS for laparoscopy-assisted total gastrectomy (LATG). A SILS™ port was inserted into an umbilical incision, while another 5 mm port was inserted at the right flank region. We performed DP-LATG on ten early gastric cancer cases consecutively from May 2011 onwards, with the surgeries all performed by a single surgeon. The results of DP-LATG were compared with the resuls of ten conventional LATGs (C-LATGs) that were performed between March 2010 and April 2011. There were no significant differences in the mean operation time (DP-LATG, 253.0 ± 26.8 min; C-LATG, 235.5 ± 20.6 min; p = 0.119), mean blood loss (33.4 ± 23.7, 39.8 ± 60.4 mL, p = 0.759), and number of lymph nodes dissected (31.6 ± 12.3, 40.9 ± 18.7, p = 0.205). There were no intraoperative complications, there was no need for additional ports, and there were no conversions to open surgery nor postoperative complications in the DP-LATG cases. We successfully and safely performed DP-LATG without incurring any notable differences from C-LATG in terms of operation time, blood loss, and number of lymph nodes dissected.

OBJECTIVE

Retinitis pigmentosa (RP) is a heterogenous group of inherited retinal degenerations caused by mutations in at least 45 genes. Recently, the FAM161A gene was identified as the causative gene for RPRP.

METHODS

We performed a clinical and molecular genetic study of a consanguineous Palestinian family with two three siblings affected with retinitis pigmentosa. DNA samples were collected from the index patient, his father, his affected sister, and two non-affected brothers. DNA sample from the index was subjected to high resolution genome-wide SNP array. Assuming identity-by-descent in this consanguineous family we applied homozygosity mapping to identify disease causing genes.

RESULTS

The index patient reported night blindness since the age of 20 years, followed by moderate disease progression with decrease of peripheral vision, the development of photophobia and later on reduced central vision. At the age of 40 his visual acuity was counting fingers (CF) for both eyes, color discrimination was not possible and his visual fields were severely constricted. Funduscopic examination revealed a typical appearance of advanced RP with optic disc pallor, narrowed retinal vessels, bone-spicule like pigmentary changes in the mid-periphery and atrophic changes in the macula. His younger affected brother (37 years) was reported with overall milder symptoms, while the youngest sister (21 years) reported problems only with night vision. Applying high-density SNP arrays we identified several homozygous genomic regions one of which included the recently identified FAM161A gene mutated in RPRP. Sequencing analysis revealed the presence of a novel homozygous nonsense mutation, c.1003C>T/p.R335X in the index patient and the affected sister.

CONCLUSIONS

We identified an RPRP family in the Palestinian population caused by a novel nonsense mutation in FAM161A. RP in this family shows a typical disease onset with moderate to rapid progression into severe visual impairment including central vision in the index and overall milder symptoms in the younger brother and sister.

The flavonoid aglycones from an illuminated parsley (Petroselinum crispum (Mill.) Nym.) cell suspension culture were identified and quantified as the flavones apigenin, luteolin and chrysoeriol and the flavonols kaempferol, quercetin and isorhamnetin. Flavonoid extracts from these cultures were purified by solid phase extraction from RP C-18 phase and given by gavage to rats. Only extract from illuminated culture increased the antioxidative capacity (AOC) of blood plasma temporarily with maximum values after 1 h. It is concluded that the course of AOC reflects changes in the plasma content of flavonoids.

OBJECTIVE

In rheumatoid arthritis (RA), predictive biomarkers for subsequent radiographic progression (RP) could improve therapeutic choices for individual patients. We previously showed that the multibiomarker disease activity (MBDA) score in patients with newly diagnosed RA identified patients at risk for RP. We evaluated the MBDA score at multiple time-points as a predictor of RP during 2 years of follow-up.

METHODS

A subset of patients with RA (N=220) from the Swedish Farmacotherapy (SWEFOT) trial were analysed for MBDA score, disease activity score of 28 joints (DAS28), C reactive protein (CRP) and erythrocyte sedimentation rate (ESR) at baseline (BL), month 3 and year 1, for predicting RP based on modified Sharp/van der Heijde scores at BL, year 1 and year 2.

RESULTS

Patients with persistently low MBDA (<30) scores or those with a decrease from moderate (30-44) to low MBDA scores, did not develop RP during 2 years of follow-up. The highest risk for RP during 2 years of follow-up (42%) was observed among patients with persistently high (>44) MBDA scores. Among methotrexate non-responders with a high MBDA score at BL or month 3, significantly more of those who received triple therapy had RP at year 2 compared with those who received antitumour necrosis factor therapy.

CONCLUSIONS

Measuring the MBDA score both before and during treatment in RA was useful for the assessment of individual patient risk for RP during 2 years of follow-up. In comparison with low CRP, ESR or DAS28, a low MBDA score at any time-point was associated with numerically lower proportions of RP.

BACKGROUND

NCT00764725.

Tolerance to the local antiallodynic effects of morphine, DPDPE ([D-Pen(2),D-Pen(5)]-Enkephalin) or JWH-015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone) after their repeated administration during neuropathic pain was evaluated. The role of the nitric oxide-cGMP-protein kinase G (PKG)-c-Jun N-terminal kinase (JNK) signaling pathway on the peripheral morphine-induced tolerance after the chronic constriction of sciatic nerve in mice was also assessed. The mechanical and thermal antiallodynic effects produced by a high dose of morphine, DPDPE or JWH-015 subplantarly administered daily from days 10 to 20 after nerve injury were estimated with the von Frey filaments and cold plate tests. The antiallodynic effects of the repeated administration of morphine combined with a sub-analgesic dose of a selective inducible nitric oxide synthase (NOS2) (L-N(6)-(1-iminoethyl)-lysine; L-NIL), L-guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ), PKG ((Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs) or JNK (anthra[1,9-cd]pyrazol-6(2H)-one; SP600125) inhibitor from days 10 to 20 after injury were also evaluated. The repeated administration of morphine, but not DPDPE or JWH-015, produced a rapid development of tolerance to its mechanical and thermal antiallodynic effects in sciatic nerve-injured mice. The co-administration of morphine with L-NIL, ODQ, Rp-8-pCPT-cGMPs or SP600125 avoided the development of morphine antiallodynic tolerance after nerve injury. These findings reveal that the repeated local administration of DPDPE or JWH-015 did not induce antinociceptive tolerance after sciatic nerve injury-induced neuropathic pain. Our data also indicate that the peripheral nitric oxide-cGMP-PKG-JNK signaling pathway participates in the development of morphine tolerance after nerve injury and propose the inactivation of this pathway as a promising strategy to avoid morphine tolerance during neuropathic pain.

In order to characterize the effect of temperature on the retention behaviour and selectivity of separation of polypeptides and proteins in reversed-phase high-performance liquid chromatography (RP-HPLC), the chromatographic properties of four series of peptides, with different peptide conformations, have been studied as a function of temperature (5-80 degrees C). The secondary structure of model peptides was based on either the amphipathic alpha-helical peptide sequence Ac-EAEKAAKEX(D/L)EKAAKEAEK-amide, (position X being in the centre of the hydrophobic face of the alpha-helix), or the random coil peptide sequence Ac-X(D/L)LGAKGAGVG-amide, where position X is substituted by the 19 L- or D-amino acids and glycine. We have shown that the helical peptide analogues exhibited a greater effect of varying temperature on elution behaviour compared to the random coil peptide analogues, due to the unfolding of alpha-helical structure with the increase of temperature during RP-HPLC. In addition, temperature generally produced different effects on the separations of peptides with different L- or D-amino acid substitutions within the groups of helical or non-helical peptides. The results demonstrate that variations in temperature can be used to effect significant changes in selectivity among the peptide analogues despite their very high degree of sequence homology. Our results also suggest that a temperature-based approach to RP-HPLC can be used to distinguish varying amino acid substitutions at the same site of the peptide sequence. We believe that the peptide mixtures presented here provide a good model for studying temperature effects on selectivity due to conformational differences of peptides, both for the rational development of peptide separation optimization protocols and a probe to distinguish between peptide conformations.

The structure of the photosynthetic reaction center (RC) from Rhodopseudomonas viridis is known to high resolution. It contains a firmly bound tetraheme cytochrome from which electrons are donated to a special pair (P) of bacteriochlorophylls, which is photooxidized upon absorption of light. Tyrosine at position 162 of the L-subunit of the reaction center (L 162 Y) is a highly conserved residue positioned halfway between P and the proximal heme group (c-559) of the cytochrome. By specific mutagenesis this residue was exchanged against the amino acids phenylalanine (F), glycine (G), methionine (M), leucine (L), tryptophan (W), threonine (T), and histidine (H). All mutants were expressed in Rps. viridis using a recently established transformation system [Laussermair & Oesterhelt (1992) EMBO J. 11, 777-783]. They were shown biochemically to synthesize all four subunits of the RC (cytochrome, subunits L, M, and H) and to assemble them correctly into the membrane. The structures of two mutants (L 162 F and L 162 T) were determined and found not to differ significantly from the wild-type structure. All mutants grew photosynthetically. The absorption spectrum of all the mutants is the same as in WT, but the redox potential of P and of c-559 was changed by the mutations. The kinetics of electron transfer from the heme group to the special pair were measured in chromatophores by flash absorption. As found earlier in the wild type (Y) several exponential components were needed to fit the data.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

The FDA approved C-11 choline PET/computed tomography (CT) for imaging patients with recurrent prostate cancer in 2012. Subsequently, the 2014 NCCN guidelines have introduced labeled choline PET/CT in the imaging algorithm of patients with suspected recurrent disease. However, there is only scarce data on the impact of labeled choline PET/CT findings on disease management. We hypothesized that labeled-choline PET/CT studies showing local or regional recurrence or distant metastases will have a direct role in selection of appropriate patient management and improve radiation planning in patients with disease that can be controlled using this mode of therapy.

METHODS

This retrospective study was approved by the Tel Aviv Sourasky and Sheba Medical Center's Helsinki ethical review committees. Patient characteristics including age, PSA, stage, prior treatments, and pre-PET choline treatment recommendations based on NCCN guidelines were recorded. Patients with biochemical failure and without evidence of recurrence on physical examination or standard imaging were offered the option of additional imaging with labeled choline PET/CT. Treatment recommendations post-PET/CT were compared with pre-PET/CT ones. Pathologic confirmation was obtained before prostate retreatment. A nonparametric χ test was used to compare the initial and final treatment recommendations following choline PET/CT.

RESULTS

Between June 2010 and January 2014, 34 labeled-choline PET/CT studies were performed on 33 patients with biochemical failure following radical prostatectomy (RP) (n=6), radiation therapy (RT) (n=6), brachytherapy (n=2), RP+salvage prostate fossa RT (n=14), and RP+salvage prostate fossa/lymph node RT (n=6). Median PSA level before imaging was 2 ng/mL (range, 0.16 to 79). Labeled choline PET/CT showed prostate, prostate fossa, or pelvic lymph node increased uptake in 17 studies, remote metastatic disease in 9 studies, and failed to identify the cause for biochemical failure in 7 scans.PET/CT altered treatment approach in 18 of 33 (55%) patients (P=0.05). Sixteen of 27 patients (59%) treated previously with radiation were retreated with RT and delayed or eliminated androgen deprivation therapy: 1 received salvage brachytherapy, 10 received salvage pelvic lymph node or prostate fossa irradiation, 2 brachytherapy failures received salvage prostate and lymph nodes IMRT, and 3 with solitary bone metastasis were treated with radiosurgery. Eleven of 16 patients retreated responded to salvage therapy with a significant PSA response (<0.2 ng/mL), 2 patients had partial biochemical responses, and 3 patients failed. The median duration of response was 500±447 days. Two of 6 patients with no prior RT were referred for salvage prostatic fossa RT: 1 received dose escalation for disease identified in the prostate fossa and another had inclusion of "hot" pelvic lymph nodes in the treatment volume.

CONCLUSIONS

These early results suggest that labeled choline PET/CT imaging performed according to current NCCN guidelines may change management and improve care in prostate cancer patients with biochemical failure by identifying patients for referral for salvage radiation therapy, improving radiation planning, and delaying or avoiding use of androgen deprivation therapy.

BACKGROUND

Radiolabeled RGD peptides that specifically target integrin α(ν)β(3) have great potential in early tumor detection through noninvasive monitoring of tumor angiogenesis. Based on previous findings of our group on radiopeptides containing positively charged aminoacids, we developed a new cyclic cRGDfK derivative, c(RGDfK)-(Orn)(3)-CGG. This new peptide availing the polar linker (Orn)(3) and the (99m)Tc-chelating moiety CGG (Cys-Gly-Gly) is appropriately designed for (99m)Tc-labeling, as well as consequent conjugation onto nanoparticles.

METHODS

A tumor imaging agent, c(RGDfK)-(Orn)(3)-[CGG-(99m)Tc], is evaluated with regard to its radiochemical, radiobiological and imaging characteristics.

RESULTS

The complex c(RGDfK)-(Orn)(3)-[CGG-(99m)Tc] was obtained in high radiochemical yield (>98%) and was stable in vitro and ex vivo. It presented identical to the respective, fully analytically characterized (185/187)Re complex retention time in RP-HPLC. In contrary to other RGD derivatives, we showed that the new radiopeptide exhibits kidney uptake and urine excretion due to the ornithine linker. High tumor uptake (3.87±0.48% ID/g at 60 min p.i.) was observed and was maintained relatively high even at 24 h p.i. (1.83±0.05 % ID/g), thus providing well-defined scintigraphic imaging. Accumulation in other organs was negligible. Blocking experiments indicated target specificity for integrin receptors in U87MG glioblastoma cells.

CONCLUSIONS

Due to its relatively high tumor uptake, renal elimination and negligible abdominal localization, the new (99m)Tc-RGD peptide is considered promising in the field of imaging α(ν)β(3)-positive tumors. However, the preparation of multifunctional SPECT/MRI contrast agents (RGD-conjugated nanoparticles) for dual modality imaging of integrin expressing tumors should be further investigated.

The effect of growing Rhodopseudomonas (Rps.) acidophila and Rps. palustris in the presence of different concentrations of the carotenoid (Car) biosynthetic inhibitor diphenylamine (DPA) has been investigated. Growth with sub-maximal concentrations of DPA induces Car limitation. The exact response to DPA is species dependent. However, both Rps. acidophila and Rps. palustris respond by preferentially incorporating the limiting amount of coloured Cars into their LH2 complexes at the expense of the RC-LH1 complexes. As inhibition by DPA becomes more severe there is an increase in the percentage of Cars with reduced numbers of conjugated C=C bonds. The effect of this changed Car composition on the structure and function of the antenna complexes has been investigated using absorption, fluorescence, CD and Raman spectroscopies. The results show that although the presence of Car molecules is important for the stability of the LH2 complexes that the overall native structure can be maintained by the presence of many different Cars.