An RNA editing-like internal uridine (U) incorporation activity (G. C. Frech, N. Bakalara, L Simpson, and A. M. Simpson, EMBO J. 14:178-187, 1995) and a 3'-terminal U addition activity (N. Bakalara, A. M. Simpson, and L. Simpson, J. Biol. Chem. 264:18679-18686, 1989) have been previously described by using a mitochondrial extract from Leishmania tarentolae. Chiral phosphorothioates were used to investigate the stereoconfiguration requirements and the stereochemical course of these nucleotidyl transfer reactions. The extract utilizes (SP)-alpha-S-UTP for both 3' and internal U incorporation into substrate RNA. The internal as well as the 3' incorporation of (SP)-alpha-S-UTP proceeds via inversion of the stereoconfiguration. Furthermore, internal U incorporation does not occur at sites containing thiophosphodiesters of the RP configuration. Our results are compatible with an enzyme cascade model for this in vitro U insertion activity involving sequential endonuclease and uridylyl transferase directly from UTP and RNA ligase steps and are incompatible with models involving the transfer of U residues from the 3' ends of guide RNAs.

OBJECTIVE

To determine the pattern of rhodopsin mutations in Chinese retinitis pigmentosa (RP) patients.

METHODS

The rhodopsin gene was examined in 101 RP patients and 190 controls from Hong Kong.

RESULTS

Three coding changes were identified: Pro347Leu, Ala299Ser, and 5211delC. Each protein sequence alteration was found in one patient. Ala299Ser also existed in two controls.

CONCLUSIONS

The C-terminal nonsense mutation may cause mis-sorting of rhodopsin protein. The finding of controls with Ala299Ser suggests this is only the third missense alteration reported that does not cause RP. The expected frequency of rhodopsin mutations in RP is <7% (2/101=2.0%, 95% confidence interval: 0.2%-7.0%).

OBJECTIVE

In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG) on cyclic nucleotide production and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in collagen (10 µg/mL)-stimulated platelet aggregation.

METHODS

Washed platelets (10(8)/mL) from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 min at 37°C in the presence of 2 mM exogenous CaCl(2) with or without EGCG or other materials, stimulated with collagen (10 µg/mL) for 5 min, and then used for the determination of intracellular cytosolic Ca(2+) ([Ca(2+)](i)), thromboxane A(2) (TXA(2)), adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), and VASP phosphorylation.

RESULTS

EGCG dose-dependently inhibited collagen-induced platelet aggregation by inhibiting both [Ca(2+)](i) mobilization and TXA(2) production. Of two aggregation-inhibiting molecules, cAMP and cGMP, EGCG significantly increased intracellular levels of cAMP, but not cGMP. EGCG-elevated cAMP level was decreased by SQ22536, an adenylate cyclase inhibitor, but not by etazolate, a cAMPspecific phosphodiesterase inhibitor. In addition, EGCG elevated the phosphorylation of VASP-Ser(157), a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser(239), a cGMP-dependent protein kinase substrate, in intact platelets and collagen-induced platelets, and VASP-Ser(157) phosphorylation by EGCG was inhibited by both an adenylate cyclase inhibitor SQ22536 and an A-kinase inhibitor Rp-8-Br-cAMPS. We have demonstrated that EGCG increases cAMP via adenylate cyclase activation and subsequently phosphorylates VASP-Ser(157) through A-kinase activation to inhibit [Ca(2+)](i) mobilization and TXA(2) production on collagen-induced platelet aggregation.

CONCLUSIONS

These results strongly indicate that EGCG is a beneficial compound elevating cAMP level in collagen-platelet interaction, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.

Oligodeoxycytidine phosphorothioate (PS-d<em>C</em>n, n = chain length), known to show virus inhibition ability by a mechanism other than the antisense one when n approximately 20, was explored for its solution structure by circular dichroism (<em>C</em>D) and ultraviolet (UV) absorption spectroscopy. For PS-d<em>C</em>4, when the strand concentration was higher than 10 microM, the respective 288-nm positive and 265-nm negative peaks appeared in the <em>C</em>D spectra at slightly acidic pHs and 0 degree <em>C</em> in the absence of salt, which is indicative of a four-stranded structure (namely, the i-motif). Strand concentration-dependent <em>C</em>D spectroscopy indicated that intermolecular association is responsible for this i-motif. The formation or i-motif was also characterized by UV absorption spectroscopy, in which the dissociation of this structure caused a sharp increase in the absorbance at 275 nm and a decrease at 305 nm. By plotting this change, the Tn values were estimated to be ca. 11 and 13 degrees <em>C</em> at 20 and 50 microM strand concentrations, respectively. Stability of the i-motif was compared between PS-d<em>C</em>, P-chiral diastereoisomers, and the Sp configuration produced a more stable structure than <em>Rp</em>. PS-d<em>C</em>20 was also investigated at physiological temperature, and the respective 288-nm positive and 265-nm negative peaks appeared at slightly acidic pH: it has been suggested that intermolecular folding was predominant above ca. 1 microM and that intramolecular folding dominated at low strand concentrations such as 0.05 microM. Gel-filtration chromatography and nondenaturing gel electrophoresis provided the supporting data for the four-stranded folding of PS-d<em>C</em>20.

Uroguanylin and guanylin are structurally related peptides that activate an intestinal form of membrane guanylate cyclase (GC-C). Guanylin was isolated from the intestine, but uroguanylin was isolated from urine, thus a tissue source for uroguanylin was sought. In these experiments, uroguanylin and guanylin were separated and purified independently from colonic mucosa and urine of opossums. Colonic, urinary, and synthetic forms of uroguanylin had an isoelectric point of approximately 3.0, eluted from CRP-HPLC) columns at 8-9% acetonitrile, elicited greater guanosine 3', 5'-cyclic monophosphate (cGMP) responses in T84 cells at pH 5.5 than pH 8, and were not cleaved and inactivated by pretreatment with chymotrypsin. In contrast, colonic, urinary, and synthetic guanylin had an isoelectric point of approximately 6.0, eluted at 15-16% acetonitrile on CRP-HPLC columns, stimulated greater cGMP responses in T84 cells at pH 8 than pH 5.5, and were inactivated by chymotrypsin, which hydrolyzed the Phe-Ala or Try-Ala bonds within guanylin. Uroguanylin joins guanylin as an intestinal peptide that may participate in an intrinsic pathway for cGMP-mediated regulation of intestinal salt and water transport. Moreover, uroguanylin and guanylin in urine may be derived from the intestinal mucosa, thus implicating these peptides in an endocrine mechanism linking the intestine with the kidney.

OBJECTIVE

This study was designed to identify pathogenic mutations causing autosomal recessive retinitis pigmentosa (RP) in consanguineous Pakistani families.

METHODS

Two consanguineous families affected with autosomal recessive RP were identified from the Punjab Province of Pakistan. All affected individuals underwent a thorough ophthalmologic examination. Blood samples were collected, and genomic DNAs were extracted. Exclusion analysis was completed, and two-point LOD scores were calculated. Bidirectional sequencing of the β subunit of phosphodiesterase 6 (PDE6β) was completed.

RESULTS

During exclusion analyses both families localized to chromosome 4p, harboring PDE6β, a gene previously associated with autosomal recessive <em>RP</em>. Sequencing of PDE6β identified missense mutations: c.1655G>A (p.R552Q) and c.1160C>T (p.P387L) in families PK<em>RP</em>161 and PK<em>RP</em>183, respectively. Bioinformatic analyses suggested that both mutations are deleterious for the native three-dimensional structure of the PDE6β protein.

CONCLUSIONS

These results strongly suggest that mutations in PDE6β are responsible for the disease phenotype in the consanguineous Pakistani families.

A single reaction product was formed during the incubation of 1.5 microM (5S,12R)-dihydroxy-6,14-cis-8,10-trans-[3H]icosatetraenoic acid (leukotriene B4, LTB4) for 30 min at 37 degrees C in 10 mM potassium phosphate buffer (pH 7.5) with 100 microM NADPH and the 150,000 X g supernatant of sonicated human polymorphonuclear leukocytes (PMN). The reaction product exhibited the same mobility on reversed-phase HPLC (RP-HPLC) and TLC as standard 20-hydroxy-LTB4 (20-OH-LTB4). When the omega-oxidation product of [3H]LTB4 was eluted from a Sep-Pak, resolved by RP-HPLC, and analyzed by GC/MS, its structure was determined to be solely 20-OH-LTB4. The Km of the 20-hydroxylase for [3H]LTB4 at its optimal pH of 7.5 was 0.22 +/- 0.08 microM (mean +/- SD, n = 4) and the Vmax was 48 +/- 11 pmol/min X mg of protein (mean +/- SD, n = 4). When the concentration of [3H]LTB4 was fixed at 1.5 microM, the Km for NADPH was 1.01 +/- 0.59 microM (mean +/- SD, n = 3). The location in the 150,000 X g supernatant of the LTB4 20-hydroxylase distinguishes it from the cytochrome P-450 system of liver, lung, and kidney microsomes and from the NADPH oxidase-cytochrome b-245 system of the human PMN. The LTB4 20-hydroxylase is either a unique cytochrome P-450 or other monooxygenase.

BACKGROUND

Understanding the dose-response of the lung in order to minimize the risk of radiation pneumonitis (RP) is critical for optimization of lung cancer radiotherapy. We propose a method to combine the dose-response relationship for RP in the landmark QUANTEC paper with known clinical risk factors, in order to enable individual risk prediction. The approach is validated in an independent dataset.

METHODS

The prevalence of risk factors in the patient populations underlying the QUANTEC analysis was estimated, and a previously published method to adjust dose-response relationships for clinical risk factors was employed. Effect size estimates (odds ratios) for risk factors were drawn from a recently published meta-analysis. Baseline values for D50 and γ50 were found. The method was tested in an independent dataset (103 patients), comparing the predictive power of the dose-only QUANTEC model and the model including risk factors. Subdistribution cumulative incidence functions were compared for patients with high/low-risk predictions from the two models, and concordance indices (c-indices) for the prediction of RP were calculated.

RESULTS

The reference dose- response relationship for a patient without pulmonary co-morbidities, caudally located tumor, no history of smoking, < 63 years old, and receiving no sequential chemotherapy was estimated as D50(0) = 34.4 Gy (95% CI 30.7, 38.9), γ50(0) = 1.19 (95% CI 1.00, 1.43). Individual patient risk estimates were calculated. The cumulative incidences of RP in the validation dataset were not significantly different in high/low-risk patients when doing risk allocation with the QUANTEC model (p = 0.11), but were significantly different using the individualized model (p = 0.006). C-indices were significantly different between the dose-only and the individualized model.

CONCLUSIONS

This study presents a method to combine a published dose-response function with known clinical risk factors and demonstrates the increased predictive power of the combined model. The method allows for individualization of dose constraints and individual patient risk estimates.

Human replication protein A (RP-A) is a three-subunit protein that is required for simian virus 40 (SV40) replication in vitro. The trypanosome homologue of RP-A has been purified from Crithidia fasciculata. It is a 1:1:1 complex of three polypeptides of 51, 28, and 14 kDa, binds single-stranded DNA via the large subunit, and is localized within the nucleus. C. fasciculata RP-A substitutes for human RP-A in the large tumor antigen-dependent unwinding of the SV40 origin of replication and stimulates both DNA synthesis and DNA priming by human DNA polymerase alpha/primase, but it does not support efficient SV40 DNA replication in vitro. This extraordinary conservation of structure and function between human and trypanosome RP-A suggests that the mechanism of DNA replication, at both the initiation and the elongation level, is conserved in organisms that diverged from the main eukaryotic lineage very early in evolution.

RP 59500, a new antibacterial agent, is a combination of two compounds, RP 54476 and RP 57669. The uptake of radiolabelled RP 59500, i.e. a mixture containing [14C]-RP 54476 plus RP 57669 or [14C]-RP 57669 plus RP 54476, by J 774 murine macrophages was evaluated by a velocity gradient centrifugation technique. After 120 min, the ratios of cellular to extracellular concentration for RP 54476 and RP 57669 were 34 and 50, respectively. The highest intracellular accumulation of RP 59500 was observed at pH 7-7.5. RP 59500 was found to accumulate less at 4 degrees C than at 37 degrees C. The uptake of RP 59500 by dead macrophages was markedly higher than that by live macrophages. As the extracellular concentration of RP 59500 was increased, the intracellular concentration of each component rose, but not proportionally. The metabolic inhibitors sodium cyanide and potassium fluoride both decreased modestly the entry of RP 57669, but not that of RP 54476, into macrophages. After removal of the extracellular antibiotic, RP 54476 and RP 57669 were released rapidly by the cells until equilibrium was established (45% of the original intracellular RP 59500 remained in the cells after 120 min). The intracellular activity of RP 59500 was assessed by incubating macrophages containing ingested Staphylococcus aureus 209P with the drug (10 x MIC: 2.5 mg/L) at 37 degrees C and determining the number of viable cell-associated bacteria. Approximately 70% of the intracellular bacteria were killed within 120 min of incubation. Thus, RP 59500 attains a high intracellular concentration and is active against intracellular S. aureus.

Both sodium nitroprusside (SNP), a nitric oxide (NO) generator, and C-type natriuretic peptide (CNP) have been found to raise cGMP levels in bovine chromaffin cells in a time- and concentration-dependent manner. The effect of these compounds on catecholamine secretion and calcium influx has also been studied, and both compounds were found to produce a slowly developing inhibitory effect on acetylcholine- or depolarization-stimulated catecholamine secretion and calcium increases without affecting the spontaneous release or the basal intracellular Ca2+ concentration. These inhibitory effects were observed only at high doses of acetylcholine or high levels of extracellular potassium and required concentrations of SNP or CNP very similar to those that increased cGMP levels. Preincubation with 100 microM zaprinast, a cGMP-phosphodiesterase inhibitor able to increase cGMP levels, mimicked the inhibitory effects of SNP and CNP. We investigated the effect of the soluble guanylate cyclase inhibitor methylene blue and the cGMP-dependent protein kinase (PKG) inhibitor 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, Rp isomer, on inhibition by SNP or CNP. Although methylene blue (10 microM) partially prevented the inhibitory effect of SNP, it did not do so for that produced by CNP, thus indicating that SNP acts through cGMP produced by the NO-activated guanylate cyclase. 8-(4-Chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, Rp isomer totally reversed both the SNP and CNP inhibitory effects. These results suggest that the activation of PKG mediates the inhibition induced by SNP and CNP. We successfully measured the PKG activity from cells preincubated with SNP or CNP, and our results show that this enzymatic activity increased with a time dependence very similar to the increase in the cGMP levels. Our results indicate that NO and CNP peptide inhibit secretagogue-stimulated catecholamine release via activation of soluble and particulate isoforms of the guanylate cyclase, respectively, presumably by inhibition of calcium entry through voltage-activated calcium channels. This inhibitory effects seems to be mediated by activation of the PKG.

Three low molecular mass polypeptides have been isolated by using the technique of organic solvent extraction of thylakoid membranes or whole cells from Rhodopseudomonas viridis. Their primary structures were determined by long liquid phase sequencer runs, combined with the isolation and sequence analysis of the C-terminal o-iodosobenzoic acid fragment and carboxypeptidase degradation. The polypeptide which consists of 58 amino-acids and is 46% homologous to the antenna polypeptide B880-alpha from Rhodospirillum rubrum was designated as B1015-alpha (1 His residue). The sequence homology between the second polypeptide, named B1015-beta (55 amino acids, 2 His residues) and B880-beta from Rs. rubrum is 52%. For the third polypeptide consisting of 36 amino acids and exhibiting a high hydrophobicity, no equivalent polypeptide has so far been found in other purple bacteria. The molar ratio of these three organic solvent soluble polypeptides from Rp. viridis was estimated to be 1:1:1. Accordingly, the 36 amino-acid polypeptide is likely to be an additional constituent of the light-harvesting complex B1015, consequently termed as B1015-gamma. According to hydrophathy profiles, the transmembrane arrangement of B1015-alpha and B1015-beta within the thylakoid membrane is supposed to be similar. B1015-gamma, however, shows a somewhat different hydropathy profile. A particular feature of this polypeptide is its high amount of aromatic amino acids. It is postulated that B1015-gamma is involved in the formation of regular arrays of light-harvesting complexes.

A method for the comprehensive profiling of intact glucosinolates (GLSs), major and minor, occurring in leaves and seeds of rocket salad (Eruca sativa L.) is presented using optimized reversed-phase liquid chromatography (RP-LC) with electrospray ionization (ESI) ion trap mass spectrometry (ITMS). ESI-ITMS in the negative mode was confirmed to be very suitable to analyze these compounds in crude extracts. After extraction from the plant material with methanol/water (70:30 v/v) at 70 degrees C, the analytes of interest were separated on a CCR367R). The results indicated the usefulness of this method for a rapid, sensitive and comprehensive profiling of the GLS family naturally occurring in extracts of crude plant matter.

OBJECTIVE

We evaluated the utility of quantitative diffusion-weighted magnetic resonance imaging (DW-MRI) for assessing both the relationship between the degree of fibrosis and the histological activity index (HAI) in chronic hepatitis (CH) cases and attempted to determine whether the apparent diffusion coefficient value (ADC) could be used as a reference for the degree of fibrosis detected by histology.

METHODS

The study population consisted of 55 CH patients (Group I) and a control group of 30 volunteers (Group II). Group I consisted of 31 CH-B (CHB), 18 CH-C (CHC) and 6 non-alcoholic steatohepatitis patients. DW-MRI of the liver with b values of 0, 500 and 1000 s/mm(2) was performed, and liver biopsies of the patients were obtained two weeks later. The ADC value, degree of liver fibrosis and HAI were compared within Group I, and the ADC values of both groups were compared with each other.

RESULTS

The ADC was lower in Group I than in Group II (P < 0.05). The ADC of the left lobe lateral (LL) (P < 0.05), left lobe medial (LM) and right lobe anterior (RA) segments (P < 0.01) in Group I were lower than those of Group II. There was no relationship between HAI and the ADC of LL, LM, RA and right lobe posterior (RP) segments in Group I. Additionally, there was no correlation between fibrosis scores and ADC in Group I, whereas there was a negative correlation between fibrosis scores and ADC values of the LL (28.3%) and RP (29.5%).

CONCLUSIONS

CH patients had lower ADC values. There was no correlation between ADC values and fibrosis stages or ADC and HAI values. Quantitative DW-MRI was not useful in determining the degree of fibrosis in liver tissue.

Wheat omega-5 gliadin has been identified as a major allergen in wheat-dependent exercise-induced anaphylaxis. We have detected seven IgE-binding epitopes in primary sequence of the protein. We newly identified four additional IgE-binding epitope sequences, QQFHQQQ, QSPEQQQ, YQQYPQQ and QQPPQQ, in three patients with wheat-dependent exercise-induced anaphylaxis in this study. Diagnosis and therapy of food allergy would benefit from the availability of defined recombinant allergens. However, because omega-5 gliadin gene has not been cloned, recombinant protein is currently unavailable. We sought to clone the omega-5 gliadin gene and produce the homogeneous recombinant protein for use in an in vitro diagnostic tool. Using a PCR-based strategy we isolated two full-length omega-5 gliadin genes, designated omega-5 and omega-5b, from wheat genomic DNA and determined the nucleotide sequences. The protein encoded by omega-5a was predicted to be 439 amino acids long with a calculated mass of 53 kDa; the omega-5b gene would encode a 393 amino acid, but it contains two stop codons indicating that omega-5b is pseudogene. The C-terminal half (178 amino acids) of the omega-5a gliadin protein, including all 11 IgE-binding epitope sequences, was expressed in Escherichia coli by means of the pET system and purified using RP-HPLC. Western blot analysis and dot blot inhibition assay of recombinant and native omega-5 gliadin purified from wheat flour demonstrated that recombinant protein had IgE-binding ability. Our results suggest that the recombinant protein can be a useful tool for identifying patients with wheat-dependent exercise-induced anaphylaxis in vitro.

The multiple memory systems hypothesis posits that different neural circuits function in parallel and may compete for information processing and storage. For example, instrumental conditioning would depend on the striatum, whereas spatial memory may be mediated by a circuit centered on the hippocampus. However, the nature of the task itself is not sufficient to select durably one system over the other. In this study, we investigated the effects of natural and pharmacological rewards on the selection of a particular memory system during learning. We compared the effects of food- or drug-induced activation of the reward system on cue-guided versus spatial learning using a Y-maze discrimination task. Drug-induced reward severely impaired the acquisition of a spatial discrimination task but spared the cued version of the task. Immunohistochemical analysis of the phosphorylated form of the cAMP response element binding (CREB) protein and c-Fos expression induced by behavioral testing revealed that the spatial deficit was associated with a decrease of both markers within the hippocampus and the prefrontal cortex. In contrast, drug reward potentiated the cued learning-induced CREB phosphorylation within the dorsal striatum. Administration of the protein kinase A inhibitor 8-Bromo-adenosine-3',5'-cyclic monophosphorothioate Rp isomer (Rp-cAMPS) into the dorsal striatum before training completely reversed the drug-induced spatial deficit and restored CREB phosphorylation levels within the hippocampus and the prefrontal cortex. Therefore, drug-induced striatal hyperactivity may underlie the declarative memory deficit reported here. This mechanism could represent an important early step toward the development of addictive behaviors by promoting conditioning to the detriment of more flexible forms of memory.

Uphill energy transfer in the LH2-containing purple bacteria Rhodopseudomonas acidophila, Rhodopseudomonas palustris, Rhodobacter sphaeroides, Chromatium vinosum and Chromatium purpuratum was studied by stationary fluorescence spectroscopy at room temperature upon selective excitation of the B800 pigments of LH2 and the B880 pigments of LH1 at 803 nm and 900 nm, respectively. The resulting fluorescence spectra differed significantly at wavelengths shorter than the fluorescence maximum but agreed at longer wavelengths. The absorption spectra of the species studied were decomposed into five bands at approx. 800, 820, 830, 850 and 880 nm using the shapes of the absorption spectra of the LH1-RC only species Rhodospirillum rubrum and the isolated B800-850 complex from Rps. acidophila strain 10050 as guide spectra. This allowed a quantification of the number of pigments in each pigment group and, consequently, the antenna size of the photosynthetic unit assuming 36 bacteriochlorophyll a molecules in an LH1-RC complex. In most of the LH2-containing purple bacterial strains the number of LH2 rings per LH1-RC was less than the idealized number of eight (Papiz et al., Trends Plant Sci. 1 (1996) 198-206), which was achieved only by C. purpuratum. Uphill energy transfer was assayed by comparing the theoretical fluorescence spectrum obtained from a Boltzmann equilibrium with the measured fluorescence spectrum obtained by 900 nm excitation. The good match of both spectra in all the purple bacteria studied indicates that uphill energy transfer occurs practically up to its thermodynamically maximal possible extent. All strains studied contained a small fraction of either poorly connected or unconnected LH2 complexes as indicated by higher fluorescence yields from the peripheral complexes than predicted by thermal equilibration or kinetic modeling. This impedes generally the quantitative analysis of blue-excited fluorescence spectra.

In vitro susceptibilities of Rickettsia rickettsii, Rickettsia conorii, and Coxiella burnetii to the new fluoroquinolone sparfloxacin (AT-4140; RP 64206) were determined. Plaque and dye uptake assays were used to measure the MICs against R. rickettsii and R. conorii. The susceptibilities of C. burnetii Nine Mile and Q 212 were determined in two acute-infection models and in two chronic-infection models. The MICs were 0.125 to 0.25 microgram/ml for R. rickettsii and 0.25 to 0.5 microgram/ml for R. conorii. Sparfloxacin (1 microgram/ml) cured cells recently infected with C. burnetii Nine Mile and Q 212 within 4 to 9 days and cured multiplying, persistently infected cells within 10 days. As previously described with other fluoroquinolones (D. Raoult, M. Drancourt, and G. Vestris, Antimicrob. Agents Chemother. 34:1512-1514, 1990), sparfloxacin failed to cure cells persistently infected with C. burnetii and blocked from dividing with cycloheximide. As determined by the dye uptake assay, no cellular toxicity was noted with sparfloxacin at up to 128 micrograms/ml. These results are consistent with those previously obtained with fluoroquinolones (D. Raoult, M. Yeaman, and O. Baca, Rev. Infect. Dis. 11[Suppl. 5]:S986, 1989), although sparfloxacin may be slightly more active.

The present study extends the utility of reversed-phase high-performance liquid chromatography (RP-HPLC) to monitor folding and stability of de novo designed synthetic two-stranded alpha-helical coiled-coils. Thus, we have compared the effect of temperature on the RP-HPLC retention behaviour of both oxidized (two identical five-heptad alpha-helical peptides linked by a disulfide bridge) and reduced coiled-coil analogues with various amino acids substituted into the hydrophobic core of the coiled-coil. We were able to correlate the RP-HPLC retention behaviour of the oxidized analogues over the temperature range of 10 to 80 degrees C with the stability of the analogues as determined by conventional thermal and chemical denaturation approaches. In addition, the contribution of a disulfide bridge to coiled-coil stability was highlighted by comparing the elution behaviour of the oxidized and reduced analogues. Overall, we demonstrate the excellent potential of "temperature profiling" by RP-HPLC to monitor differences in oligomerization state and protein stability.

Poliovirus has been studied as a live recombinant vaccine vector because of its attractive characteristics. The genetic instability, however, has hampered recombinant polioviruses (PVs) from being developed as an appropriate vaccine. A variety of different foreign inserts were cloned directly into our poliovirus Sabin 1-based RPS-Vax vector system, resulting in the production of recombinant PVs. The genetic stability of each recombinant PV was examined during 12 rounds of consecutive passage. It was found that the genetic stability of the recombinants was not well correlated with their insert size. Instead, elevated stability was frequently observed in recombinants with inserts of high G/C contents. Furthermore, a comparative study using different constructs of the human immunodeficiency virus env gene revealed that the internal deletion of the unstable insert was seemingly caused by the presence of the adjacent A/T-rich region. The instability of these inserts was completely remedied by (i) increasing the G/C contents and (ii) replacing the local A/T-rich region with the G/C-rich codon without a change of the amino acid. This means that stability is closely associated with the G/C content and the G/C distribution pattern. To see whether these findings can be applied to the design of genetically stable recombinant PV, we have reconstructed the heteromultimeric insert based on our design architecture, including the above-mentioned G/C rules and the template/ligation-free PCR protocol. The heteromultimeric insert was very unstable, as expected, but the manipulated insert with the same amino acid sequence showed complete genetic stability, not only in vitro, but also in vivo. Even though this guideline was established with our RPS-Vax vector system, to some extent, it can also be applied to other live viral vaccine vectors.

Radical prostatectomy (RP) is a commonly used procedure in the treatment of clinically localized prostate cancer. For this report, the authors critically analyzed the factors associated with recovery of erectile function after surgery. A systematic review of the literature using the Medline and CancerLit databases was conducted. Keywords for the literature search included prostate cancer, radical prostatectomy, erectile dysfunction, impotence, treatment, and prophylaxis. Accurate patient selection (based on patient age, preoperative erectile function, and comorbidity profile) and adequate surgical technique (ie, the preservation of neurovascular bundles) were the major determinants of postoperative erectile function. Moreover, better results were achieved when an appropriate pharmacologic treatment using either oral or local approaches was given. Therefore, the authors concluded that, if patients are stratified correctly according to preoperative, intraoperative, and postoperative factors, then a satisfactory functional recovery may be expected after surgery. For these reasons, an ideal multivariate model predicting the restoration of erectile function after surgery should include patient, surgeon, and postsurgical treatment variables. The authors also concluded that the stratification of patients with regard to their risk of developing erectile dysfunction after surgery was feasible based on several parameters, which should be taken into account for correct patient treatment and counseling. To address this objective, accurate tools for predicting the likelihood of complete functional recovery after surgery are needed. Cancer 2009;115(13 suppl):3150-9. (c) 2009 American Cancer Society.

Isolated interphase lamin C, obtained from Ehrlich ascites tumor cells, was digested by Lys-C endoproteinase, the resulting peptides separated by reversed-phase HPLC and subjected to microsequencing in order to identify phosphorylation sites in interphase and following phosphorylation in vitro by cdc2-kinase, protein kinase C (PKC) and protein kinase A (PKA), respectively. Nuclear lamin C showed partial phosphorylation of Ser392 and Ser409, and possibly Ser407 in interphase. Phosphorylation was increased in response to cdc2-kinase at Ser390 and Ser392 and to PKC at Ser572. The N-terminal peptide (aa 1-32) containing consensus sequences for the 3 kinases was phosphorylated by cdc2-kinase, PKC and PKA. The sequence data suggests that multiple molecular switches via lamina modification control the dynamic behaviour of the nucleoskeleton during the cell cycle.

The effect of ethanol on receptor-mediated phospholipase C-linked signal transduction processes was investigated in isolated rat hepatocytes. Pretreatment of the cells with ethanol (6-300 mM) markedly inhibited a subsequent stimulation of phospholipase C by vasopressin, angiotensin II, or epidermal growth factor. By contrast, the effects of the alpha 1-adrenergic agonist phenylephrine and of glucagon were not affected by ethanol pretreatment. Ethanol inhibited the agonist-induced decrease in polyphosphoinositides, the formation of inositol phosphates, and the increase in cytosolic free Ca2+ levels, as detected with the intracellular Ca2+ indicator indo-1. The effects of ethanol were concentration dependent and were pronounced at low concentrations of agonists but were not significant at saturating levels. Pretreatment of the cells with the protein kinase C inhibitor H7 partly prevented the inhibition by ethanol of vasopressin-induced phospholipase C activation. By contrast, pretreatment of the cells with (Rp)-adenosine cyclic 3':5'-phosphorothioate [Rp)-cAMP-S), a competitive inhibitor of protein kinase A, potentiated the inhibitory effect of ethanol on the Ca2+ mobilization by vasopressin. (Rp)-cAMP-S similarly potentiated the inhibition of phospholipase C by the protein kinase C-activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The kinase A inhibitor also made the Ca2+ mobilization by phenylephrine sensitive to ethanol, indicating that the formation of cAMP in the cells played a role in suppressing the sensitivity to ethanol. Pretreatment of the cells with ethanol enhanced the inhibitory effects of TPA on the vasopressin-induced phospholipase C activation at all concentrations of the hormone; however, these synergistic effects were prevented when TPA was added prior to ethanol, a condition that prevents the activation of phospholipase C by ethanol. The data indicate that ethanol causes desensitization of the receptor-mediated phospholipase C secondary to the ethanol-induced activation of phospholipase C and activation of protein kinase C. Ethanol treatment also affects the sensitivity of the phospholipase C system to control by protein kinases A and C. The data indicate that ethanol can affect the control of intracellular signal transduction processes in liver cells under physiologically relevant conditions.

OBJECTIVE

Glucagon is a 29 amino acid peptide hormone that exhibits degradation via both chemical and physical pathways. The objective of the studies reported herein was to identify the degradation products and scheme for glucagon hydrolysis in acidic solutions.

METHODS

Solutions of glucagon in 0.01 N HCl (pH 2.5) were degraded at 60 degrees C for 70 h. One isocratic and two gradient RP-HPLC methods were developed to separate the degradation products. Structure elucidation of the separated peaks was achieved using amino acid sequencing, amino acid analysis, and mass spectrometry. Degradation was carried out in the pH range 1.5-5 to check for changes in degradation scheme with pH. Authentic samples of degradation products were degraded under similar acidic conditions to confirm precursor successor relationships in the degradation scheme.

RESULTS

Sixteen major degradation products were isolated and identified. The major pathways of degradation were found to be aspartic acid cleavage at positions 9, 15, and 21 and glutaminyl deamidation at positions 3, 20, and 24. Cleavage occurred on both sides of Asp-15 but only on the C-terminal side of Asp-9 and Asp-21. Deamidation of the Asn residue at position 28 was not detected.