We investigated the effect of lysophosphatidylcholine (lysoPtdCho) and palmitoylcarnitine (PamCar), ischemia-induced amphipathic lipid metabolites, on the inward rectifier K+ channel in guinea-pig ventricular cells, under whole-cell and cell-attached configurations with patch-clamp techniques. (a) Both lysoPtdCho (10-50 microM) and PamCar (10-50 microM) depolarized the resting membrane potential (RP), retarded the repolarization of action potential, provoked spontaneous action potential discharges from oscillatory afterpotentials, and eventually caused a sudden rise of the RP to plateau levels. (b) These lysoPtdCho- or PamCar-induced depolarizations of RP were due to a decrease in the inward rectifier K+ current (IK1), and the sudden rise of the RP could be accounted for by a crossover of N-shaped current-voltage relationship on the voltage axis (zero current line) more than once. (c) Single-channel studies in the cell-attached mode revealed that lysoPtdCho (5-100 microM) decreased the conductance of the single IK1 channel with little change in its open probability, whereas PamCar (10-50 microM) did so by decreasing the open probability, with the channel conductance unaltered. (d) A short-chain acylcarnitine, l-propionylcarnitine (PpCar, 100 microM), prevented the depressant effect of lysoPtdCho (50 microM), but not of PamCar (50 microM), on the IK1. (e) Both lysoPtdCho and PamCar produced identical electrophysiological alterations on the membrane potential and IK1 in whole-cell recordings. However, molecular mechanisms involved in the effects of these toxic metabolites on single IK1 channels differ.

Structural studies of the high molecular mass (HMM) glutenin subunits 1Bx7 (from cvs Hereward and Galatea) and 1Bx20 (from cv. Bidi17) of bread wheat were conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). For all three proteins, MALDI-TOFMS analysis showed that the isolated fractions contained a second component with a mass about 650 Da lower than the major component. The testing and correction of the gene-derived amino acid sequences of the three proteins were performed by direct MALDI-TOFMS analysis of their tryptic peptide mixture. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimizing the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP-HPLC/ESI-MS analysis of the tryptic digest. This resulted in coverage of about 98% of the sequences. In contrast to the gene-derived data, the results obtained demonstrate the insertion of the sequence QPGQGQ between Trp716 and Gln717 of subunit 1Bx7 (cv. Galatea) and a possible single amino acid substitution within the T20 peptide of subunit 1Bx20. Moreover, the mass spectrometric data demonstrated that the lower mass components present in all the fractions correspond to the major components but lack about six amino acid residues, which are probably lost from the protein C-terminus. Finally, the results obtained provide evidence for the lack of glycosylation or other post-translational modifications of these subunits.

Heterozygous truncating mutations in the RPRP) cases. To examine the role of RPRP, we screened 101 unrelated Chinese RP patients (unselected for mode of inheritance) and 190 elderly normal control subjects for sequence changes in the coding exons for the 2156 amino acid RPRPRP among Chinese. The mutation was R677X, the most common found in Americans. Five other known sequence changes were found. In addition, nine novel sequence alterations were identified: 746G>A (R249H), 1437G>T (M479I), 2116G>C (G706R), 3024G>A (Q1008Q), 3188G>A (Q1063R), 5797C)T (R1933X), 6423A>G (I2141M), and the variants 6542C)T and 6676T>A, both in the 3' untranslated region. One control subject and three members of a non-RP family were heterozygous for R1933X, which is therefore likely to be a non-disease-causing variant. The most C-terminal truncation previously reported was due to Tyr1053 (1-bp del) and occurred in RP patients. Thus the presence of a normal level of at least part of RPRP. Hum Mutat 17:436, 2001.

Mutations in P<em>RP</em>F3, a gene encoding the essential pre-mRNA splicing factor Hprp3p, have been identified in patients with autosomal dominant retinitis pigmentosa type 18 (<em>RP</em>18). Patients with <em>RP</em>18 have one of two single amino acid substitutions, Pro493Ser or Thr494Met, at the highly conserved Hprp3p <em>C</em>-terminal region. Pro493Ser occurs sporadically, whereas Thr494Met is observed in several unlinked <em>RP</em> families worldwide. The latter mutation also alters a potential recognition motif for phosphorylation by casein kinase II (<em>C</em>KII). To understand the molecular basis of <em>RP</em>18, we examined the consequences of Thr494Met mutation on Hprp3p molecular interactions with components of the U4/U6.U5 small nuclear ribonucleoprotein particles (snRNPs) complex. Since numerous mutations causing human diseases change pre-mRNA splice sites, we investigated whether Thr494Met substitution affects the processing of P<em>RP</em>F3 mRNA. We found that Thr494Met does not affect P<em>RP</em>F3 mRNA processing, indicating that the mutation may exert its effect primarily at the protein level. We used small hairpin RNAs to specifically silence the endogenous P<em>RP</em>F3 while simultaneously expressing HA-tagged Thr494Met. We demonstrated that the <em>C</em>- but not N-terminal region of Hprp3p is indeed phosphorylated by <em>C</em>KII in vitro and in cells. <em>C</em>KII-mediated Hprp3p phosphorylation was significantly reduced by Thr494Met mutation. <em>C</em>onsequently, the Hprp3p <em>C</em>-terminal region is rendered partially defective in its association with itself, Hprp4p, and U4/U6 snRNA. Our findings provide new insights into the biology of Hprp3p and suggest that the loss of Hprp3p phosphorylation at Thr494 is a key step for initiating Thr494Met aberrant interactions within U4/U6 snRNP complex and that these are likely linked to the <em>RP</em>18 phenotype.

OBJECTIVE

More than 20 mutations associated with retinitis pigmentosa (RP) have been identified in the retinitis pigmentosa 1 (RPC-terminal of the RPRPCX) domain of RPC-terminal of the protein affects its function is still not known. This study aims to get a better understanding of the RPRP patients.

METHODS

Peripheral blood was taken from 72 RP patients. Together with 101 RP patients and 190 control subjects previously reported, mutation screening was performed by polymerase chain reaction (PCR) and direct sequencing. Statistical analysis was performed using SPSS.

RESULTS

Two novel missense sequence changes, D984G and CG, at the 3' untranslated region were found. They were not found in 190 control subjects. D984G causes RP. It creates two possible N-myristoylation sites according to PROSITE. CRP in the family. It abolishes an N-myristoylation site. R872H, a previously reported polymorphism, was predominantly present in control subjects (P=0.001).

CONCLUSIONS

Our results suggest that disruption of the <em>C</em>-terminal of <em>RP</em>1 may be associated with the development of <em>RP</em>, and the possible involvement of the <em>RP</em>1 polypeptide downstream of its D<em>C</em>X domain in normal <em>RP</em>1 function.

OBJECTIVE

In this study, we investigated the effects of caffeic acid (CAFA), a phenolic acid, on Ca(2＋)-antagonistic cyclic nucleotides associated with the phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) and vasodilator-stimulated phosphoprotein (VASP) and the thromboxane A2 (TXA2)-associated microsomal cyclooxygenase-1 (COX-1) activity in collagen (10 μg/mL)-stimulated platelet aggregation.

METHODS

Washed platelets (10(8)/mL) obtained from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 minutes at 37℃ in the presence of 2 mM exogenous CaCl2 with or without CAFA or other materials, stimulated with collagen (10 μg/mL) for 5 minutes, then used to determine the levels of intracellular cytosolic Ca(2＋) ([Ca(2＋)]i), TXA2, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), COX-1 activity, VASP and IP3R phosphorylation.

RESULTS

CAFA dose-dependently inhibited collagen-induced platelet aggregation and suppressed the production of TXA2, an aggregation-inducing autacoid associated with the strong inhibition of COX-1 in platelet microsomes exhibiting cytochrome C reductase activity. CAFA dose-dependently inhibited collagen-elevated [Ca(2＋)]i mobilization, which was increased by a cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS, but not a cGMP-dependent protein kinase (G-kinase) inhibitor, Rp-8-Br-cGMPS. In addition, CAFA significantly increased the formation of cAMP and cGMP, intracellular Ca(2＋)-antagonists that function as aggregation-inhibiting molecules. CAFA increased IP3R (320 kDa) phosphorylation, indicating the inhibition of IP3-mediated Ca(2＋) release from internal stores (i.e. the dense tubular system) via the IP3R on collagen-activated platelets. Furthermore, CAFA-induced IP3R phosphorylation was strongly inhibited by an A-kinase inhibitor, Rp-8-Br-cAMPS, but only mildly inhibited by a G-kinase inhibitor, Rp-8-Br-cGMPS. These results suggest that the inhibition of [Ca(2＋)]i mobilization by CAFA is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CAFA elevated the phosphorylation of VASP-Ser(157), an A-kinase substrate, but not the phosphorylation of VASP-Ser(239), a G-kinase substrate. We demonstrate that CAFA increases cAMP and subsequently phosphorylates both IP3R and VASP-Ser(157) through A-kinase activation to inhibit [Ca(2＋)]i mobilization and TXA2 production via the inhibition of the COX-1 activity.

CONCLUSIONS

These results strongly indicate that CAFA is a potent beneficial compound that elevates the level of cAMP-dependent protein phosphorylation in collagen-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.

The venom proteome of the monocled cobra, Naja kaouthia, from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A2 (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l-amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a 'toxicity score', were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening.

An RP-HPLC method for the determination of daidzein, genistein, formononetin and biochanin A in red clover (Trifolium pratense L.) was developed and validated. The compounds are quantified after hydrolytic extraction using an internal standard. On a base-deactivated C(18) column good separation of the analytes, also from accompanying substances, and excellent peak shape are achieved by gradient elution with aqueous sulfuric acid and acetonitrile. The method was applied to the analysis of different red clover cultivars.

In contrast to the many studies on the venoms of scorpions, spiders, snakes and cone snails, up to now there has been no report of the proteomic analysis of sea anemones venoms. In this work we report for the first time the peptide mass fingerprint and some novel peptides in the neurotoxic fraction (Fr III) of the sea anemone Bunodosoma cangicum venom. Fr III is neurotoxic to crabs and was purified by rp-HPLC in a C-18 column, yielding 41 fractions. By checking their molecular masses by ESI-Q-Tof and MALDI-Tof MS we found 81 components ranging from near 250 amu to approximately 6000 amu. Some of the peptidic molecules were partially sequenced through the automated Edman technique. Three of them are peptides with near 4500 amu belonging to the class of the BcIV, BDS-I, BDS-II, APETx1, APETx2 and Am-II toxins. Another three peptides represent a novel group of toxins (~3200 amu). A further three molecules (~ approximately 4900 amu) belong to the group of type 1 sodium channel neurotoxins. When assayed over the crab leg nerve compound action potentials, one of the BcIV- and APETx-like peptides exhibits an action similar to the type 1 sodium channel toxins in this preparation, suggesting the same target in this assay. On the other hand one of the novel peptides, with 3176 amu, displayed an action similar to potassium channel blockage in this experiment. In summary, the proteomic analysis and mass fingerprint of fractions from sea anemone venoms through MS are valuable tools, allowing us to rapidly predict the occurrence of different groups of toxins and facilitating the search and characterization of novel molecules without the need of full characterization of individual components by broader assays and bioassay-guided purifications. It also shows that sea anemones employ dozens of components for prey capture and defense.

The multicomponent venom of the spider Cupiennius salei was separated by three different chromatographic strategies to facilitate subsequent analysis of peptidic venom components by tandem mass spectrometry (MALDI-TOF-MS and ESI-MS), Edman degradation and amino acid analysis: (a) desalting of the crude venom by RP-HPLC only, (b) chromatographic separation of the crude venom into 42 fractions by RP-HPLC, and (c) multidimensional purification of the crude venom by size exclusion and cation exchange chromatography and RP-HPLC. A total of 286 components were identified in the venom of C. salei by mass spectrometry and the sequence of 49 new peptides was determined de novo by Edman degradation and tandem mass spectrometry; 30 were C-terminally amidated. The novel peptides were assigned to two main groups: (a) short cationic peptides and (b) Cys-containing peptides with the inhibitor cystine knot motif. Bioinformatics revealed a limited number of substantial similarities, namely with the peptides CpTx1 from the spider Cheiracantium punctorium and U3-ctenitoxin-Asp1a from the South American fishing spider (Ancylometes sp.) and with sequences from a Lycosa singoriensis venom gland transcriptome analysis. The results clearly indicate that the quality of the data is strongly dependent on the chosen separation strategy. The combination of orthogonal analytical methods efficiently excludes alkali ion and matrix adducts, provides indispensable information for an unambiguous identification of isomasses, and results in the most comprehensive repertoire of peptides identified in the venom of C. salei so far.

A cell surface antigen that is expressed by normal and 95% of transformed colonic epithelium and is recognized by the monoclonal antibody A33 (Welt, S., Divgi, C. R., Real, F. X., Yeh, S. D., Garin-Chesa, P., Finstad, C. L., Sakamoto, J., Cohen, A., Sigurdson, E. R., Kemeny, N., Carswell, E. A., Oettgen, H. F., and Old, L. J. (1990) J. Clin. Oncol. 8, 1894-1906) has been purified to homogeneity from the human colonic carcinoma cell line LIM1215. The A33 protein was purified from Triton X-114 extracts of LIM1215 cells under nondenaturing conditions. These extracts were applied sequentially to Green-Sepharose HE-4BD, Mono-Q HR 10/10, Superose 12 HR 10/30, and micropreparative Brownlee Aquapore RP 300. The purification was monitored by biosensor analysis using surface plasmon resonance detection with a F(ab')2 fragment of the humanized A33 monoclonal antibody immobilized on the sensor surface and Western blot analysis following SDS-polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions using humanized A33 monoclonal antibody. The purified A33 antigen has a Mr on SDS-PAGE of 43,000 under nonreducing conditions. By contrast, the purified protein displayed a Mr of approximately 180,000 under native conditions on both size exclusion chromatography and native PAGE, possibly due to the formation of a homotetramer. N-terminal amino acid sequence analysis of the purified protein identified 34 amino acid residues of a unique sequence: ISVETPQDVLRASQGKSVTLPXTYHTSXXXREGLIQWD. A polyclonal antibody was raised against a synthetic peptide corresponding to residues 2-20 of this sequence. The antipeptide serum recognized the purified protein using Western blot analysis under both nonreducing (Mr 43,000) and reducing (Mr 49,000) conditions.

BACKGROUND

The authors previously found that, although the total percentage of prostate needle biopsy cores with carcinoma was a significant predictor of prostate specific antigen (PSA) failure among men undergoing radical prostatectomy (RP), there was a trend toward a lower risk of recurrence in patients with positive bilateral biopsies, suggesting that high-volume, unilateral disease was a worse predictor of outcome than an equivalent number of positive cores distributed over two lobes. In the current study, the authors sought to compare the total percentage of cores with carcinoma directly with the percentage of cores from the more involved or dominant side of the prostate with carcinoma for their ability to predict outcome among men who underwent RP.

METHODS

A retrospective survey of 535 patients from the Shared Equal Access Regional Cancer Hospital database who underwent RP at 4 different equal-access medical centers between 1988 and 2002 was undertaken. The total percentage of cores positive was compared with the percentage of cores positive from the dominant and nondominant sides for their ability to predict biochemical recurrence after RP. The best predictor then was compared with the standard clinical variables PSA, biopsy Gleason score, and clinical stage in terms of ability to predict time to PSA recurrence after RP using multivariate analysis.

RESULTS

The adverse pathologic features of positive surgical margins and extracapsular extension were significantly more likely to be ipsilateral to the dominant side on the prostate biopsy. The percentage of cores positive from the dominant side provided slightly better prediction (concordance index [C] = 0.636) for PSA failure than the total percentage of cores positive (C = 0.596) and markedly better than the percentage of cores from the nondominant side (C = 0.509). Cutoff points for percentage of cores positive from the dominant side were identified (< 34%, 34-67%, and>> 67%) that provided significant risk stratification for PSA failure (P < 0.001). On multivariate analysis, the percentage of cores positive from the dominant side was the strongest independent predictor of PSA recurrence (P < 0.001). Biopsy Gleason score (P = 0.017) also was a significant, independent predictor of recurrence. There was a trend, which did not reach statistical significance, toward an association between greater PSA values and biochemical failure (P = 0.052). Combining the PSA level, biopsy Gleason score, and percentage of cores positive from the dominant side of the prostate resulted in a model that provided a high degree of prediction for PSA failure (C = 0.671).

CONCLUSIONS

The percentage of cores positive from the dominant side of the prostate was a slightly better predictor of PSA recurrence than was the total percentage of cores positive. Using the percentage of cores from the dominant side along with the PSA level and the biopsy Gleason score provided significant risk stratification for PSA failure.

The relationship of virus load to clinical disease progression in HIV-infected children remains to be elucidated. In this study, HIV-1 proviral DNA load was determined in peripheral blood mononuclear cells (PBMCs) by the quantitative competitive DNA polymerase chain reaction assay (QC-PCR) in 47 HIV-infected children subdivided by age (group I, < or = 2 years; group II,>> or = 5 years), who were further categorized to include 12 rapid progressors (RP, age < or = 2 years, Centers for Disease Control [CDC] defined clinical category C and/or immune category 3, or death before age 2 years) and slow progressors (SP, age>> or = 5 years, excluding CDC categories C and/or immune category 3). Significantly higher mean proviral copies/10(3) PBMCs were detected in group I versus group II (75.4 +/- 104.3 and 13.0 +/- 17.8 respectively, p < 0.0001) and in RP (158.0 +/- 118.2) as compared to either SP (11.8 +/- 18.8, p < 0.0001) or other age-matched infected children (20.3 +/- 38.8, p < 0.0001). Thus HIV-infected children appear to have a higher cell-associated virus load early in life, especially in association with rapid disease progression.

The ubiquitin-proteasome pathway is a major proteolytic system in eukaryotic cells and regulates various cellular processes. The 26 S proteasome, the central enzyme of this pathway, consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. Growing evidence indicates that proteasome assembly is assisted by a variety of chaperones. In particular, it has been reported recently that Nas2, Nas6, Rpn14, and Hsm3 bind specific Rpt subunits, thereby contributing to the formation of 19 S RP. Rpn14 transiently binds to the C-terminal domain of the Rpt6 subunit (Rpt6-C) during maturation of the ATPase ring of 19 S RP. In this study, we determined the crystal structure of yeast Rpn14 at 2.0 A resolution, which revealed that this chaperone consists of a unique N-terminal domain with unknown function and a C-terminal domain assuming a canonical seven-bladed beta-propeller fold. The Rpt6-binding site on Rpn14 was predicted based on structural comparison with the complex formed between Nas6 and Rpt3-C. The top face of Rpn14 exhibits a highly acidic surface area, whereas the putative interacting surface of Rpt6-C is basic. By inspection of structural data along with genetic and biochemical data, we determined the specific residues of Rpn14 and Rpt6 for complementary charge interactions that are required for 19 S RP assembly.

The aspartate residue (Asp 32) located in the complementarity-determining region (CDR) of a recombinant humanized monoclonal antibody (MAb I) is highly susceptible to the isomerization reaction. The modification of Asp 32 residue due to the isomerization reaction results in a significant reduction in the binding affinity of MAb I to IgE. The binding of a MAb I therapeutic to IgE is important for its desired pharmacological effect. In earlier investigations, we demonstrated that the conformational flexibility and residue exposure are factors that are responsible for the observed reactivity of Asp 32 in MAb I. This report explores the role of cosolutes such as glycerol and sucrose in the modulation of Asp 32 reactivity in MAb I. These cosolutes are routinely incorporated in injectable pharmaceutical formulations. The reactivity of the Asp residue in MAb I in these different cosolute-based formulations was compared to its reactivity in a peptide model VDYDG comprising residues 29-33 of MAb I. The formulations of MAb I and VDYDG containing varying concentrations of glycerol and sucrose were incubated at 50 degrees C for a period of 5-7 days. The isomerization of the Asp residue in VDYDG and MAb I was monitored using rp-HPLC and hydrophobic interaction chromatography (HIC), respectively. Structural analysis of MAb I using differential scanning calorimetry (DSC) demonstrated that the structural stability of MAb I was increased in formulations containing glycerol and sucrose. However, the stability of Asp 32 in MAb I was significantly decreased in these formulations. This research suggests that a formulation approach that relies purely on enhancing the structural stability of proteins through addition of these cosolutes could result in problems associated with the chemical stability of these biomolecules.

Therapy of vancomycin-resistant enterococcal infections is an increasing problem for many large medical centers. One promising agent for use against Enterococcus faecium is RP 59500 (Quinupristin/Dalfopristin). To assess the potential for emergence of resistant strains during clinical trials with this new compound, we collected and tested 11 vancomycin-resistant and three vancomycin-susceptible E. faecium strains. The strains were exposed to doubling dilutions of RP 59500, beginning at drug concentrations ranging from 0.25 to 16 micrograms/ml agar. A saturated swab with approximately 3 x 10(7) organisms/ml was spread as a lawn on agar containing RP 59500 and incubated at 35 degrees C for 48 h. Growth on the highest drug-containing plate was used to prepare the inoculum for the next series of resistance-selection experiments. After the final passage, the range of the highest RP 59500 plate concentration with growth was 32-512 micrograms/ml (initial resistance frequency ranging from 1 x 10(-6) to>> 1 x 10(-4). After the selection of resistant strains, the organisms were passed twice weekly on antimicrobial agent-free media to determine resistance stability in the absence of drug. Resistance in strains with an RP 59500 MIC of>> or = 16 micrograms/ml was stable after repeated passage for 4 weeks. These results indicate that stable resistance to RP 59500 can be selected in E. faecium when the organism is exposed to increasing drug concentrations only slightly above the minimum inhibitory concentration (MIC). This stable resistance is seen in strains exhibiting an MIC>> or = 16 micrograms/ml, and unstable in those with resistance where the RP 59500 MIC is less than eight times the original drug MIC. Our observation suggests more than one mechanism of resistance is likely present when stable resistance to RP 59500 develops.

In the present study, possible mechanisms involved in the tetanus-induced potentiation of gamma-aminobutyric acid-A (GABA-A) receptor-mediated inhibitory postsynaptic currents (IPSCs) were investigated using the whole cell voltage-clamp technique on CA1 neurons in rat hippocampal slices. Stimulations (100 Hz) of the stratum radiatum, while voltage-clamping the membrane potential of neurons, induces a long-term potentiation (LTP) of evoked fast IPSCs while increasing the number but not the amplitude of spontaneous IPSCs (sIPSCs). The potentiation of fast IPSCs was input specific. During the period of IPSC potentiation, postsynaptic responses produced by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride and baclofen, GABA-A and GABA-B agonists respectively, were not significantly different from control. CGP 36742, a GABA-B antagonist, blocked the induction of tetanus-induced potentiation of evoked and spontaneous IPSCs, while GTPgammaS, an activator of G proteins, substitution for GTP in the postsynaptic recording electrode did not occlude potentiation. Since GABA-B receptors work through G proteins, our results suggest that pre- but not postsynaptic GABA-B receptors are involved in the potentiation of fast IPSCs. A tetanus delivered when GABA-A responses were completely blocked by bicuculline suggests that GABA-A receptor activation during tetanus is not essential for the induction of potentiation. Rp-cAMPs, an antagonist of protein kinase A (PKA) activation, blocks the induction of potentiation of fast IPSCs. Forskolin, an activator of PKA, increases baseline evoked IPSCs as well as the number of sIPSCs, and a tetanic stimulation during this enhancement uncovers a long-term depression of the evoked IPSC. Sulfhydryl alkylating agents, N-ethylmaleimide and p-chloromercuribenzoic acid, which have been found to presynaptically increase GABA release and have been suggested to have effects on proteins involved in transmitter release processes occurring in nerve terminals, occlude tetanus-induced potentiation of evoked and spontaneous IPSCs. Taken together our results suggest that LTP of IPSCs originates from a presynaptic site and that GABA-B receptor activation, cyclic AMP/PKA activation and sulfhydryl-alkylation are involved. Plasticity of IPSCs as observed in this study would have significant implications for network behavior in the hippocampus.

Raynaud's phenomenon (RP) is a disorder characterized by episodic periods of vasoconstriction typically provoked by exposure to cold. Phosphodiesterase 5 (PDE5) inhibitors may improve digital blood flow and clinical symptoms in patients with RP, but the mechanisms are unknown. We examined the hypothesis that a PDE5 inhibitor, tadalafil, attenuates cold-induced vasoconstriction. Additionally, we examined whether tadalafil reduced vascular dysfunction following ischemia, thus altering the response to repeated cooling. We conducted a double-blind, placebo-controlled crossover study in 20 subjects with RP on two separate study days, when subjects received either placebo or tadalafil (10 mg). Digital blood flow (flux) was measured by laser Doppler flowmetry at rest and during two graduated local heat and cold exposure cycles. Temperature-response curves were evaluated by E(max) (maximal flux during heating), E(min) (minimal flux during cooling), and ET(50) and ET(90) (the local temperature at which flux decreased by 50% and 90% of E(max)-E(min), respectively). Tadalafil did not increase baseline flux (81.0+/-73.0 vs 91.3+/-114.0 arbitrary unit (AU), P=0.57), E(max) (280.0+/-107.6 vs 279.5+/-119.8 AU, P=0.94), ET(50) (25.4+/-4.4 vs 26.6+/-5.7 degrees C, P=0.62), or ET(90) (21.2+/-3.9 vs 21.8+/-5.0 degrees C, P=0.78), (cycle 1 values presented). There were no differences between cycles on either study day. In conclusion, in patients with RP, single-dose tadalafil does not increase digital blood flow at baseline or in response to heating, nor does it attenuate cold-induced vasoconstriction. Furthermore, it does not precondition the endothelium to resist a second cooling challenge. The clinical benefit in patients with RP treated with PDE5 inhibitors probably involves mechanisms other than acute inhibition of cold-induced vasoconstriction.

Citrullination is a protein PTM of arginine residues catalyzed by peptidylarginine deiminase. Protein citrullination has been detected in the CNS and associated with a number of neurological diseases. However, identifying citrullinated proteins from complex mixtures and pinpointing citrullinated residues have been limited. Using RP LC and high-resolution MS, this study determined in vitro citrullination sites of glial fibrillary acid protein (GFAP), neurogranin (NRGN/RCCC, and Glu-C digests. The relative ratio of citrullination was estimated by simultaneous identification of citrullinated and unmodified peptides from Alzheimer's and control brain samples. The site occupancy of citrullination at the residue R68 of NRGN ranged from 1.6 to 9.5%. Compared to CID, higher-energy collisional dissociation (HCD) mainly produced protein backbone fragmentation for citrullinated peptides. CID-triggered HCD fragmentation is an optimal approach for the identification of citrullinated peptides in complex protein digests.

While primary Raynaud phenomenon (RP) only rarely leads to complications, secondary RP when associated with systemic sclerosis (SSc) frequently results in necrosis, ulcers or even gangrene. Therefore timely therapeutic intervention is required. Management of RP includes physical therapy, avoidance of triggers, and a variety of medications, depending on the disease severity. On the basis of published studies and of our own experience we suggest: a) for primary and uncomplicated secondary RP, calcium channel blockers in adequate doses; b) for SSc-associated secondary RP with hypertension, cardial or renal involvement, drugs which interfere with angiotensin; c) in case of concomitant depression, exploitation of the vasodilatory effects of serotonin reuptake inhibitors, d) in presence of ulcers or marked trophic alterations, intravenous administration of iloprost, e) for recalcitrant cases and endangered digits, sildenafil, f) for prevention of severe relapses, the endothelin-receptor antagonist bosentan. The drugs mentioned in c-f are not approved for RP and used off-label. In patients with secondary SSc-associated RP, timely therapy can reduce the percentage of complications such as ulcers or amputations. In addition, timely treatment of RP possibly retards ensuing fibrotic processes.

The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31-8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine 3',5'-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules. alpha-Melanocyte stimulating hormone (alpha-MSH), like PKC activators, overcame melatonin-induced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after alpha-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of Rp-cAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase.

1. The kinetics of the degradation of the kinins bradykinin and Met-Lys-bradykinin, angiotensins I and II and the tachykinin substance P by PMNL-collagenase (MMP 8), PMNL-gelatinase (MMP 9) and by the recombinant catalytic domain of MMP 8 (rcd-PMNL-c) was examined by RP-HPLC. The resulting fragments were identified by automated Edman degradation or by amino acid analysis. 2. The initial degradation rates of substance P at a substrate concentration of 25 microM were 5 min-1 for MMP 9 and 150 min-1 for MMP 8. The kinetic constants KM and kcat were determined by concentration-dependent measurements. For MMP 8/substance P the constants were KM = 78 +/- 14 microM and kcat = 412 +/- 67 min-1. For MMP 9/substance P the constants were KM = 91 +/- 15 microM and kcat = 25 +/- 4 min-1. Both enzymes cleaved substance P between Gln6 and Phe7 and between Gly9 and Leu10. 3. Under the same conditions, MMP 8 degraded angiotensin I at an initial rate of 20 h-1, resulting mainly in the vasoactive fragments angiotensin II and angiotensin(1-7). At a substrate concentration of 25 microM and an enzyme/substrate ratio of 1:100, angiotensin II was degraded very slowly (19% in 24 h) by MMP 8. Under these conditions, MMP 9 degraded angiotensin I to a lesser extent than MMP 8 (25% in 24 h) and was unable to cleave angiotensin II. 4. Under the same conditions, bradykinin and Met-Lys-bradykinin were cleaved by PMNL-collagenase at a rate of 20% in 24 h, producing BK(1-7) and BK(1-8). PMNL-gelatinase was unable to cleave the kinins under these conditions. 5. In all cases, rcd-PMNL-c produced the same fragments as wild type PMNL-collagenase, but at a significantly lower rate.

The reactivity of recombinant pea cytosolic ascorbate peroxidase (rAPX) towards H2O2, the nature of the intermediates and the products of the reaction have been examined using UV/visible and EPR spectroscopies together with HPLC. Compound I of rAPX, generated by reaction of rAPX with 1 molar equivalent of H2O2, contains a porphyrin pi-cation radical. This species is unstable and, in the absence of reducing substrate, decays within 60 s to a second species, compound I*, that has a UV/visible spectrum [lambda(max) (nm) = 414, 527, 558 and 350 (sh)] similar, but not identical, to those of both horseradish peroxidase compound II and cytochrome c peroxidase compound I. Small but systematic differences were observed in the UV/visible spectra of compound I* and authentic rAPX compound II, generated by reaction of rAPX with 1 molar equivalent H2O2 in the presence of 1 molar equivalent of ascorbate [lambda(max) (nm) = 416, 527, 554, 350 (sh) and 628 (sh)]. Compound I* decays to give a 'ferric-like' species (lambda(max) = 406 nm) that is not spectroscopically identical to ferric rAPX (lambda(max) = 403 nm) with a first order rate constant, k(decay)' = (2.7 +/- 0.3) x 10(-4) s(-1). Authentic samples of compound II evolve to ferric rAPX [k(decay) = (1.1 +/- 0.2) x 10(-3) s(-1)]. Low temperature (10 K) EPR spectra are consistent with the formation of a protein-based radical, with g values for compound I* (g parallel = 2.038, g perpendicular = 2.008) close to those previously reported for the Trp191 radical in cytochrome c peroxidase (g parallel = 2.037, g perpendicular = 2.005). The EPR spectrum of rAPX compound II was essentially silent in the g = 2 region. Tryptic digestion of the 'ferric-like' rAPX followed by RP-HPLC revealed a fragment with a new absorption peak near 330 nm, consistent with the formation of a hydroxylated tryptophan residue. The results show, for the first time, that rAPX can, under certain conditions, form a protein-based radical analogous to that found in cytochrome c peroxidase. The implications of these data are discussed in the wider context of both APX catalysis and radical formation and stability in haem peroxidases.

OBJECTIVE

We evaluated the attitude in using chemotherapy near the end of life in advanced pancreatic adenocarcinoma (PAC). Clinical and laboratory parameters recorded at last chemotherapy administration were analyzed, in order to identify risk factors for imminent death.

METHODS

Retrospective analysis of patients who underwent at least one line of palliative chemotherapy was made. Data concerning chemotherapy (regimens, lines, and date of last administration) were collected. Clinical and laboratory factors recorded at last chemotherapy administration were: performance status, presence of ascites, hemoglobin, white blood cell (WBC), platelets, total bilirubin, albumin, LDH, C-reactive protein (C-rp), and Ca 19.9.

RESULTS

We analyzed 231 patients: males/females, 53/47 %; metastatic/locally advanced disease, 80/20 %; and median age, 66 years (range 32-85). All patients died due to disease progression. Median overall survival was 6.1 months (95 % CI 5.1-7.2). At the last chemotherapy delivery, performance status was 0-1 in 37 % and 2 in 63 %. Fifty-nine percent of patients received one chemotherapy line, while 32, 8, and 1 % had second-, third-, and fourth line, respectively. The interval between last chemotherapy administration and death was <4 weeks in 24 %, ≥4-12 in 47 %, and >12 in 29 %. Median survival from last chemotherapy to death was 7.5 weeks (95 % CI 6.7-8.4). In a univariate analysis, ascites, elevated WBC, bilirubin, LDH, C-rp and Ca 19.9, and reduced albumin were found to predict shorter survival; however, none of them remained significant in a multivariate analysis.

CONCLUSIONS

A significant proportion of patients with advanced PAC received chemotherapy within the last month of life. The clinical and laboratory parameters recorded at last chemotherapy delivery did not predict shorter survival.