The effect of heat processing, storage time and temperature on the migration of bisphenol A (BPA) from organosol and epoxy can coatings to a fatty-food simulant and tuna was determined. Analyses of BPA were performed by RP-HPLC with fluorescence detection. Four migration experiments, performed between 2000 and 2003, using cans with organosol, epoxy and a combination of both types of coatings were performed under different processing conditions and storage times. Migration levels as high as 646.5 microg kg(-1) BPA from an organosol coating of tuna fish cans were found using a fatty-food simulant following the heat processing of the simulant-filled cans. Levels ranging from 11.3 to 138.4 microg kg(-1) BPA from tuna cans coated with an epoxy resin migrated to the fatty-food simulant during 1 year at 25 degrees C. Levels of BPA migration into a fatty-food simulant from thermally processed and stored tuna cans coated with a combination of organosol and epoxy resins and from vegetable cans coated with an epoxy resin were below the limit of quantitation of 10.0 microg kg(-1). Migration of BPA to tuna ranged from <7.1 to 105.4 microg kg(-1) during long-term storage at 25 degrees C. BPA levels in tuna cans purchased from three local supermarkets ranged from <7.1 to 102.7 microg kg(-1). The highest migration levels were found following heat processing at temperatures as high as 121 degrees C and at times as long as 90 min. Coatings from different can batches can give different levels of BPA migration. The migration levels of BPA found in this work are below the present European Union migration limit, except the 646.5 microg kg(-1) found after the commercial heating process was applied to the simulant-filled cans coated with the organosol resin.

Direct PCR sequencing with boronated nucleotides provides an alternative to current PCR sequencing methods. The positions of boranophosphate-modified nucleotides incorporated randomly into DNA during PCR can be revealed directly by exonuclease digestion to give sequencing ladders. Cytosine nucleotides, however, are especially sensitive to exonuclease digestion and provide suboptimal sequencing ladders. Therefore, a series of 5-substituted analogs of 2'-deoxycytidine 5'-(alpha-P-borano)triphosphates (dCTPalphaB) were synthesized with the hope of increasing the nuclease resistance of deoxycytosine residues and thereby enhancing the deoxycytosine band intensities. These dCTP analogs contain a boranophosphate modification at the alpha-phosphate group in 2'-deoxycytidine 5'-triphosphate (dCTP) as well as a 5-methyl, 5-ethyl, 5-bromo or 5-iodo substitution for the 5-hydrogen of cytosine. The two diastereomers of each new dCTP derivative were separated by reverse phase HPLC. The first eluted diastereomer (putatively Rp) of each dCTP analog was a substrate for T7 DNA polymerase (Sequenase) and had an incorporation efficiency similar to normal dCTP and dCTPalphaB, with the 5-iodo-dCTPalphaB analog being the least efficient. Substitution at the C-5 position of cytosine by alkyl groups (ethyl and methyl) markedly enhanced the dCTPalphaB resistance towards exonuclease III (5-Et-dCTPalphaB >5-Me-dCTPalphaB>>dCTPalphaB approximately 5-Br-dCTPalphaB >5-I-dCTPalphaB), thereby generating DNA sequences that better define the deoxycytosine positions. The introduction of modified dCTPalphaB should increase the utility of direct DNA sequencing with boronated nucleoside 5'-triphosphates.

RP-HPLC fractionation of the electrically stimulated skin secretion of the arboreal South American frog Hypsiboas albopunctatus ("spotted treefrog") led to the isolation of a cytolytic C-terminally amidated peptide. This novel peptide, named hylin a1 (Hy-a1), consists of 18 amino acid residues (IFGAILPLALGALKNLIK-NH(2)). The alpha-helical structure of the synthetic hylin a1 peptide was confirmed by CD spectroscopy in the presence of 60% (v/v) TFE. The synthetic peptide displayed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria including Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa and also against fungi (Candida albicans, C. krusei, C. parapsilosis and Cryptococcus neoformans). Hylin a1 was also able to disrupt human erytrocytes (HC(50)=18 microM). Similarity analysis using PSI-BLAST revealed 50-44% of identity to maximins Hv, H16, H15 and H10 from Bombina maxima and also to hylins b1 and b2 (Hy-b1 and Hy-b2) from Hypsiboas lundii (synonym: Hyla biobeba).

The apparent conflict between literature evidence for (i) radical pair (RP) stabilization in adenosylcobalamin (AdoCbl)-dependent enzymes and (ii) the manifestation of magnetic field sensitivity due to appreciable geminate recombination of the RP has been reconciled by pre-steady-state magnetic field effect (MFE) investigations with ethanolamine ammonia lyase (EAL). We have shown previous stopped-flow MFE studies to be insensitive to magnetically induced changes in the net forward rate of C-Co homolytic bond cleavage. Subsequently, we observed a magnetic-dependence in the continuous-wave C-Co photolysis of free AdoCbl in 75% glycerol but have not done so in the thermal homolysis of this bond in the enzyme-bound cofactor in the presence of substrate. Consequently, in the enzyme-bound state, the RP generated upon homolysis appears to be stabilized against the extent of geminate recombination required to observe an MFE. These findings have strong implications for the mechanism of RP stabilization and the unprecedented catalytic power of this important class of cobalamin-dependent enzymes.

The conventional concept is that the insulin-like growth factor binding proteins (IGFBPs) are cysteine-rich proteins, with conserved N- and C-domains, that are capable of binding insulin-like growth factors (IGFs) with high affinity. This dogma was recently challenged by the discovery of a group of cysteine-rich proteins that share important structural similarities with the IGFBPs, but have demonstrably lower affinity for IGFs. It is therefore proposed that these IGFBP-related proteins (IGFBP-rPs) and the IGFBPs constitute an IGFBP superfamily. We speculate that the IGFBP superfamily is derived from an ancestral gene/protein that was critically involved in the regulation of cell growth and was capable of binding IGF peptides. Over the course of evolution, some members (IGFBPs) evolved into high-affinity IGF binders and others (IGFBP-rPs) into low-affinity IGF binders, thereby conferring on the IGFBP superfamily the ability to influence cell growth by both IGF-dependent and IGF-independent means.

Nitric oxide (NO) has been recently shown to modulate in vitro motility, viability, the acrosome reaction (AR), and metabolism of spermatozoa in various mammalian species, but the mechanism or mechanisms through which it influences sperm functions has not been clarified. In human capacitated spermatozoa, both the intracellular cGMP level and the percentage of AR-positive cells were significantly increased after 4 h of incubation with the NO donor, sodium nitroprusside (SNP). SNP-induced AR was significantly reduced in the presence of the soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ; this block was bypassed by adding 8-bromo-cGMP, a cell-permeating cGMP analogue, to the incubation medium. Finally, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, two inhibitors of the cGMP-dependent protein kinases (PKGs), inhibited the SNP-induced AR. Furthermore, SNP-induced AR did not occur in Ca2+ -free medium or in the presence of the protein kinase C (PKC) inhibitor, calphostin C. This study suggests that the AR-inducing effect of exogenous NO on capacitated human spermatozoa is accomplished via stimulation of an NO-sensitive sGC, cGMP synthesis, and PKG activation. In this effect the activation of PKC is also involved, and the presence of extracellular Ca2+ is required.

OBJECTIVE

To investigate the role of WDR36 and P53 sequence variations in POAG susceptibility.

METHODS

The authors performed a case-control genetic association study in 268 unrelated Spanish patients (POAG1) and 380 control subjects matched for sex, age, and ethnicity. WDR36 sequence variations were screened by either direct DNA sequencing or denaturing high-performance liquid chromatography. P53 polymorphisms p.R72P and c.97-147ins16bp were analyzed by single-nucleotide polymorphism (SNP) genotyping and PCR, respectively. Positive SNP and haplotype associations were reanalyzed in a second sample of 211 patients and in combined cases (n = 479).

RESULTS

The authors identified almost 50 WDR36 sequence variations, of which approximately two-thirds were rare and one-third were polymorphisms. Approximately half the variants were novel. Eight patients (2.9%) carried rare mutations that were not identified in the control group (P = 0.001). Six Tag SNPs were expected to be structured in three common haplotypes. Haplotype H2 was consistently associated with the disease (P = 0.0024 in combined cases). According to a dominant model, genotypes containing allele P of the P53 p.R72P SNP slightly increased glaucoma risk. Glaucoma susceptibility associated with different WDR36 genotypes also increased significantly in combination with the P53 RP risk genotype, indicating the existence of a genetic interaction. For instance, the OR of the H2 diplotype estimated for POAG1 and combined cases rose approximately 1.6 times in the two-locus genotype H2/RP.

CONCLUSIONS

Rare WDR36 variants and the P53 p.R72P polymorphism behaved as moderate glaucoma risk factors in Spanish patients. The authors provide evidence for a genetic interaction between WDR36 and P53 variants in POAG susceptibility, although this finding must be confirmed in other populations.

Mutations in almost 200 genes are associated with hereditary retinal diseases. Of these diseases, retinitis pigmentosa (RP) is the most common and is genetically and clinically highly heterogeneous. At least 62 genes are associated with RP and mutations in these genes account for approximately half of the cases of disease. In the present study, mutations in the CHM gene, which are known to associate with choroideremia, were identified in six of 157 families with retinitis pigmentosa by whole exome sequencing. No potential pathogenic mutations in the 62 RP‑associated genes were found in the six families. Sanger sequencing confirmed the mutations in CHM, including four novel (c.558_559delTT, c.964G>T, c.966delA, c.1166+2T>G) and two known (c.703‑1G>A and c.1584_1587delTGTT) mutations. Available clinical data suggest an atypical phenotype of choroideremia in these patients compared to that of Caucasians. Overlapping clinical features and atypical phenotypic variation may contribute to the confusion of one another. Awareness of the phenotypic variation and careful clinical examination may facilitate proper clinical diagnosis and genetic counseling of complicated hereditary retinal diseases. Whole exome sequencing therefore is useful in the identification of genetic cause for less clarified hereditary retinal diseases and enriches our understanding of phenotypic variations of gene mutation.

A- and C-fiber evoked ventral root potential (VRP) responses have been examined in isolated spinal cord preparations maintained in vitro that were taken from young rats in which behavioral hyperalgesia (thermal and mechanical) was induced following UV irradiation of one hindpaw. Evoked VRPs were compared with those in naive untreated animals. The duration of both the A- and C-fiber evoked VRP was significantly increased in UV-treated animals. The amplitude of the summated VRP evoked by repeated low-frequency (1.0-5.0 Hz) C-fiber stimulation, a measure of windup, was significantly greater in UV-treated animals. In UV-treated animals, repeated low-frequency (1.0-5.0 Hz) stimulation of A-fiber inputs to the spinal cord also evoked a significant summated VRP, which was not observed in spinal cords from untreated animals. In naive animals the prolonged VRP evoked following single shock C-fiber stimulation was significantly antagonized by the NMDA receptor antagonist D-AP5 and the NK2 receptor antagonist MEN, 10376 but not by the NK1 receptor antagonists CP-96,345 or RP,67580. Summated VRPs evoked by repeated C-fiber stimulation in naive animals were significantly antagonized only by D-AP5. In hyperalgesic animals the prolonged VRP evoked by C-fiber stimulation was significantly reduced by NK1, NK2, and NMDA antagonists. The summated VRP was also significantly reduced by these antagonists. In both untreated and UV-irradiated animals the single shock evoked A-fiber ventral root response was significantly antagonized only by D-AP5. However, the summated VRP evoked by repeated A-fiber stimulation in UV-treated animals was also significantly reduced by NMDA, NK1, and NK2 receptor antagonists. The present study has demonstrated enhanced A- and C-fiber evoked responses in the rat spinal cord in vitro following induction of a peripheral injury by UV irradiation and which was associated with behavioral hyperalgesia to thermal and mechanical stimuli. Under this condition, repetitive stimulation of A-fiber primary afferents was capable of producing an enhancement of spinal excitability similar to that evoked by C-fibers in normal animals. Furthermore, we have observed the expression of an NK1 receptor component to the C-fiber evoked response following the establishment of the peripheral injury. The enhanced ventral root responses and changes in receptor sensitivity may contribute to the phenomenon of central sensitization and may be directly related to the behavioral hyperalgesia observed. Moreover, these findings may be relevant to the mechanisms of enhanced central excitability that occur in clinical conditions of inflammatory hyperalgesia and neuropathic pain.

Cylindrical iliac crest biopsies were obtained from 16 patients with autosomal dominant osteopetrosis after intravital double labeling with tetracycline, and compared with normal age- and sex-matched controls. Ten patients had the radiological type I (5 women, 5 men, aged 17-62 years, mean 42) characterized by diffuse, symmetrical osteosclerosis and enlarged thickness of the cranial vault. Six patients had type II (2 women, 4 men, aged 22-44 years, mean 36), where "Rugger Jersey Spine" and endobone are characteristic findings. Structural studies of cortical and trabecular bone were performed, and trabecular bone resorption and formation rates were studied using dynamic histomorphometry. The total biopsy length (C. Wi) were increased in type I (p less than 0.05), and unchanged in type II. Both types showed increased cortical width (Ct. Wi) (p less than 0.01 and p less than 0.05, respectively), and decreased fractional width of cancellous bone (Cn.Wi/C.Wi) (p less than 0.01 and p less than 0.05). The fractional trabecular bone volume (BV/TV) and trabecular thickness (Tb. Th) were both significantly increased in type I (p less than 0.05), while resorptive and formative indices of trabecular bone remodeling were normal. No difference was found in trabecular bone balance, which was slightly positive in both patients and controls. In type II osteopetrosis the eroded surfaces (OS/BS) were significantly increased (p less than 0.01), as was the total resorptive period RP) (p less than 0.05). The resorption depth (R.D.) was normal, while the resorption rate (MRR) was insignificantly decreased. Many big multinucleated osteoclasts were seen in this type suggesting defective resorptive function.(ABSTRACT TRUNCATED AT 250 WORDS)

Accurate and sensitive measurement techniques are a key issue in the quantification of the microvascular and endothelial dysfunction in systemic sclerosis (SSc). Thermal hyperemia comprises two separate mechanisms: an initial peak that is axon reflex mediated; and a sustained plateau phase that is nitric oxide dependent. The main objective of our study was to test whether thermal hyperemia in patients with SSc differed from that in patients with primary Raynaud's phenomenon (RP) and healthy controls. In a first study, we enrolled 20 patients suffering from SSc, 20 patients with primary RP and 20 healthy volunteers. All subjects were in a fasting state. Post-occlusive hyperemia, 0.4 mg sublingual nitroglycerin challenge and thermal hyperemia were performed using laser Doppler flowmetry on the distal pad of the third left finger. In a second study, thermal hyperemia was performed in 10 patients with rheumatoid arthritis and 10 patients with primary RP. The thermal hyperemia was dramatically altered in terms of amplitude and kinetics in patients with SSc. Whereas 19 healthy volunteers and 18 patients with primary RP exhibited the classic response, including an initial peak within the first 10 minutes followed by a nadir and a second peak, this occurred only in four of the SSc patients (p < 0.0001). The 10 minutes thermal peak was 43.4 (23.2 to 63), 42.6 (31 to 80.7) and 27 (14.7 to 51.4) mV/mm Hg in the healthy volunteers, primary RP and SSc groups, respectively (p = 0.01), while the 44 degrees C thermal peak was 43.1 (21.3 to 62.1), 42.6 (31.6 to 74.3) and 25.4 (15 to 52.4) mV/mm Hg, respectively (p = 0.01). Thermal hyperemia was more sensitive and specific than post-occlusive hyperhemia for differentiating SSc from primary RP. In patients with rheumatoid arthritis, thermal hyperemia was also altered in terms of amplitude. Thermal hyperemia is dramatically altered in patients with secondary RP in comparison with subjects with primary RP. Further studies are required to determine the mechanisms of this altered response, and whether it may provide additional information in a clinical setting.

Nitrogen fixation is catalysed by some photosynthetic bacteria. This paper presents a phylogenetic comparison of a nitrogen fixation gene (nifH) with the aim of elucidating the processes underlying the evolutionary history of Rhodopseudomonas palustris. In the NifH phylogeny, strains of Rps. palustris were placed in close association with Rhodobacter spp. and other phototrophic purple non-sulfur bacteria belonging to the alpha-Proteobacteria, separated from its close relatives Bradyrhizobium japonicum and the phototrophic rhizobia (Bradyrhizobium spp. IRBG 2, IRBG 228, IRBG 230 and BTAi 1) as deduced from the 16S rRNA phylogeny. The close association of the strains of Rps. palustris with those of Rhodobacter and Rhodovulum, as well as Rhodospirillum rubrum, was supported by the mol% G+C of their nifH gene and by the signature sequences found in the sequence alignment. In contrast, comparison of a number of informational and operational genes common to Rps. palustris CGA009, B. japonicum USDA 110 and Rhodobacter sphaeroides 2.4.1 suggested that the genome of Rps. palustris is more related to that of B. japonicum than to the Rba. sphaeroides genome. These results strongly suggest that the nifH of Rps. palustris is highly related to those of the phototrophic purple non-sulfur bacteria included in this study, and might have come from an ancestral gene common to these phototrophic species through lateral gene transfer. Although this finding complicates the use of nifH to infer the phylogenetic relationships among the phototrophic bacteria in molecular diversity studies, it establishes a framework to resolve the origins and diversification of nitrogen fixation among the phototrophic bacteria in the alpha-Proteobacteria.

The purpose of this study was to examine whether rCGRP has effects on TNF-alpha produced by mouse resident peritoneal macrophages. Macrophages were obtained from the peritoneal exudate of male Balb/c mouse. The cells were plated on culture dishes at a density of 2.5x10(5) cells per well and allowed to adhere for 2 hr. Pretreatment with rCGRP (10 nM-1 microM) for 24 hr, the macrophages were cultured with LPS 1 microg/ml for another 24 h. The medium was harvested for measuring TNF-alpha by ELISA kits. The results showed that rCGRP had no direct effects on TNF-alpha production, but it inhibited LPS-induced TNF-alpha production in a concentration-dependent manner. When rCGRP was at a concentration of 1 microM, the LPS-induced TNF-alpha production was inhibited by 39%. The effect of rCGRP was reversed by hCGRP(8-37) (10 microM), an antagonist of CGRP1 receptor. The LPS-induced TNF-alpha production from macrophages was also inhibited by forskolin 3 microM, an activator of adenylate cyclase. Furthermore, pretreatment with H-89 1 microM or Rp-cAMPS 100 microM, the inhibitors of cAMP-dependent protein kinase, the effect of rCGRP was abolished. These data suggest that the LPS-induced TNF-alpha production is inhibited by rCGRP via activation of cAMP responses in mouse resident peritoneal macrophages.

OBJECTIVE

To characterize the visual phenotype caused by mutations in the BTB-Kelch protein, KLHL7, responsible for the RPRP).

METHODS

Comprehensive ophthalmic testing included visual acuity, static visual field, kinetic visual field, dark adaptometry, full-field electroretinography, spectral-domain optical coherence tomography, and fundus photography. Longitudinal visual function data (range, 15-27 years) were available for some of the affected individuals.

RESULTS

We report a phenotypic assessment of 3 unrelated families, each harboring different KLHL7 mutations (c.458C>T, c.449G>A, and c.457G>A). The fundi showed classic signs of RP. Best-corrected visual acuity was 20/50 or better in at least one eye up to age 65 years. Static and kinetic visual fields showed concentric constriction to central 10° to 20° by age 65 years; 2 patients with Goldmann perimetry exhibited bilateral visual field retention in the far periphery. Both rod and cone full-field electroretinographic amplitudes were substantially lower than normal, with a decline rate of 3% per year in cone 31-Hz flicker response. Rod and cone activation and inactivation variables were abnormal. Spectral-domain optical coherence tomography indicated retention of foveal inner segment-outer segment junction through age 65 years.

CONCLUSIONS

Mutations in KLHL7 are associated with a late-onset form of autosomal dominant retinal degeneration that preferentially affects the rod photoreceptors. Full-field electroretinographic findings, including recovery kinetics, are consistent with those observed in other forms of autosomal dominant RP.

CONCLUSIONS

The phenotypes are similar among patients with 3 types of KLHL7 mutations (c.458C>T, c.449G>A, and c.457G>A). Strong retention of foveal function and bilateral concentric constriction of visual fields with far periphery sparing may guide mutation screening in autosomal dominant RP.

The effects of the synthetic GH-releasing peptides, GHRP-2 and GHRP-6, on phosphatidylinositol (PI) hydrolysis and cAMP production have been examined in human pituitary somatotropinomas with and without adenylyl cyclase-activating gsp oncogenes. Both peptides dose-dependently stimulated the rate of PI hydrolysis and GH secretion by cell cultures of both types of somatotropinoma. GHRP-2 was considerably more potent than GHRP-6. The effects on GH secretion were reduced or abolished by phloretin, an inhibitor of protein kinase C, and W7, an inhibitor of calmodulin. However, antagonism of the GHRH-receptor and of protein kinase A with (N-Ac-Tyr1,D-Arg2)GRF-(1-29)-NH2 and Rp-adenosine-3',5'-cyclic monophosphothioate, respectively, did not alter the stimulatory effects of GHRP-2 and GHRP-6 on GH secretion. The effect of GHRP-2 and/or GHRP-6 on cAMP production was studied in 15 tumors, seven of which possessed constitutive adenylyl cyclase activity as evidenced by presence of gsp oncogenes. Both peptides stimulated cAMP production in the latter but not former types of tumor. Moreover, GHRP-2 and GHRP-6 potentiated the stimulation of cAMP production induced by GHRH and pituitary adenylate cyclase-activating polypeptide in tumors without gsp oncogenes. These results demonstrate that GHRP-2 and GHRP-6 exert identical effects on human pituitary somatotropinomas, except for differences in potency. Additionally, under conditions of adenylyl cyclase activity above basal levels (i.e. through stimulation of G2-protein coupled receptors or because of gsp oncogene expression), cAMP production can be increased even further by GHRP, providing evidence for cross-talk between the PI and adenylyl cyclase transduction systems in pituitary cells.

Two new x-type high-molecular-weight glutenin subunits with similar size to 1Dx5, designated 1Dx5*t and 1Dx5.1*t in Aegilops tauschii, were identified by SDS-PAGE, RP-HPLC, and MALDI-TOF-MS. The coding sequences were isolated by AS-PCR and the complete ORFs were obtained. Allele 1Dx5*t consists of 2481 bp encoding a mature protein of 827 residues with deduced Mr of 85,782 Da whereas 1Dx5.1*t comprises 2526 bp encoding 842 residues with Mr of 87,663 Da. The deduced Mr's of both genes were consistent with those determined by MALDI-TOF-MS. Molecular structure analysis showed that the repeat motifs of 1Dx5*t were correspondingly closer to the consensus compared to 1Dx5.1*t and 1Dx5 subunits. A total of 11 SNPs (3 in 1Dx5*t and 8 in 1Dx5.1*t) and two indels in 1Dx5*t were identified, among which 8 SNPs were due to C-T or A-G transitions (an average of 73%). Expression of the cloned ORFs and N-terminal sequencing confirmed the authenticities of the two genes. Interestingly, several hybrid clones of 1Dx5*t expressed a slightly smaller protein relative to the authentic subunit present in seed proteins; this was confirmed to result from a deletion of 180 bp through illegitimate recombination as well as an in-frame stop codon. Network analysis demonstrated that 1Dx5*t, 1Dx2t, 1Dx1.6t, and 1Dx2.2* represent a root within a network and correspond to the common ancestors of the other Glu-D-1-1 alleles in an associated star-like phylogeny, suggesting that there were at least four independent origins of hexaploid wheat. In addition to unequal homologous recombination, duplication and deletion of large fragments occurring in Glu-D-1-1 alleles were attributed to illegitimate recombination.

Antimicrobial peptides are components of innate immunity that is the first-line defense against invading pathogens for a wide range of organisms. Here, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucine-rich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named leptoglycin. The amino acid sequence of the peptide purified by RP-HPLC (C(18) column) was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation: GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimal inhibitory concentrations (MICs) of 8 microM, 50 microM, and 75 microM respectively, but it did not show antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) and dermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activity was observed at the 2-200 microM range concentration. The amino acid sequence of leptoglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusual central proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides. Taken together, these results suggest that this natural antimicrobial peptide could be a tool to develop new antibiotics.

Bioactive peptides and tryptic digests of various proteins were separated under acidic and alkaline conditions by ion-pair-reversed-phase high-performance liquid chromatography (RP-HPIPC) in 200 microm I.D. monolithic, poly(styrene-divinylbenzene)-based capillary columns using gradients of acetonitrile in 0.050% aqueous trifluoroacetic acid, pH 2.1, or 1.0% triethylamine-acetic acid, pH 10.6. Chromatographic performances with mobile phases of low and high-pH were practically equivalent and facilitated the separation of more than 50 tryptic peptides of bovine serum albumin within 15-20 min with peak widths at half height between 4 and 10 s. Neither a significant change in retentivity nor efficiency of the monolithic column was observed during 17-day operation at pH 10.6 and 50 degrees C. Upon separation by RP-HPIPC at high-pH, peptide detectabilities in full-scan negative-ion electrospray ionization mass spectrometry (negESI-MS) were about two to three times lower as compared to RP-HPIPC at low-pH with posESI-MS detection. Tandem mass spectra obtained by fragmentation of deprotonated peptide ions in negative ion mode yielded interpretable sequence information only in a few cases of relatively short peptides. However, in order to obtain sequence information for peptides separated with alkaline mobile phases, tandem mass spectrometry (MS/MS) could be performed in positive ion mode. The chromatographic selectivities were significantly different in separations performed with acidic and alkaline eluents, which facilitated the fractionation of a complex peptide mixture obtained by the tryptic digestion of 10 proteins utilizing off-line, two-dimensional RP-HPIPC at high pH x RP-HPIPC at low pH and subsequent on-line identification by posESI-MS/MS.

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.

A novel ion-pair reversed phase electrospray ionization (IP-RP-ESI) liquid chromatography-mass spectrometry (LC-MS) method has been developed for identification and quantification of Bcl-2 antisense phosphorothioate oligonucleotides G3139 and metabolites in plasma. This method utilized solid phase extraction for desalting and matrix removal and detection by an ion trap mass spectrometer. Resolution was accomplished on a micro CC. Five G3139 metabolites were identified in plasma and urine from treated patients and rats. A cassette HPLC-MS/MS quantification method for G3139 and three metabolites was developed and validated with a limit of quantification (LOQ) of 17.6 nM in human and rat plasma with acceptable precision and accuracy. Plasma pharmacokinetics of G3139 and metabolites in these species were described.

Recognition of polyubiquitylated substrates by the proteasome is a highly regulated process that requires polyubiquitin receptors. We show here that the concentrations of the proteasomal and extraproteasomal polyubiquitin receptors change in a developmentally regulated fashion. The stoichiometry of the proteasomal p54/Rpn10 polyubiquitin receptor subunit, relative to that of other regulatory particle (RP) subunits falls suddenly at the end of embryogenesis, remains low throughout the larval stages, starts to increase again in the late third instar larvae and remains high in the pupae, adults and embryos. A similar developmentally regulated fluctuation was observed in the concentrations of the Rad23 and Dsk2 extraproteasomal polyubiquitin receptors. Depletion of the polyubiquitin receptors at the end of embryogenesis is due to the emergence of a developmentally regulated selective proteolytic activity. To follow the fate of subunit p54/Rpn10 in vivo, transgenic Drosophila melanogaster lines encoding the N-terminal half (NTH), the C-terminal half (CTH) or the full-length p54/Rpn10 subunit were established in the inducible Gal4-UAS system. The daughterless-Gal4-driven whole-body expression of the full-length subunit or its NTH did not produce any detectable phenotypic changes, and the transgenic products were incorporated into the 26S proteasome. The transgene-encoded CTH was not incorporated into the 26S proteasome, caused third instar larval lethality and was found to be multi-ubiquitylated. This modification, however, did not appear to be a degradation signal because the half-life of the CTH was over 48 hours. Accumulation of the CTH disturbed the developmentally regulated changes in subunit composition of the RP and the emergence of the selective proteolytic activity responsible for the depletion of the polyubiquitin receptors. Build-up of subunit p54/Rpn10 in the RP had already started in 84-hour-old larvae and reached the full complement characteristic of the non-larval developmental stages at the middle of the third instar larval stage, just before these larvae perished. Similar shifts were observed in the concentrations of the Rad23 and Dsk2 polyubiquitin receptors. The postsynthetic modification of CTH might be essential for this developmental regulation, or it might regulate an essential extraproteasomal function(s) of subunit p54/Rpn10 that is disturbed by the expression of an excess of CTH.

OBJECTIVE

To measure the time and subjective quality of individual steps of robot-assisted radical prostatectomy (RARP), as RARP performed by trainees has recently become the most common technique of RP in the USA, and although outcomes from expert surgeons are reported, limited data are available to document training experiences.

METHODS

The patients studied were from a prospective cohort of 178 participants (124 with training data). Transperitoneal RARP was performed by one faculty surgeon and one assistant from a rotation of four urological oncology fellows and three residents. RARP was divided into 11 steps, and staff times were recorded for each step. Trainee times and quality scores were recorded for each step, the later defined as grade A equal to staff (A+, no verbal coaching); B, minor corrections; and C, major corrections. Short-term outcomes were recorded to assess the safety of the training.

RESULTS

The mean (range) console time/case of trainees was 40 (10-123) min. The median console time for a complete case by faculty and by trainees (pooled group) was 128 and 231 min, respectively, an increase in 81%. Individual trainee-performed steps increased in time (compared to staff) by a median range of 50-177%, and the incidence of quality grades < A of 9-100%. Trainee quality grades for basic tissue-dissection steps were higher than for advanced tissue dissection and suturing. There was no downgrading for a major correction. Analysis of short-term outcomes suggested acceptable results in a training environment. The study is limited by no available validated training measurement tools, and a low frequency of beginner trainees advancing to more difficult steps during the rotation.

CONCLUSIONS

During the initial exposure of trainees to RARP of <40 cases, we measured time and subjective quality grading of basic steps, and introduction to advanced steps. Training requires more procedure time, but does not appear to diminish expected outcomes.

Dopaminergic and glutamatergic signalling cascades are integrated in striatal medium spiny neurones by cyclic AMP response-element binding protein and Elk-1 phosphorylation. Phosphorylated cyclic AMP response-element binding protein and phosphorylated Elk-1 contribute to c-fos expression by binding to the calcium and cyclic AMP response-element and the serum response element, respectively, in the c-fos promoter. The role of cyclic AMP and mitogen-activated protein kinase signalling cascades in glutamate-induced cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression was investigated using semiquantitative immunocytochemistry in vivo. Intracerebroventricular infusion of the sodium channel blocker, tetrodotoxin, decreased the glutamate-induced increase in phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Intracerebroventricular infusion of the mitogen-activated and extracellular signal-regulated kinase inhibitor, PD98059, the p38 mitogen-activated protein kinase inhibitor, SB203580, or the cyclic AMP inhibitor, Rp-8-Br-cAMPS, decreased glutamate-induced phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Simultaneous infusion of glutamate and Sp-8-Br-cAMPS, a cyclic AMP analogue, augmented induction of Fos immunoreactivity but not phosphorylated cyclic AMP response-element binding protein or phosphorylated Elk-1 immunoreactivity. These data indicate that cyclic AMP and mitogen-activated protein kinase signalling cascades are necessary for glutamate to induce cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression in the striatum. Furthermore, neuronal activity plays an important role in glutamate-induced signalling cascades in vivo.

Peptide P(11)-4 (QQRFEWEFEQQ) was designed to self-assemble to form beta-sheets and nematic gels in the pH range 5-7 at concentrations>> or =12.6 mM in water. This self-assembly is reversibly controlled by adjusting the pH of the solvent. It can also self-assemble into gels in biological media. This together with its biocompatibility and biodegradability make P(11)-4 an attractive building block for the fabrication of nanoscale materials with uses in, for example, tissue engineering. A limitation to large-scale production of such peptides is the high cost of solid phase chemical synthesis. We describe expression of peptide P(11)-4 in the bacterium Escherichia coli from constructs carrying tandem repeats of the peptide coding sequence. The vector pET31b+ was used to express P(11)-4 repeats fused to the ketosteroid isomerase protein which accumulates in easily recoverable inclusion bodies. Importantly, the use of auto-induction growth medium to enhance cell density and protein expression levels resulted in recovery of 2.5 g fusion protein/L culture in both shake flask and batch fermentation. Whole cell detergent lysis allowed recovery of inclusion bodies largely composed of the fusion protein. Cyanogen bromide cleavage followed by reverse phase HPLC allowed purification of the recombinant peptide with a C-terminal homoserine lactone (rP(11)-4(hsl)). This recombinant peptide formed pH dependent hydrogels, displayed beta-structure measured by circular dichroism and fibril formation observed by transmission electron microscopy.