We report novel chemical properties of the ribozyme derived from the smallest group I intron (subgroup ICC). These features may be due to the two GA3 tetraloop interactions postulated in the intron and the high GC content of the secondary structure. The second order rate constant for the Azoarcus ribozyme, ((k(cat)/Km)S = 8.4 +/- 2.1 x 10(-5) M(-1) min(-1)) is close to that found for the related ribozyme derived from the pre-tRNA(Ile) of the cyanobacterium Anabaena PCCRp-phosphorothioate at the cleavage site and the 1000-fold decrease in cleavage rate with a deoxyribonucleoside leaving group.

Numerous top-down kinetic models have been constructed to describe the cell cycle. These models have typically been constructed, validated and analyzed using model species (molecular intermediates and proteins) and phenotypic observations, and therefore do not focus on the individual model processes (reaction steps). We have developed a method to: (a) quantify the importance of each of the reaction steps in a kinetic model for the positioning of a switch point [i.e. the restriction point (RP)]; (b) relate this control of reaction steps to their effects on molecular species, using sensitivity and co-control analysis; and thereby (c) go beyond a correlation towards a causal relationship between molecular species and effects. The method is generic and can be applied to responses of any type, but is most useful for the analysis of dynamic and emergent responses such as switch points in the cell cycle. The strength of the analysis is illustrated for an existing mammalian cell cycle model focusing on the RP [Novak B, Tyson J (2004) J Theor Biol230, 563-579]. The reactions in the model with the highest RP control were those involved in: (a) the interplay between retinoblastoma protein and E2F transcription factor; (b) those synthesizing the delayed response genes and cyclin D/Cdk4 in response to growth signals; (c) the E2F-dependent cyclin E/Cdk2 synthesis reaction; as well as (d) p27 formation reactions. Nine of the 23 intermediates were shown to have a good correlation between their concentration control and RP control. Sensitivity and co-control analysis indicated that the strongest control of the RP is mediated via the cyclin E/Cdk2:p27 complex concentration. Any perturbation of the RP could be related to a change in the concentration of this complex; apparent effects of other molecular species were indirect and always worked through cyclin E/Cdk2:p27, indicating a causal relationship between this complex and the positioning of the RP.

OBJECTIVE

Fundus autofluorescence (FAF), as an index of lipofuscin accumulation in the retinal pigment epithelium (RPE), provides indirect information on the level of metabolic activity of the RPE and thus the integrity of the RPE/photoreceptor complex. To investigate whether the photoreceptor/RPE complex is still viable in patients with Leber congenital amaurosis (LCA), FAF imaging was performed.

METHODS

Three patients with LCA (patients A, B, and C; ages, 24, 15, and 37 years, respectively) were enrolled and one patient with RP with preserved visual acuity (age, 28 years) was included as a control. The diagnosis was based on history, visual function, and Ganzfeld electroretinography (International Society for Clinical Electrophysiology of Vision [ISCEV] standard). FAF was recorded with a confocal scanning laser ophthalmoscope (cSLO; Heidelberg Retina Angiograph; Heidelberg Engineering, Heidelberg, Germany).

RESULTS

All patients with LCA had vision reduced to perception of light and had undetectable ERGs. FAF was normal in patient A. In patient B, there was a parafoveal ring of mildly increased FAF. The midperiphery showed mildly decreased FAF. Patient C showed a parafoveal ring of moderately increased FAF. FAF was moderately decreased along the arcades and the midperiphery. The patient with RP showed a parafoveal ring of moderately increased FAF and severely decreased FAF eccentric to the macula including the periphery.

CONCLUSIONS

The FAF findings in these patients with LCA suggest that there is continuous metabolic demand from the photoreceptors and that the RPE/photoreceptor complex is, at least in part, anatomically intact, but the photoreceptors have lost function. These findings may have implications for future treatment. It is notable that more than 20 years of severe visual impairment associated with LCA can be associated with normal FAF, indicating that photoreceptor function may be rescuable.

Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors (GRPr) with high affinity and specificity. The GRPr is overexpressed on a variety of human cancer cells, including prostate, breast, lung, and pancreatic cancers. The specific aim of this study was to develop (99m)Tc-radiolabeled BBN analogues that maintain high specificity for the GRPr in vivo. A preselected synthetic sequence via solid-phase peptide synthesis (SPPS) was designed to produce N(3)S-BBN (N(3)S = dimethylglycyl-l-seryl-l-cysteinylglycinamide) conjugates with the following general structure: DMG-S-C-G-X-Q-W-A-V-G-H-L-M-(NH(2)), where the spacer group, X = 0 (no spacer), omega-NH(2)(CH(2))(2)COOH, omega-NH(2)(CH(2))(4)COOH, omega-NH(2)(CH(2))(7)COOH, or omega-NH(2)-(CH(2))(10)COOH. The new BBN constructs were purified by reversed phase-HPLC (RP-HPLC). Electrospray mass spectrometry (ES-MS) was used to characterize the nonmetalated BBN conjugates. Re(V)-BBN conjugates were prepared by the reaction of Re(V)gluconate with N(3)S-X-BBN[7-14]NH(2) (X = 0 carbons, beta-Ala (beta-alanine), 5-Ava (5-aminovaleric acid), 8-Aoc (8-aminooctanoic acid), and 11-Aun (11-aminoundecanoic acid)) with gentle heating. Re-N(3)S-5-Ava-BBN[7-14]NH(2) was also prepared by the reaction of [Re(V)dimethylglycyl-l-seryl-l-cysteinylglycinamide] with 5-Ava-BBN[7-14]NH(2). ES-MS was used to determine the molecular constitution of the new Re(V) conjugates. The (99m)Tc conjugates were prepared at the tracer level by each the prelabeling, post-conjugation and pre-conjugation, postlabeling approaches from the reaction of Na[(99m)TcO(4)] with excess SnCl(2), sodium gluconate, and corresponding ligand. The (99m)Tc and Re(V) conjugates behaved similarly under identical RP-HPLC conditions. In vitro and in vivo models demonstrated biological integrity of the new conjugates.

A method is described for the quantitative determination of free and bound solute concentrations in the cytoplasm of intact cells. The method includes (a) introduction of a gelatin gel reference phase (RP) into the cytoplasm; (b) diffusion of dissolved substances between cytoplasm and RP, (c) cell quenching to - 196 degrees C to prevent subsequent solute redistributions, (d) ultra-low temperature microdissection to isolate RP and cytoplasm samples, and (e) analysis of isolates for solute and water content. In normal oocytes of the salamander, Desmognathus ochrophaeus, free or RP Na+ and K+ are 21.0 +/- 1.1 and 128.8 +/- 2.4 mu eq/ml, respectively, and vary stoichiometrically in altered oocytes. Overall cytoplasmic concentrations are 75.2 +/- 2.7 mu eq Na+/ml and 88.6 +/- 1.5 mu eq K+/ml. Cytoplasmic chemical activities are 16.2 mu eq Na+/ml and 99.2 mu eq K+/ml, corresponding to activity coefficients of 0.22 and 1.12, respectively. The results demonstrate unambiguously that (a) oocytes actively transport Na+ and K+, and (b) cytoplasm has important binding properties which differentiate it from an ordinary aqueous solution. These cytoplasmic properties are investigated in the following paper.

BACKGROUND

Gene silencing by RNA interference (RNAi) is a powerful tool for functional genomics. Although RNAi was first described in Caenorhabditis elegans, several nematode species are unable to mount an RNAi response when exposed to exogenous double stranded RNA (dsRNA). These include the satellite model organisms Pristionchus pacificus and Oscheius tipulae. Available data also suggest that the RNAi pathway targeting exogenous dsRNA may not be fully functional in some animal parasitic nematodes. The genus Panagrolaimus contains bacterial feeding nematodes which occupy a diversity of niches ranging from polar, temperate and semi-arid soils to terrestrial mosses. Thus many Panagrolaimus species are adapted to tolerate freezing and desiccation and are excellent systems to study the molecular basis of environmental stress tolerance. We investigated whether Panagrolaimus is susceptible to RNAi to determine whether this nematode could be used in large scale RNAi studies in functional genomics.

RESULTS

We studied two species: Panagrolaimus sp. PS1159 and Panagrolaimus superbus. Both nematode species displayed embryonic lethal RNAi phenotypes following ingestion of Escherichia coli expressing dsRNA for the C. elegans embryonic lethal genes Ce-lmn-1 and Ce-ran-4. Embryonic lethal RNAi phenotypes were also obtained in both species upon ingestion of dsRNA for the Panagrolaimus genes ef1b and rps-2. Single nematode RT-PCR showed that a significant reduction in mRNA transcript levels occurred for the target ef1b and rps-2 genes in RNAi treated Panagrolaimus sp. 1159 nematodes. Visible RNAi phenotypes were also observed when P. superbus was exposed to dsRNA for structural genes encoding contractile proteins. All RNAi phenotypes were highly penetrant, particularly in P. superbus.

CONCLUSIONS

This demonstration that Panagrolaimus is amenable to RNAi by feeding will allow the development of high throughput methods of RNAi screening for P. superbus. This greatly enhances the utility of this nematode as a model system for the study of the molecular biology of anhydrobiosis and cryobiosis and as a possible satellite model nematode for comparative and functional genomics. Our data also identify another nematode infraorder which is amenable to RNAi and provide additional information on the diversity of RNAi phenotypes in nematodes.

We review herein our experience in the management of bleeding esophageal varices in cirrhotic patients and consider our findings in light of the dramatic changes in the treatment of cirrhosis resulting from the more widespread use of orthotopic liver transplantation (OLT). It does not seem realistic, at present, to propose OLT as the only effective treatment of variceal bleeding for a variety of reasons, and there remains a large group of patients who are noncompliant or unsuitable for liver transplantation. We propose that initial bleeding be controlled by endoscopic sclerotherapy, thereby allowing careful evaluation to be made electively. Grade A patients appear to be managed best by a reduced-size portacaval shunt (RPS) with prospects of good survival and few complications. Grade B patients can be managed by either sclerotherapy, RPS, or OLT, depending upon individual circumstances. Grade C patients are best managed by liver transplantation, again with excellent survival. In those grade C patients not deemed suitable for OLT (especially alcoholic patients), long-term endoscopic sclerotherapy is the best option. Changes in patient status may sometimes require revision of the treatment decision.

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a staphylocidal peptide released by activated platelets. This peptide initiates its microbicidal activity by membrane permeabilization, with ensuing inhibition of intracellular macromolecular synthesis. RP-1 is a synthetic congener modeled on the C-terminal microbicidal alpha-helix of tPMP-1. This study compared the staphylocidal mechanisms of RP-1 with those of tPMP-1, focusing on isogenic tPMP-1-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains for the following quantitative evaluations: staphylocidal efficacy; comparative MIC; membrane permeabilization (MP) and depolarization; and DNA, RNA, and protein synthesis. Although the proteins had similar MICs, RP-1 caused significant killing of ISP479C (<50% survival), correlating with extensive MP (>95%) and inhibition of DNA and RNA synthesis (>90%), versus substantially reduced killing of ISP479R (>80% survival), with less MP (55%) and less inhibition of DNA or RNA synthesis (70 to 80%). Interestingly, RP-1-induced protein synthesis inhibition was equivalent in both strains. RP-1 did not depolarize the cell membrane and caused a relatively short postexposure growth inhibition. These data closely parallel those previously reported for tPMP-1 against this strain set and exemplify how synthetic molecules can be engineered to reflect structure-activity relationships of functional domains in native host defense effector molecules.

Cell membranes of the human epidermoid cell line A431 express classical bradykinin (BK) B2 receptors, as assessed by [3H]BK binding studies. Furthermore, stimulation by BK induced a time-dependent modulation of protein kinase C (PKC) activity in A431 cells: a rapid activation (t1/2 approximately 1 min) is followed by a slow inhibition (t1/2 approximately 20 min) of PKC translocation measured by [3H]phorbol 12,13-dibutyrate binding. In addition, BK stimulated both adenylate cyclase activity in A431 membranes and accumulation of intracellular cyclic AMP (cAMP) in intact cells in a retarded manner. A possible BK-induced activation of the cAMP pathway mediated via PKC, phospholipase D, prostaglandins or Ca2+/calmodulin was excluded. A 35 kDa protein was found in A431 membranes to be specifically phosphorylated in the presence of both BK and protein kinase A (PKA). An anti-alpha s-antibody, AS 348, abolished stimulation of adenylate cyclase activity in response to BK, cholera toxin and isoprenaline, strongly suggesting the involvement of Gs proteins in the BK action. The BK-activated cAMP signalling system might be important for the observed inactivation of PKC slowly evoked by BK: the BK-induced rapid activation of PKC is decreased by dibutyryl cAMP, and the slow inhibition of PKC is prevented by an inhibitor of PKA, adenosine 3':5'-monophosphothioate (cyclic, Rp isomer). The inhibition of PKC translocation might be exerted directly at the level of PKC activation, since stimulation of phosphoinositide hydrolysis by BK was affected by neither dibutyryl cAMP nor forskolin. Thus our results provide the first evidence that A431 cells BK is able to activate two independent signal-transduction pathways via a single class of B2 receptors but two different G proteins. The lagging stimulation of the cAMP signalling pathway via Gs might serve to switch off PKC, which is rapidly activated via Gq-mediated stimulation of phosphoinositide hydrolysis.

OBJECTIVE

To identify the cause of retinitis pigmentosa (RP) in UTAD003, a large, six-generation Louisiana family with autosomal dominant retinitis pigmentosa (adRP).

METHODS

A series of strategies, including candidate gene screening, linkage exclusion, genome-wide linkage mapping, and whole-exome next-generation sequencing, was used to identify a mutation in a novel disease gene on chromosome 10q22.1. Probands from an additional 404 retinal degeneration families were subsequently screened for mutations in this gene.

RESULTS

Exome sequencing in UTAD003 led to identification of a single, novel coding variant (c.2539G>A, p.Glu847Lys) in hexokinase 1 (HK1) present in all affected individuals and absent from normal controls. One affected family member carries two copies of the mutation and has an unusually severe form of disease, consistent with homozygosity for this mutation. Screening of additional adRP probands identified four other families (American, Canadian, and Sicilian) with the same mutation and a similar range of phenotypes. The families share a rare 450-kilobase haplotype containing the mutation, suggesting a founder mutation among otherwise unrelated families.

CONCLUSIONS

We identified an HK1 mutation in five adRP families. Hexokinase 1 catalyzes phosphorylation of glucose to glucose-6-phosphate. HK1 is expressed in retina, with two abundant isoforms expressed at similar levels. The Glu847Lys mutation is located at a highly conserved position in the protein, outside the catalytic domains. We hypothesize that the effect of this mutation is limited to the retina, as no systemic abnormalities in glycolysis were detected. Prevalence of the HK1 mutation in our cohort of RP families is 1%.

N-oleoyldopamine (OLDA), a bioactive lipid originally found in the mammalian brain, is an endovanilloid that selectively activates the transient receptor potential vanilloid type 1 (TRPV1) channel. This study tests the hypothesis that OLDA protects the heart against ischemia and reperfusion (I/R) injury via activation of the TRPV1 in wild-type (WT) but not in gene-targeted TRPV1-null mutant (TRPV1(-/-)) mice. Hearts of WT or TRPV1(-/-) mice were Langendorffly perfused with OLDA (2 x 10(-9) M) in the presence or absence of CGRPCGRP) receptor antagonist; RP-67580 (1 x 10(-6) M), a selective neurokinin-1 receptor antagonist; chelerythrine (5 x 10(-6) M), a selective protein kinase C (PKC) antagonist; or tetrabutylammonium (TBA, 5 x 10(-4) M), a nonselective K(+) channel antagonist, followed by 35 min of global ischemia and 40 min of reperfusion (I/R). Left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), coronary flow (CF), and left ventricular peak positive dP/dt (+dP/dt) were evaluated after I/R. OLDA improved recovery of cardiac function after I/R in WT but not TRPV1(-/-) hearts by increasing LVDP, CF, and +dP/dt and by decreasing LVEDP. CGRPRP-67580, chelerythrine, or TBA abolished the protective effect of OLDA in WT hearts. Radioimmunoassay showed that the release of substance P (SP) and CGRP after OLDA treatment was higher in WT than in TRPV1(-/-) hearts, which was blocked by chelerythrine or TBA. Thus OLDA exerts a cardiac protective effect during I/R injury in WT hearts via CGRP and SP release, which is abolished by PKC or K(+) channel antagonists. The protective effect of OLDA is void in TRPV1(-/-) hearts, supporting the notion that TRPV1 mediates OLDA-induced protection against cardiac I/R injury.

A new α-conotoxin LsIA was isolated from the crude venom of Conus limpusi using assay-guided RP-HPLC fractionation. Synthetic LsIA was a potent antagonist of α3β2, α3α5β2 and α7 nAChRs, with half-maximal inhibitory concentrations of 10, 31 and 10 nM, respectively. The structure of LsIA determined by NMR spectroscopy comprised a characteristic disulfide bond-stabilized α-helical structure and disordered N-terminal region. Potency reductions of up to 9-fold were observed for N-terminally truncated analogues of LsIA at α7 and α3β2 nAChRs, whereas C-terminal carboxylation enhanced potency 3-fold at α3β2 nAChRs but reduced potency 3-fold at α7 nAChRs. This study gives further insight into α-conotoxin pharmacology and the molecular basis of nAChR selectivity, highlighting the influence of N-terminal residues and C-terminal amidation on conotoxin pharmacology.

According to the fetal programming hypothesis, impaired intrauterine development results in insulin resistance and associated metabolic disturbances. Recently, we reported increased body fat, a forerunner of insulin resistance, in the pups of mineral-restricted rat dams. To identify the causative mineral(s), the effect of magnesium restriction was assessed. Female weanling WNIN rats (n = 21) consumed ad libitum for 9 wk a 70% magnesium-restricted diet or were pair-fed a control (C) diet (n = 7). After 9 wk, they were mated with control males. Control dams and pups were fed the control diet throughout, whereas 7 Mg-restricted dams were switched to the control diet at parturition and their pups weaned onto the control diet (RP). Pups of the remaining 14 restricted dams were weaned onto the control diet (RW) or the Mg-restricted diet (R). All groups had 8 male pups from weaning. Pups were studied on postnatal d 90 and 180. R pups weighed less than C pups at weaning, but both RP and RW pups caught up with controls by d 90. At this time, R pups were neither insulin resistant nor glucose intolerant, but had a higher percentage of body fat and plasma triglycerides and lower lean body and fat-free mass than C pups. These variables were partially corrected in both RP and RW pups. On postnatal d 180, R, RP, and RW pups were insulin resistant and had a lower insulin response to a glucose challenge than C pups; however, glucose tolerance was impaired only in RW pups. Thus, maternal magnesium restriction irreversibly increases body fat and induces insulin resistance in pups by 6 mo of age, whereas additional perinatal Mg deficiency impairs glucose tolerance.

Studies have suggested that integration of kinase and phosphatase activities maintains the steady-state L-type Ca(2+) current in ventricular myocytes, a balance disrupted in failing hearts. As we have recently reported that the PP1/PP2A inhibitor calyculin A evokes pronounced increases in L-type I(Ca), the goal of this study was to identify the counteracting kinase and phosphatase that determine 'basal'I(Ca) in isolated mouse ventricular myocytes. Whole-cell voltage-clamp studies, with filling solutions containing 10 mm EGTA, revealed that calyculin A (100 nm) increased I(Ca) at test potentials between -42 and +49 mV (44% at 0 mV) from a holding potential of -80 mV. It also shifted the V(0.5) (membrane potential at half-maximal) of both activation (from -17 to -25 mV) and steady-state inactivation (from -32 to -37 mV) in the hyperpolarizing direction. The broad-spectrum protein kinase inhibitor, staurosporine (300 nm), was without effect on I(Ca) when added after calyculin A. However, by itself, staurosporine decreased I(Ca) throughout the voltage range examined (50% at 0 mV) and blocked the response to calyculin A, indicating that the phosphatase inhibitor's effects depend upon an opposing kinase activity. The PKA inhibitors Rp-cAMPs (100 microm in the pipette) and H89 (1 microm) failed to reduce basal I(Ca) or to block the calyculin A-evoked increase in I(Ca). Likewise, calyculin A was still active with 10 mm intracellular BAPTA or when Ba(2+) was used as the charge carrier. These data eliminate roles for protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMKII) as counteracting kinases. However, the protein kinase C (PKC) inhibitors Ro 31-8220 (1 microm) and Gö 6976 (200 nm) decreased steady-state I(Ca) and blunted the effect of calyculin A. PP2A is not involved in this regulation as intracellular applications of 10-100 nm okadaic acid or 500 nm fostriecin failed to increase I(Ca). However, PP1 is important, as dialysis with 2 microm okadaic acid or 500 nm inhibitor-2 mimicked the increases in I(Ca) seen with calyculin A. These in situ studies identify constitutive activity of PP1 and the counteracting activity of certain isoforms of PKC, in pathways distinct from receptor-mediated signalling cascades, as regulatory components that determine the steady-state level of cardiac L-type I(Ca).

1. The effects of the vasoconstrictor angiotensin II (Ang II) on whole-cell ATP-sensitive K+ currents (IK,ATP) of smooth muscle cells isolated enzymatically from rat mesenteric arteries were investigated using the patch clamp technique. 2. Ang II, at a physiological concentration (100 nM), reduced IK,ATP activated by 0.1 mM internal ATP and 10 microM levcromakalim by 36.4 +/- 2.3%. 3. The protein kinase C (PKC) activator 1-oleoyl-2-acetyl-sn-glycerol (OAG, 1 microM) reduced IK,ATP by 44.1 +/- 2.7%. GDP beta S (1 mM), included in the pipette solution, abolished the inhibition by Ang II, while that by OAG was unaffected. 4. Pretreatment with the PKC inhibitors staurosporine (100 nM) or calphostin C (500 nM) prevented the Ang II-induced inhibition of IK,ATP. 5. Ang II inhibition was unaffected by cell dialysis with PKA inhibitor peptide (5 microM), and the PKA inhibitor Rp-cAMPS (100 microM) did not reduce IK,ATP. 6. Our results suggest that Ang II modulates KATP channels through activation of PKC but not through inhibition of PKA.

OBJECTIVE

The aim of this study was to prospectively compare diffusion-weighted magnetic resonance imaging (DWI) and [(11)C]choline positron emission tomography/computed tomography (PET/CT) with computed tomography (CT) for preoperative lymph node (LN) staging in prostate cancer (PCa) patients.

METHODS

Between June 2010 and May 2012, CT, DWI and [(11)C]choline PET/CT were performed preoperatively in 33 intermediate- and high-risk PCa patients undergoing radical prostatectomy (RP) and extended pelvic lymph node dissection (ePLND) including obturator fossa and internal, external and common iliac fields. Patient- and field-based performance characteristics for all three imaging techniques based on histopathological results are reported. Imaging techniques were compared by means of the area under the curve (AUC).

RESULTS

LN metastases were detected in 92 of 1,012 (9%) LNs from 14 of 33 (42%) patients. On patient-based analysis, sensitivity, specificity and accuracy for CT were 57, 68 and 64%, respectively, for DWI were 57, 79 and 70%, respectively, and for [(11)C]choline PET/CT were 57, 90 and 76%, respectively. On field-based analysis, these numbers for CT were 47, 94 and 88%, respectively, for DWI were 56, 97 and 92%, respectively, and for [(11)C]choline PET/CT were 62, 96 and 92%, respectively. Neither DWI nor [(11)C]choline PET/CT performed significantly better than CT on pairwise comparison of patient- and field-based results.

CONCLUSIONS

All three imaging techniques exhibit a rather low sensitivity with less than two thirds of LN metastases being detected on patient- and field-based analysis. Overall diagnostic efficacy did not differ significantly between imaging techniques, whereas distinct performance characteristics, esp. patient-based specificity, were best for [(11)C]choline PET/CT followed by DWI and CT.

The effect of nitric oxide (NO) on Na+/H+ exchange (NHE) activity was investigated utilizing Caco-2 cells as an experimental model. Incubation of Caco-2 cells with 10(-3) M S-nitroso-N-acetylpenicillamine (SNAP), a conventional donor of NO, for 20 min resulted in a approximately 45% dose-dependent decrease in NHE activity, as determined by assay of ethylisopropylamiloride-sensitive 22Na uptake. A similar decrease in NHE activity was observed utilizing another NO-specific donor, sodium nitroprusside. SNAP-mediated inhibition of NHE activity was not secondary to a loss of cell viability. NHE3 activity was significantly reduced by SNAP (P < 0.05), whereas NHE2 activity was essentially unaltered. The effects of SNAP were mediated by the cGMP-dependent signal transduction pathway as follows: 1) LY-83583 and 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), specific inhibitors of soluble guanylate cyclase, blocked the inhibitory effect of SNAP on NHE; 2) 8-bromo-cGMP mimicked the effects of SNAP on NHE activity; 3) the SNAP-induced decrease in NHE activity was counteracted by a specific protein kinase G inhibitor, KT-5823 (1 microM); 4) chelerythrine chloride (2 microM) or calphostin C (200 nM), specific protein kinase C inhibitors, did not affect inhibition of NHE activity by SNAP; 5) there was no cross activation by the protein kinase A-dependent pathway, as the inhibitory effects of SNAP were not blocked by Rp-cAMPS (25 microM), a specific protein kinase A inhibitor. These data provide novel evidence that NO inhibits NHE3 activity via activation of soluble guanylate cyclase, resulting in an increase in intracellular cGMP levels and activation of protein kinase G.

This study deals with the effect of altitudinal variation on the content of phenolic compounds in three traditional herbal plants, which are also consumed as food in Central Europe. Herbs of Calluna vulgaris (L.) HULL, flowers and fruits of Sambucus nigra L., and berries of Vaccinium myrtillus L. collected in the Naturpark Solktaler (Austria) were extracted using accelerated solvent extraction (ASE). Identification and quantification of the constituents in the polar extracts (methanol 80%, v/v) were achieved by means of RP-HPLC-PDA and/or LC-PDA-MS analysis with external standards. 3,5- O-Dicaffeoylquinic acid was identified in flowers of S. nigra for the first time. Rising concentrations of flavonoids and especially flavonol-3- O-glycosides with adjacent hydroxyl groups in ring B in C. vulgaris and S. nigra with increasing altitude were observed. Anthocyanins from the berries of both S. nigra and V. myrtillus occurred in decreasing amounts with rising altitude. C. vulgaris showed the best radical scavenging capacity based on the DPPH assay.

OBJECTIVE

To date, no biomarkers have been found to predict, before treatment, which patients will develop radiation pneumonitis (RP), a potentially fatal toxicity, after chemoradiation for lung cancer. We investigated potential associations between single nucleotide polymorphisms (SNPs) in HSPB1 and risk of RP after chemoradiation for non-small cell lung cancer (NSCLC).

METHODS

Subjects were patients with NSCLC treated with chemoradiation at 1 institution. The training data set comprised 146 patients treated from 1999 to July 2004; the validation data set was 125 patients treated from August 2004 to March 2010. We genotyped 2 functional SNPs of HSPB1 (rs2868370 and rs2868371) from all patients. We used Kaplan-Meier analysis to assess the risk of grade ≥2 or ≥3 RP in both data sets and a parametric log-logistic survival model to evaluate the association of HSPB1 genotypes with that risk.

RESULTS

Grade ≥3 RP was experienced by 13% of those with CG/GG and 29% of those with CC genotype of HSPB1 rs2868371 in the training data set (P=.028); corresponding rates in the validation data set were 2% CG/GG and 14% CC (P=.02). Univariate and multivariate analysis confirmed the association of CC of HSPB1 rs2868371 with higher risk of grade ≥3 RP than CG/GG after adjustment for sex, age, performance status, and lung mean dose. This association was validated both in the validation data set and with Harrell's C statistic.

CONCLUSIONS

The CC genotype of HSPB1 rs2868371 was associated with severe RP after chemoradiation for NSCLC.

1. A study has been made, in guinea-pig isolated trachealis, of the effects of charybdotoxin in modulating (a) the activity of large conductance K(+)-channels, (b) the spontaneous electrical activity of intact cells and (c) the mechanical effects of some bronchodilator drugs. 2. Single smooth muscle cells were isolated from guinea-pig trachealis by enzymic digestion and were studied by the patch clamp recording technique. Recordings were made from outside-out plasmalemmal patches when the medium bathing the external surface of the patches contained 1.2 mM Ca2+ and 6 mM K+ while that bathing the cytosolic surface contained 0.1 microM Ca2+ and 140 mM K+. Charybdotoxin (100 nM), applied to the external surface of patches held at 0 mV, abolished the unitary currents associated with the opening of large conductance K(+)-channels. 3. Opened segments of guinea-pig trachea were used for the simultaneous recording of membrane potential and tension changes. In these experiments charybdotoxin (100 nM) caused the conversion of spontaneous electrical slow waves into spike-like action potentials. This effect was accompanied by a very small reduction in resting membrane potential. 4. Tissue bath recording showed that charybdotoxin (100 nM) increased the spontaneous mechanical tone of the tissue, antagonized (2.8 fold in each case) the relaxant actions of isoprenaline and theophylline but did not antagonize the relaxant actions of cromakalim or RP 49356. 5. It is concluded that charybdotoxin is an effective inhibitor of large conductance K(+)-channels in guinea-pig trachealis cells. The ability of charybdotoxin to convert spontaneous slow waves into spike-like action potentials suggests that the large, charybdotoxin-sensitive, K+-channels play an important role in determining the strong outward rectifying behaviour of the cells. The ability of charybdotoxin to antagonize isoprenaline and theophylline, but not to antagonize cromakalim and RP 49356, suggests that opening of the large conductance, charybdotoxin-sensitive K+-channel is implicated in the action of the former but not the latter pair of bronchodilator drugs.

We previously reported that a small peptide based on amino acids 143-153 of the c-Jun N-terminal kinase (JNK)-binding domain of JIP-1 functioned as an in vitro inhibitor of JNK activity. This peptide (TI-JIP: RP-KRPTTLNLF) resembles the kinase-interaction motif (KIM = (K/R)(2-3)X(1-6)(L/I)X(L/I)), which is common to upstream activators, downstream substrates, phosphatases, and scaffold proteins present in MAPK cascades. In this study, we characterized the mechanism of JNK inhibition by this peptide and further investigated the biochemical features of this peptide resulting in potent JNK inhibition. We also tested various KIM-based peptides for their ability to inhibit JNK activity. TI-JIP was found to be competitive with respect to the phosphoacceptor substrate c-Jun (K(I) = 0.39 +/- 0.08 microm), and exhibit mixed (non-competitive) inhibition with respect to ATP. All seven substitutions of Pro-5 we tested significantly reduced the JNK inhibition, as did altering the Pro-5 to Leu-8 spacing. When we independently tested eight substitutions of either Thr-6 or Thr-7, only one substitution in each position was well tolerated. Furthermore, peptides based on the KIMs from other proteins were significantly less potent JNK inhibitors than TI-JIP, including a peptide from the JNK interactor Sab that contained all critical inhibitory residues present in TI-JIP. Therefore, despite having previously identified Arg-4, Pro-5, Leu-8, and Leu-10 in TI-JIP as independently critical for mediating JNK inhibition, we find their presence in other 11-mer peptides is not sufficient for JNK inhibition. TI-JIP is therefore a unique KIM-based inhibitor of JNK activity.

The effect of varying brain temperature on neutrophil accumulation in brain and the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) on cerebrovascular endothelium after controlled cortical impact (CCI) was studied in rats. Sprague Dawley rats were anesthetized and subjected to CCI to the left parietal cortex. Ten minutes after CCI, brain temperature was modulated and maintained at 32 degrees C, 37 degrees C, or 39 degrees C (n = 8 per group) for 4 h. Rats were then decapitated and immunohistochemistry on brain sections was performed using monoclonal antibodies (MoAb) that recognize neutrophils (RP-3), ICAM-1 (TM-8, Athena Neurosciences), or MoAb that react with E-selectin (La-Roche). Each of these markers was quantified in 100 x fields. Neutrophil accumulation was also quantified with myeloperoxidase (MPO) assay. Absolute neutrophil count (ANC) was measured in blood samples before and 1 h and 4 h after CCI. Neutrophil accumulation in injured brain was decreased in rats maintained at 32 degrees C vs 39 degrees C (4-fold difference as assessed by immunohistochemistry, p < 0.05; 8-fold difference as assessed by MPO assay, p < 0.05). Peripheral blood ANC was not affected by temperature. E-selectin was induced on cerebrovascular endothelium after CCI (p < 0.05), but was only decreased modestly at 32 degrees C versus 39 degrees C (p = 0.11). ICAM-1 was not upregulated on cerebrovascular endothelium at this early time following CCI. Neutrophil accumulation is directly dependent on brain temperature during the initial 4 h after CCI. This appears to be mediated by mechanisms other than effects of temperature on E-selectin or ICAM-1 expression or systemic ANC.

The regulatory particle (RP) of the 26S proteasome contains a heterohexameric ring of AAA-ATPases (RPT1-6) that unfolds and inserts substrates into the core protease (CP) for degradation. Through genetic analysis of the Arabidopsis thaliana gene pair encoding RPT2, we show that this subunit plays a critical role in 26S proteasome assembly, histone dynamics, and plant development. rpt2a rpt2b double null mutants are blocked in both male and female gamete transmission, demonstrating that the subunit is essential. Whereas rpt2b mutants are phenotypically normal, rpt2a mutants display a range of defects, including impaired leaf, root, trichome, and pollen development, delayed flowering, stem fasciation, hypersensitivity to mitomycin C and amino acid analogs, hyposensitivity to the proteasome inhibitor MG132, and decreased 26S complex stability. The rpt2a phenotype can be rescued by both RPT2a and RPT2b, indicative of functional redundancy, but not by RPT2a mutants altered in ATP binding/hydrolysis or missing the C-terminal hydrophobic sequence that docks the RPT ring onto the CP. Many rpt2a phenotypes are shared with mutants lacking the chromatin assembly factor complex CAF1. Like caf1 mutants, plants missing RPT2a or reduced in other RP subunits contain less histones, thus implicating RPT2 specifically, and the 26S proteasome generally, in plant nucleosome assembly.

Pyruvate,orthophosphate (Pi) dikinase (PPDK) is best recognized as a chloroplastic C(4) cycle enzyme. As one of the key regulatory foci for controlling flux through this photosynthetic pathway, it is strictly and reversibly regulated by light. This light/dark modulation is mediated by reversible phosphorylation of a conserved threonine residue in the active-site domain by the PPDK regulatory protein (RP), a bifunctional protein kinase/phosphatase. PPDK is also present in C(3) plants, although it has no known photosynthetic function. Nevertheless, in this report we show that C(3) PPDK in leaves of several angiosperms and in isolated intact spinach (Spinacia oleracea) chloroplasts undergoes light-/dark-induced changes in phosphorylation state in a manner similar to C(4) dikinase. In addition, the kinetics of this process closely resemble the reversible C(4) process, with light-induced dephosphorylation occurring rapidly (< or =15 min) and dark-induced phosphorylation occurring much more slowly >> or =30-60 min). In intact spinach chloroplasts, light-induced dephosphorylation of C(3) PPDK was shown to be dependent on exogenous Pi and photosystem II activity but independent of electron transfer from photosystem I. These in organello results implicate a role for stromal pools of Pi and adenylates in regulating the reversible phosphorylation of C(3)-PPDK. Last, we used an in vitro RP assay to directly demonstrate ADP-dependent PPDK phosphorylation in desalted leaf extracts of the C(3) plants Vicia faba and rice (Oryza sativa). We conclude that an RP-like activity mediates the light/dark modulation of PPDK phosphorylation state in C(3) leaves and chloroplasts and likely represents the ancestral isoform of this unusual and key C(4) pathway regulatory "converter" enzyme.