Initiation of translation of genomic RNA (gRNA) of hepatitis C virus (HCV) is provided by a highly structured fragment in its 5'-untranslated region, the so-called Internal Ribosome Entry Site (IRES). In this work, the exposed NH2-groups of proteins in the 40S subunit of the human ribosome and in its binary complexes with RNA transcripts corresponding to the full-size HCV IRES or its fragments were probed using the N-hydroxysuccinimide derivative of the fluorescent dye Cy3. Comparison of efficiencies of modification of ribosomal proteins in free subunits and in their binary complexes with the RNA transcripts revealed ribosomal proteins involved in the HCV IRES binding. It was found that binding of the 40S subunits with the RNA transcript corresponding to full-size HCV IRES results in a decrease in modification levels of ribosomal protein (rp) S27 and, to a lesser extent of rpS10; also, a noticeable decrease in the efficiency of labeling of proteins RACK1/S2/S3a was observed. When a fragment of HCV IRES containing the initial part of the open reading frame (ORF) of the viral gRNA was deleted, the level of rpS10 modification became the same as in free subunits, whereas the levels of modification of rpS27 and the RACK1/S2/S3a group remained virtually unchanged compared to those observed in the complex of 40S subunit with the full-size HCV IRES. Binding of 40S subunits to a fragment of the HCV IRES lacking an ORF and domain II increased the modification level of the RACK1/S2/S3a proteins, while the efficiencies of labeling of rpS10 and rpS27 remained the same as upon the deletion of the ORF fragment. Comparison of these results with known structural and biochemical data on the organization of 40S subunit and the location of the HCV IRES on it revealed structural elements of the IRES contacting exposed lysine residues of the above-mentioned ribosomal proteins. Thus, it was found that the majority of exposed lysine residues of rpS27 are involved in the binding of the HCV IRES region formed by the junction of subdomains IIIa, IIIb, and IIIc with the central stalk of domain III, and that several lysine residues of rpS10 participate in the binding of the HCV IRES region corresponding to the initial part of the ORF of the viral gRNA. In addition, we concluded that lysine residues of rpS3a are involved in the binding of domains II and III of HCV IRES.

An injectable hyaluronic acid (HA) microhydrogel was successfully developed as a novel drug carrier for controlled release formulation of protein drugs. HA hydrogels were prepared by the disulfide bond formation of thiolated HA (HA-SH). EPO was loaded in situ during HA-SH hydrogel preparation using an accelerating agent of sodium tetrathionate. The gelation time was drastically reduced from a day to 30 min when sodium tetrathionate was added for HA-SH hydrogel preparation. In vitro release of EPO in PBS at 37 degrees C showed that EPO was rapidly released for 3 days with an initial burst and then slowly up to 9 days from HA-SH hydrogels. HA-SH microhydrogels were prepared by the reactive spray drying of diluted HA-SH precursor solution. The mean particle size was approximately 2.3 mum and the water content after spray drying was approximately 14%. Ellman's test showed that sodium tetrathionate contributed not only for rapid crosslinking reaction but also for the reduction of residual free thiol content in HA-SH microhydrogels after spray drying. EPO recovery from HA-SH microhydrogels after degradation with hyaluronidase SD was higher than 95%. The released EPO appeared to be intact from the analysis with RP-HPLC. According to in vivo release test of EPO from HA-SH microhydrogels in Sprague Dawley (SD) rats, elevated plasma concentration of EPO higher than 0.1 ng/mL, which is a critical minimal concentration for EPO efficacy, was maintained up to 7 days. There was no adverse effect during and after the in vivo tests.

BACKGROUND

Mitochondrial superoxide radical (O(2)(•¯)) production increases after cardiac ischemia/reperfusion (IR). Ischemic preconditioning (IPC) preserves mitochondrial function and attenuates O(2)(•¯) production, but the mechanism is unknown. Mitochondrial membrane potential (mΔΨ) is known to affect O(2)(•¯) production; mitochondrial depolarization decreases O(2)(•¯) formation. We examined the relationship between O(2)(•¯) production and mΔΨ during IR and IPC.

METHODS

Rat hearts were subjected to Control or IPC. Mitochondria were isolated at end equilibration (End EQ), end ischemia (End I), and end reperfusion (End RP). mΔΨ was measured using a tetraphenylphosphonium electrode. Mitochondrial O(2)(•¯) production was measured by electron paramagnetic resonance using DMPO spin trap. Cytochrome c levels were measured using high-pressure liquid chromatography.

RESULTS

IPC preserved mΔΨ at End I (-156 ± 5 versus -131 ± 6 mV, P < 0.001) and End RP (-168 ± 2 versus -155 ± 2 mV, P < 0.05). At End RP, IPC attenuated O(2)(•¯) production (2527 ± 221 versus 3523 ± 250 AU/mg protein, P < 0.05). IPC preserved cytochrome c levels (351 ± 14 versus 269 ± 16 picomoles/mg protein, P < 0.05) at End RP, and decreased mitochondrial cristae disruption (10% ± 4% versus 33% ± 7%, P < 0.05) and amorphous density formation (18% ± 4% versus 28% ± 1%, P < 0.05).

CONCLUSIONS

We conclude that IPC preserves mΔΨ, possibly by limiting disruption of mitochondrial inner membrane. IPC also decreases mitochondrial O(2)(•¯) production and preserves mitochondrial ultrastructure after IR. While it was previously held that slight decreases in mΔΨ decrease O(2)(•¯) production, our results indicate that preservation of mΔΨ is associated with decreased O(2)(•¯) and preservation of cardiac function in IPC. These findings indicate that the mechanism of IPC may not involve mΔΨ depolarization, but rather preservation of mitochondrial electrochemical potential.

We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freund's adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.

Mercury is one of the most investigated natural elements and potential contaminants in the environment. Antioxidants have long been known to reduce the free radical-induced oxidative damage. Considering the antioxidant properties of melatonin, this study was aimed to evaluate the effect of melatonin on antioxidant system of rat epididymal sperm in vitro. Sperm samples were dispersed in RPS medium (pH 6.9) and incubated with mercury in the form of mercuric chloride (MC) at three different concentrations (1 microM, 10 microM, 100 microM), melatonin (MLT) at a concentration (100 microM) and mercuric chloride+melatonin (100 microM each) for 3h at 32 degrees C. Sperm viability and motility were assessed every 30 min during the 3-h incubation period. An aliquot of sperm sample was homogenised, centrifuged and used for the assay of superoxide dismutase, glutathione peroxidase, glutathione reductase, TBARS assay to detect lipid peroxidation and hydrogen peroxide generation assay. Samples treated with mercury showed a dose-dependent decrease in motility while there was no significant decrease in sperm viability. In mercury-incubated sperm, the activity of superoxide dismutase, glutathione peroxidase and glutathione reductase decreased significantly while TBARS levels and H2O2 generation were increased in a dose-dependent manner. Co-incubation of sperm with mercury and melatonin exhibited no significant changes in the levels of motility, viability and antioxidant indices as compared to untreated controls. The results suggest that graded doses of mercury elicit depletion of antioxidant defense system in sperm without altering the viability and melatonin treatment was found to significantly inhibit oxidative damage caused by mercury.

Cellular potassium extrusion is now considered a natural protective mechanism following myocardial ischemia, and newly synthetized molecules mimicking cellular extrusion of K+ (potassium channel activators) appear promising for cardioprotection, although the underlying mechanisms for their beneficial effects have not been fully characterized. Indeed, the cardioprotective efficacy of K+ channel activators at low temperature or in the presence of the high K+ content of standard cardioplegic solution has never been addressed. Therefore the cardioprotective interaction of the thioformamide K+ channel activator aprikalim (RP 52891) and high K+ content, cold cardioplegia was studied in isolated ischemic rabbit hearts. Isolated hearts were perfused according to the Langendorff procedure at a constant pressure (85 cmH2O; 1 cmH2O = 98.1 Pa); systolic and diastolic left ventricular pressures, coronary flow, and heart rate were monitored throughout the study. Cardiac temperature was monitored through a thermocouple microprobe positioned in the left ventricular free wall. Global ischemia was carried out by completely shutting off the perfusate flow for 90 min, and reperfusion was monitored for 30 min. Several groups of isolated hearts (n = 6 per group) were treated before ischemia with either cold cardioplegia (St-Thomas' Hospital cardioplegic solution, 4 degrees C), aprikalim (10 microM), or glibenclamide (1 microM) alone, or with one of the following combinations: cold cardioplegia + aprikalim, cold cardioplegia + glibenclamide, or cold cardioplegia + both aprikalim and glibenclamide. A 10 microM infusion of aprikalim significantly increased coronary flow (33 to 63 mL/min, +90%) without negative chronotropic or inotropic effects.(ABSTRACT TRUNCATED AT 250 WORDS)

The discovery of quorum-sensing (QS) systems regulating antibiotic resistance and virulence factors (VFs) has afforded a novel opportunity to prevent bacterial pathogenicity. Dietary molecules have been demonstrated to attenuate QS circuits of bacteria. But, to our knowledge, no study exploring the potential of colostrum hexasaccharide (CHS) in regulating QS systems has been published. In this study, we analyzed CHS for inhibiting QS signaling in Staphylococcus aureus. We isolated and characterized CHS from mare colostrum by high-performance thin-layer chromatography (HPTLC), reverse-phase high-performance liquid chromatography evaporative light-scattering detection (RP-HPLC-ELSD), (1)H and (13)C nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS). Antibiofilm activity of CHS against S. aureus and its possible interference with bacterial QS systems were determined. The inhibition and eradication potentials of the biofilms were studied by microscopic analyses and quantified by 96-well-microtiter-plate assays. Also, the ability of CHS to interfere in bacterial QS by degrading acyl-homoserine lactones (AHLs), one of the most studied signal molecules for Gram-negative bacteria, was evaluated. The results revealed that CHS exhibited promising inhibitory activities against QS-regulated secretion of VFs, including spreading ability, hemolysis, protease, and lipase activities, when applied at a rate of 5 mg/ml. The results of biofilm experiments indicated that CHS is a strong inhibitor of biofilm formation and also has the ability to eradicate it. The potential of CHS to interfere with bacterial QS systems was also examined by degradation of AHLs. Furthermore, it was documented that CHS decreased antibiotic resistance in S. aureus. The results thus give a lead that mare colostrum can be a promising source for isolating a next-generation antibacterial.

We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.

The production of the inflammatory mediator paf-acether (paf) from human epidermal cells was investigated in vitro. Human epidermal cells, freshly isolated from normal skin or in culture, were incubated in Tyrode's buffer containing 0.25% lipid-free bovine serum albumin in the presence of 2 microM calcium ionophore A23187, at 37 degrees C, for 1 to 60 min. Paf production slightly began at the first min of stimulation, was significant after 10 min, reached a maximum at 20 min (251 +/- 25 pg/l X 10(6) cells, mean +/- 1 SD), and decreased thereafter. About 50% of the paf amount produced by epidermal cells was recovered in supernatants. Addition of the non-acetylated paf precursor 1-O-octadecyl-sn-glycero-3-phosphocholine, i.e., lyso-paf, at 0.1 microM to epidermal cells during A23187-stimulation did not alter this production. In contrast, addition of acetyl-coenzyme A at 0.1 mM enhanced paf production by 5 times. The material produced by epidermal cells was identical to synthetic paf because: 1) the aggregation of aspirin-treated and ADP-insensitive washed rabbit platelets it induced was inhibited by BN 52021, an antagonist of the paf putative receptor; 2) the factor was inactivated by phospholipase A2 but was insensitive to lipase from Rhizopus arrhizus; 3) it exhibited the same retention time as synthetic paf during standard and reverse-phase (RP) high-pressure liquid chromatography (HPLC) elution. The paf precursors, i.e., lyso-paf and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, were also detected in epidermal cells, stimulated with A23187 or not. As determined by RP-HPLC analysis and confirmed by gas chromatography analysis, these precursors and the paf produced by epidermal cells exhibited more than 90% of a hexadecyl chain at the sn-1 position of the molecule. The present results demonstrate the synthesis and release of paf by normal human epidermal cells. Paf production within the epidermis might account for the development of cutaneous inflammation and the pathogenesis of many skin disorders.

RNA polymerases can synthesize RNA containing phosphorothioate linkages in which a sulfur replaces one of the nonbridging oxygens. Only the Rp isomer is generated during transcription. A Rp phosphorothioate at the 5' splice-site of the Tetrahymena group I intron does not inhibit splicing (McSwiggen, J.A. and Cech, T.R. (1989) Science 244, 679). Transcription of mutants in which the first base of the 3' exon, U+1, was mutated to C or G, in the presence, respectively, of either cytosine or guanosine thiotriphosphate, introduced a phosphorothioate at the 3' splice-site. In both cases exon ligation was blocked. In the phosphorothioate substituted U+1G mutant, a new 3' splice-site was selected one base downstream of the correct site; despite the fact that the correct site was selected with very high fidelity in unsubstituted RNA. In contrast, the exon ligation reaction was successfully performed in reverse using unsubstituted intron RNA and ligated exons containing an Rp phosphorothioate at the exon junction site. Chirality was reversed during transesterification as in 5' splice-site cleavage (vide supra). This suggests that one non-bridging oxygen is particularly crucial for both splicing reactions.

OBJECTIVE

Lipoxygenases (LOX) are lipid-peroxidating enzymes that are implicated in the pathogenesis of a variety of inflammatory disorders such as arthritis, psoriasis, and asthma. 15-LOX catalyzes the oxygenation of free arachidonic acid to 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), which is reduced to 15-hydroxyeicosatetraenoic acid (15-HETE). The biological role of 15-HETE is less clear. We sought to determine if cultured human rheumatoid synovial cells were able to express 15-LOX mRNA, leading to the synthesis of 15-HETE, and to examine the effect of different cytokines on 15-LOX activity.

METHODS

Adherent synovial cells were obtained by enzymatic digestion of rheumatoid synovium, isolated from patients with rheumatoid arthritis (RA) undergoing hip synovectomy. Between passages 4 and 8, reticulocyte-type 15-LOX expression in these cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) in situ and confirmed by classical RT-PCR analysis followed by enzymatic digestion. The PCR fragment was purified, amplified, and sequenced. Cultured synovial cells were incubated with or without different cytokines and exogenous [1-(14)C] arachidonic acid metabolism of synoviocytes was analyzed by reverse phase high performance liquid chromatography (RP-HPLC).

RESULTS

RT-PCR results showed that human RA type B synoviocytes expressed a reticulocyte-type 15-LOX. By sequence analysis, the PCR fragment (474 bp) was determined to be 100% identical to that of reticulocyte-type 15-LOX cDNA. Other results associated specific inflammatory cytokines with the activity of 15-LOX in these cells. RP-HPLC analysis showed that interleukin 4 (IL-4) increased 15-HETE production (2.4-fold); we also observed an increase in 15-HETE production (1.2-fold) after incubation of the cells with IL-1beta.

CONCLUSIONS

Human RA type B synoviocytes are able to express 15-LOX mRNA leading to the synthesis of 15-HETE, which is modulated by various cytokines that play a major role in the pathophysiology of RA, especially IL-4 and IL-1.

Glutamatergic neurotransmission is associated with release of arachidonic acid (AA) from membrane phospholipids of both neurons and astrocytes. Since free AA has been shown to enhance glutamate-mediated synaptic transmission, it can be postulated that glutamate release and AA formation constitute a positive feed-back mechanism for sustained excitatory neurotransmission. In the present study, we examined whether the glutamate-evoked release of AA could be modulated by peptides. Using mouse cortical neurons in primary cultures, we show that the release of AA evoked by glutamate is potentiated by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide (PACAP). This effect is mediated through the activation of PACAP I receptors. However, several arguments show that this potentiating mechanism does not involve the cAMP/PKA pathway. 1) Increasing intracellular cAMP by either cholera toxin, forskolin, or 8-Br-cAMP treatments does not affect the glutamate-evoked release of AA; 2) potentiation of the glutamate response by PACAP is not prevented by the PKA inhibitor 8-Br-Rp-cAMPS. Also, an involvement of the phospholipase C protein kinase C pathways is unlikely since inhibitors of both phospholipase C (i.e. U-73122) and protein kinase C (i.e. Ro 31-8220) do not affect the potentiation of the glutamate response by PACAP. These observations indicate an effect mediated by PACAP I receptors, which does not involve the second messenger pathways classically associated with activation of this type of receptors. Furthermore, results indicate that this potentiating mechanism mediated by PACAP I receptor acts at a level downstream of the glutamate receptor-mediated calcium influx.

Protein kinase C has been previously shown both to phosphorylate and to desensitize the ability of the human 5-HT1A receptor to inhibit adenylyl cyclase [Raymond, J. R. (1991) J. Biol. Chem. 266, 14747-14753]. In this study, we examined the effects of short-term treatment with protein kinase A activators on coupling to the inhibition of adenylyl cyclase and on phosphorylation of the human serotonin 5-HT1A receptor in CHO cells that stably express 1200 fmol of receptor/mg of protein. Forskolin induced a concentration- and time-dependent phosphorylation of the receptor that was detectable at 5 min and maximal at 15-30 min with a half-maximal concentration of 10-20 microM. Phosphorylation was also induced by Sp-cAMPS or dibutyryl-cAMP, and blocked by Rp-cAMPS and a pseudosubstrate inhibitor of PKA, but not by heparin (inhibitor of receptor kinase) or sphingosine (inhibitor of PKC). The stoichiometry of phosphorylation induced by forskolin was 1 mol of phosphate per mole of receptor. PKA activators did not induce a measurable desensitization of 5-HT1A receptor-inhibited adenylyl cyclase activity. However, forskolin augmented the desensitization caused by a submaximal concentration of phorbol 12-myristate 13-acetate (300 nM PMA) as evidenced by a rightward shift of the concentration-response curve for 5-HT, and approximately doubled the amount of phosphate incorporated into the receptor by PMA. Forskolin did not augment desensitization or increase the degree of phosphorylation induced by a maximal concentration of PMA (5 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Bidirectional fluxes of Cl- across isolated and stripped goldfish intestinal epithelium mounted in Ussing-type chambers increased after addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), suggesting an increase of the paracellular permeability for Cl-. Confirming this, the addition of 8-Br-cAMP to the stripped intestine reduced the diffusion potential generated by isosmotic serosal or mucosal replacement of part of the NaCl by mannitol. The addition of the protein kinase C (PKC) activator 4 beta-phorbol 12,13-dibutyrate (PDB), 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), or the Ca2+ ionophore A23187 was without effect on the Cl- permeability. The cAMP-specific reduction of the diffusion potential was used to screen the epithelium for the presence of receptors coupled to adenylyl cyclase. The results indicate the presence of a serotonin (5-HT) receptor, positively coupled to adenylyl cyclase but insensitive to 5-HT1-, 5-HT2-, 5-HT3-, and nonclassical 5-HT4-receptor antagonists. Addition of vasoactive intestinal polypeptide (VIP) also reduced the diffusion potential in a dose-dependent way. Epinephrine restored the diffusion potential after its reduction by 5-HT or VIP. This effect could be mimicked by the partial alpha 2-adrenergic receptor agonist clonidine and blocked by the alpha 2-antagonists yohimbine and idazoxan. The Rp diastereoisomer of cAMP, (Rp)adenosine 3',5'-cyclic phosphorothioate [(Rp)cAMPS], counteracted the effect of VIP. The results indicate that in goldfish enterocytes VIP and 5-HT reduce the ion selectivity of the tight junctions through elevation of cAMP and that activation of alpha 2-adrenergic receptors antagonize these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

We report the simultaneous determination of the carboxylic acids related to the tricarboxylic acid (TCA) cycle, which plays an important role in producing adenosine triphosphate (ATP) and generating energy in mitochondria. Seven carboxylic acids from the TCA cycle, and pyruvic acid and 2-methylsuccinic acid, as an internal standard, were derivatized with a fluorescent reagent for carboxyl groups, 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ), in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 4-N,N-dimethyaminopyridine as the coupling reagents, at 60 degrees C for 120 min. Subsequently, the excess DBD-PZ was removed efficiently using a cation-exchange cartridge, SDB-RPS (Empore). These fluorescent derivatives were separated well from each other on an octadecyl silica column (TSKgel ODS-80Ts, 250 x 4.6 mm, i.d.) with an eluent of acetonitrile-water containing 1% formic acid at a flow rate of 0.8 mL/min, and were detected fluorometrically at 560 nm, with excitation at 450 nm. The validation data were satisfactory in the range of 2.5-100 microm citric acid, isocitric acid, 2-oxoglutaric acid, succinic acid and fumaric acid. The detection limit (S/N = 3) for citric acid was 2 fmol on the column. The structures of these derivatives were confirmed by high-performance liquid chromatography-mass spectrometry, which proved that their carboxylic groups were completely labeled with DBD-PZ, except for oxaloacetic acid. This HPLC method was successfully applied to the analysis of TCA cycle metabolites in rat urine. The method will also be useful for metabolome research, such as for target analyses of metabolites with carboxyl groups, not only in urine but also in cells and organs.

The association of retinitis pigmentosa (RP) and microphthalmia has been reported in a number of familial and isolated cases. Here, the results of genetic analysis in a familial case of early RP associated with nanophthalmos are described. Two affected sibs were ascertained from an endogamous population in Mexico. A genome-wide linkage analysis was performed by means of an Affymetrix 250K microarray. Five large regions of homozygosity were demonstrated. The largest interval comprised 15.08 Mb at chromosome 1q31-32.1 and contained the Crumbs homologue-1, CRB1, a gene responsible for a number of recessive retinal dystrophies. Nucleotide sequence analysis demonstrated a c.1125C>G transversion in CRB1 exon 5, predicting a novel p.Tyr375X variant. To our knowledge this is the first instance in which a CRB1 mutation has been associated with early RP and nanophthalmos. Our results suggest a role for CRB1 in promoting axial growth of the eye. Clinical analysis of additional subjects with retinal dystrophies due to CRB1 mutations will help to identify if the high hyperopia, a frequently observed trait in these subjects, could be related to decreased eye axial length (nanophthalmos).

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to stimulate melanotroph secretion, and PACAP-like immunoreactivity and expression of PACAP type I receptor messenger RNA have been identified in the pituitary pars intermedia (PI). The present study showed that PACAP messenger RNA is also expressed in the PI. To examine the mechanism of PACAP action in the PI, cytosolic Ca2+ concentrations ([Ca2+]i) and ionic currents were measured in acutely dissociated rat melanotrophs. In about 40% of the melanotrophs studied, PACAP induced an increase in [Ca2+]i, which was suppressed by extracellular Ca2+ removal; extracellular Na+ replacement; the blocker of L-type Ca2+ channels, nicardipine; or the secreto-inhibitory neurotransmitter, dopamine. The PACAP-induced [Ca2+]i increase was mimicked by activators of protein kinase A (PKA) and protein kinase C (PKC), Sp-diastereomer of cAMP and 1-oleoyl-2-acetyl-sn-glycerol, and was reduced by inhibitors of PKA and PKC, Rp-diastereomer of cAMP and staurosporine. Patch-clamp analysis revealed that PACAP caused inward currents with a reversal potential of -0.8 mV and facilitated voltage-dependent Ba2+ currents. It further revealed that PACAP-induced inward currents were mimicked by 1-oleoyl-2-acetyl-sn-glycerol and inhibited by staurosporine, and that Sp-diastereomer of cAMP facilitated Ba2+ currents. These results suggest that PACAP potentiates Ca2+ entry mechanisms of rat melanotrophs by activation of nonselective cation channels via PKC and facilitation of voltage-dependent Ca2+ channels via PKA.

By challenging specific receptors, melatonin synthesized and released by photoreceptors regulates various physiological functions in the vertebrate retina. Here, we studied modulatory effects of melatonin on K+ currents of rod-dominant ON type bipolar cells (Rod-ON-BCs) in rat retinal slices by patch-clamp techniques. Double immunofluorescence experiments conducted in isolated cell and retinal section preparations showed that the melatonin MT₂ receptor was expressed in somata, dendrites and axon terminals of rat Rod-ON-BCs. Electrophysiologically, application of melatonin selectively inhibited the tetraethylammonium (TEA)-sensitive K+ current component, but did not show any effect on the 4-aminopyridine (4-AP)-sensitive component. Consistent with the immunocytochemical result, the melatonin effect was blocked by co-application of 4-phenyl-2-propionamidotetralin (4-P-PDOT), a specific MT₂ receptor antagonist. Neither protein kinase A (PKA) nor protein kinase G (PKG) seemed to be involved because both the PKA inhibitor Rp-cAMP and the PKG inhibitor KT5823 did not block the melatonin-induced suppression of the K+ currents. In contrast, application of the phospholipase C (PLC) inhibitor U73122 or the protein kinase C (PKC) inhibitor bisindolylmaleimide IV (Bis IV) eliminated the melatonin effect, and when the Ca²+ chelator BAPTA-containing pipette was used, melatonin failed to inhibit the K+ currents. These results suggest that suppression of the TEA-sensitive K+ current component via activation of MT₂ receptors expressed on rat Rod-ON-BCs may be mediated by a Ca²+-dependent PLC/inositol 1,4,5-trisphosphate (IP₃/PKC signaling pathway.

OBJECTIVE

To report a prospective, controlled, non-randomized patient study to determine the systemic response to extraperitoneal laparoscopic (eLRP) and open retropubic radical prostatectomy (RRP).

METHODS

In all, 403 patients who had eLRP (163) or open RRP (240) were recruited; patients in both groups had similar preoperative staging. In addition to peri-operative variables (operative duration, complications, blood loss, transfusion rate, hospitalization, catheterization), oncological data (Gleason score, pathological stage, positive margins) were also compared. The extent of the systemic response to surgery-induced tissue trauma was measured in all patients, by assessing the levels of acute-phase markers C-reactive protein (CRP), serum amyloid A (SAA), interleukin-6 (IL-6) and IL-10 before, during and after RP.

RESULTS

The duration of surgery, transfusion rate, hospital stay and duration of catheterization were comparable with those in previous studies. There was an increase in IL-6, CRP and SAA but no change in IL-10, and no differences between eLRP and RRP over the entire period assessed.

CONCLUSIONS

The invasiveness of eLRP could not be substantiated objectively based on the variables measured in this study. The surgical trauma and associated invasiveness of both methods were equivalent.

Ganoderma lucidum is a well-known mushroom with various pharmacological effects that has been used for health and longevity purposes. The objective of this study was to investigate the anti-invasive effect of lucidenic acids isolated from a new G. lucidum strain (YK-02) against human hepatoma carcinoma (HepG(2)) cells. Triterpenoid components in the ethanol extract of G. lucidum (YK-02) were separated by means of a semi-preparative RP HPLC. Four major peaks were separated and crystallized from triterpenoids fraction, and were identified as lucidenic acids A, B, C, and N according to their spectroscopic values of (1)H NMR and MS. Treatment of the lucidenic acids (50 microM) in the presence of 200 nM phorbol 12-myristate 13-acetate (PMA) after 24 h of incubation all resulted in significant inhibitory effects on PMA-induced MMP-9 activity and invasion of HepG(2 )cells. The results indicate that the lucidenic acids isolated from G. lucidum (YK-02) are anti-invasive bioactive components on hepatoma cells.

Adenylyl (beta,gamma-methylene)diphosphonic acid (AMPPCP) labeled with deuterium at the adenine ring ([8-2H]AMPPCP) and at the beta,gamma-methylene group (AMPPCD2P), as well as adenosine 5'-monophosphate labeled at the adenine ring ([8-2H]AMP), was synthesized and used for deuterium nuclear magnetic resonance (NMR) determination of effective correlation times (tau c) of the free nucleotide and the complexes with adenylate kinase (AK). Extensive and rigorous control experiments and theoretical analysis were performed to justify the validity of the experimental approaches, particularly the fast exchange condition, and the reliability of the tau c values obtained. For the free nucleotide, the results suggest that the phosphonate group of free AMPPCP possesses appreciable local mobility relative to the adenine ring and that complexation with Mg2+ greatly reduced such a local mobility. For the complexes with AK, effective tau c values of 7, 15, 28, 28, and 27 ns were obtained for AMPPCD2P, MgAMPPCD2P, [8-2H]AMPPCP, Mg[8-2H]AMPPCP, and [8-2H]AMP, respectively. These results suggest that the adenine ring of substrates is rigidly bound in all cases, that the phosphonate chain of AMPPCP possesses considerable local mobility, and that Mg2+ reduces such local mobility but does not totally immobilize it. The local dynamics of the analogues bound to AK was correlated with local binding energies for the binding of MgAMPPCP and MgATP to AK estimated from the binding studies by proton NMR and other techniques, in conjunction with the binding theory of Jencks [Jencks, W. P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4046-4050]. The results suggest that no general correlation exists between the local rigidity of portions of a bound substrate and the corresponding (ground state) local binding energy contributed by these portions. In particular, the adenosine moiety contributes little to the binding energy despite the fact that the adenine ring is rigidly bound; the triphosphate (PPPi) moiety behaves oppositely; Mg2+ immobilizes the triphosphate chain but does not enhance binding. Finally, isomers of the substitution-inert beta,gamma-bidentate Cr(III) complexes of adenosine 5'-triphosphate (CrATP) were used to probe two unresolved catalytic problems implicitly related to the local mobility of the phosphonate chain of AMPPCP in the AK-MgAMPPCP complex. The first problem concerns the result of electron paramagnetic resonance (EPR) studies that (Rp)- but not (Sp)-[beta-17O]ATP caused a line broadening in the Mn(II) EPR spectrum of the AK-MnATP complex [Kalbitzer, H. R., Marquetant, R., Connolly, B. A., & Goody, R. S. (1983) Eur. J. Biochem. 133, 221-227].(ABSTRACT TRUNCATED AT 400 WORDS)

Reactions of ferric and ferrous cytochromes c' from four photosynthetic bacteria (Rhodobacter capsulatus ATCC 11166, Rhodopseudomonas palustris ATCC 17001, Rhodospirillum rubrum ATCC 11170, and Chromatium vinosum ATCC 17899) with nitric oxide have been investigated by electronic absorption and electron paramagnetic resonance spectroscopies. The heme iron(III) of these ferric cytochromes c' has been recently reported to be in a quantum mechanically admixed (S = 5/2, 3/2) state [Fujii, S., Yoshimura, T., Kamada, H., Yamaguchi, K., Suzuki, S., Shidara, S. and Takakuwa, S. (1995) Biochim. Biophys. Acta 1251, 161-169]. The affinity of ferric cytochromes c' for NO among these bacterial species (C. vinosum>> Rps. palustris approximately Rb. capsulatus>>) R. rubrum) was apparently related to the S = 3/2 content in the or der. In the reaction of ferrous cytochrome c' with NO, six- and five-coordinated nitrosylhemes, which represent species with and without a ligand at the axial position trans to nitrosyl group, have been formed. The content of six-coordinated nitrosylheme in NO-ferrous cytochrome c' has been determined to be Rb. capsulatus approximately Rps. palustris>> C. vinosum < R rubrum, suggesting that a stability of iron-to-histidine bond decreases with this order. The NO reactions of ferric and ferrous cytochromes c' from photosynthetic bacteria have been compared with those of cytochromes c' from denitrifying bacteria.

A number of DNA helicases have been isolated from mammalian cells, but their abilities to stimulate DNA replication accompanied with DNA unwinding have not been addressed so far. We constructed a model DNA replication system using the yeast autonomously replicating sequence (ARS) as the replication origin. In this system, SV40 T antigen as a DNA helicase assembles to the replication origin where the DNA duplex is unwound by torsional stress due to the negative supercoiling of template DNA, which leads to bidirectional DNA replication from the origin. We report here that DNA helicase B isolated from mouse FM3A cells can greatly stimulate DNA synthesis in this replication system in place of SV40 T antigen. DNA synthesis was dependent on the presence of single-stranded DNA binding protein (RP-A), DNA polymerase alpha/primase from mouse cells, and Escherichia coli DNA gyrase. DNA gyrase was required not only at elongation as a DNA swivelase but also at initiation to increase negative superhelical density of template DNA with the assistance of RP-A. A mammalian DNA fragment containing a replication initiation zone upstream of the c-myc gene as well as the yeast ARS fragment acted as a cis-element in this system using DNA helicase B. Both DNA helicase B and SV40 T antigen have the ability to extensively unwind the template DNA in the presence of RP-A and DNA gyrase, which may be crucial for stimulation of DNA synthesis in this system.

The aim of this work was the purification and identification of the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate recovered from a cuttlefish industrial manufacturing effluent. This process consisted on the ultrafiltration of cuttlefish softening wastewater, with a 10 kDa cut-off membrane, followed by the hydrolysis with alcalase of the retained fraction. Alcalase produced ACE inhibitors reaching the highest activity (IC₅₀ = 76.8 ± 15.2 μg mL⁻¹) after 8 h of proteolysis. Sequential ultrafiltration of the 8 h hydrolysate with molecular weight cut-off (MWCO) membranes of 10 and 1 kDa resulted in the increased activity of each permeate, with a final IC₅₀ value of 58.4 ± 4.6 μg mL⁻¹. Permeate containing peptides lower than 1 kDa was separated by reversed-phase high performance liquid chromatography (RP-HPLC). Four fractions (A-D) with potent ACE inhibitory activity were isolated and their main peptides identified using high performance liquid chromatography coupled to an electrospray ion trap Fourier transform ion cyclotron resonance-mass spectrometer (HPLC-ESI-IT-FTICR) followed by comparison with databases and de novo sequencing. The amino acid sequences of the identified peptides contained at least one hydrophobic and/or a proline together with positively charged residues in at least one of the three C-terminal positions. The IC₅₀ values of the fractions ranged from 1.92 to 8.83 μg mL⁻¹, however this study fails to identify which of these peptides are ultimately responsible for the potent antihypertensive activity of these fractions.