Incubation of big endothelin-1 (big ET-1(1-39] with either the cytosolic or membrane fraction obtained from cultured endothelial cells, resulted in an increase in immunoreactive-endothelin (IR-ET), which was markedly inhibited by metal chelators. Phosphoramidon, a metalloproteinase inhibitor, specifically suppressed the membrane fraction-induced increase in IR-ET, whereas the increase in IR-ET observed with the cytosolic fraction was not influenced by phosphoramidon. Reverse-phase (RP)-HPLC of the incubation mixture of big ET-1 with the cytosolic or membrane fraction revealed one major IR-ET component corresponding to the elution position of synthetic ET-1(1-21). Simultaneously, immunoreactivities like the C-terminal fragment (CTF22-39) of big ET-1 were present, as deduced from the RP-HPLC coupled with the radioimmunoassay for CTF. Our results indicate the presence of two types of metalloproteinases, which convert big ET-1 to ET-1 via a single cleavage between Trp21 and Val22, in vascular endothelial cells.

OBJECTIVE

A simple weighted prognostic algorithm, based on capillaroscopy and autoantibodies, is developed to classify patients at different risk of transition from isolated RP to SSc within 5 years from the screening visit.

METHODS

Two hundred and eighty-eight of 768 patients with isolated RP who underwent capillaroscopy were recruited. The prognostic contributions of capillaroscopic findings (giant loops, haemorrhages and the number of capillaries) and SSc-associated autoantibodies (ACAs, anti-topo I and ANAs) were assessed in a semi-parametric regression models suitable for competing risks. A prognostic index was built by a bagging technique. A structured tree approach was used to extract simple classificatory rules that can be directly interpreted.

RESULTS

Thirty-four transitions from isolated RP to SSc and 42 to other CTDs were observed. All of the chosen variables had a substantial prognostic impact. A complex non-linear prognostic pattern was observed for capillaries, with the risk of developing SSc increasing as the number of loops decreased. The presence of ANAs had a strong impact on prognosis [hazard ratio (HR) = 9.70], which was increased by the presence of ACA (HR = 3.94; P < 0.001). A weighted prognostic classification for the development of SSc was constructed using capillary number, giant loops and ANAs. The prognostic discrimination was satisfactory (Harrell's C-index = 0.86).

CONCLUSIONS

Our prognostic model is based on easy-to-obtain features (i.e. the number of capillaries, giant loops and ANAs) and could be used to facilitate clinical decision making in the screening phase, and may also have important implications for stratifying patients into risk groups for future clinical assessment.

Enterococcus faecalis strain EFS2, isolated from the surface of a traditional cheese, produced a bacteriocin active against Gram-positive bacteria including Listeria spp. and some Staphylococcus aureus strains. The bacteriocin, named enterococcin EFS2, has been purified to homogeneity by ammonium sulphate precipitation and reversed-phase high performance liquid chromatography (RP-HPLC). The molecular weight was determined by mass spectrometry to be 7149.6. The amino acid composition of enterococcin EFS2 revealed that it contained 67 amino acid residues and had a blocked amino-terminal end. Enterococcin EFS2 induced viability loss, efflux of K+ ions and ATP, and cell lysis. Kinetic study of bactericidal activity of enterococcin EFS2 on Listeria innocua strain LIN11 indicated slower cell destruction than by nisin. At pH 7.0, the activity of enterococcin EFS2 was the highest at 35 degrees C and was lost at 15 degrees C. The bacteriocin was more active against L. innocua strain LIN11 in broth adjusted to pH 6.0, 7.0 and 8.0 than to pH 4.5 at 30 degrees C.

OBJECTIVE

To investigate the genetic basis for autosomal recessive severe early-onset retinitis pigmentosa (RP) in a consanguineous Israeli Muslim Arab family.

METHODS

Haplotype analysis for all known genes underlying autosomal recessive RP was performed. Mutation screening of the underlying gene was done by direct sequencing. An in vitro splicing assay was used to evaluate the effect of the identified mutation on splicing.

RESULTS

Haplotype analysis indicated linkage to the Tubby-like protein 1 (TULP)1 gene. Direct sequencing revealed a homozygous single base insertion, c.1495+2_1495+3insT, located in the conserved donor splice-site of intron 14. This mutation co-segregated with the disease, and was not detected in 114 unrelated Israeli Muslim Arab controls. We used an in vitro splicing assay to demonstrate that this mutation leads to incorrect splicing.

CONCLUSIONS

To date, 22 distinct pathogenic mutations of TULP1 have been reported in patients with early-onset RP or Leber congenital amaurosis. Here we report a novel splice-site mutation of TULP1, c.1495+2_1495+3insT, underlying autosomal recessive early-onset RP in a consanguineous Israeli Muslim Arab family. This report expands the spectrum of pathogenic mutations of the TULP1 gene.

OBJECTIVE

To assess the national trends and comparative effectiveness of the various treatments for pediatric ureteropelvic junction obstruction (UPJO).

METHODS

Within the Nationwide Inpatient Sample, a weighted estimate of 35,275 pediatric patients (<19 years; 1998-2010) with UPJO underwent open pyeloplasty (OP), laparoscopic pyeloplasty (LP), robot-assisted pyeloplasty (RP, ≥October 2008) or endopyelotomy (EP). National trends in utilization and comparative effectiveness were evaluated.

RESULTS

Minimally invasive pyeloplasty (RP+LP, MIP) utilization began to increase in 2007; MIP accounted for 16.9% of cases (2008-2010). EP accounted for 1.4% of all cases from 1998 to 2010. On individual multivariate models (relative to OP): (a) no significant differences were noted between groups for intraoperative complications; (b) RP and LP had equivalent risks of postoperative complications developing (vs OP), but EP had a significantly higher risk of postoperative complications; (c) RP and EP had significantly higher risks of necessitating transfusions; (d) RP, LP, and EP had higher overall risks of greater hospital charges; (e) RP had a lower risk of greater length of stay, while EP had a higher risk (LP and OP were equivalent).

CONCLUSIONS

OP continues to be the predominant treatment for patients with UPJO. RP was the most common MIP modality in every age group. Compared with OP patients, RP patients had equivalent risk for intraoperative and postoperative complications, lower risk for greater length-of-stay, but higher risks for transfusions and greater hospital charges. LP patients had higher overall hospital charges, but no mitigating benefits relative to OP. EP fared poorly on most outcomes.

Retinitis pigmentosa (RP) is a degenerative retinopathy leading to visual impairment in more than 1.5 million patients worldwide. Splice site (SS) mutations cause various diseases including RP. Most exonic donor splice-site (DS) mutations are reported at the last nucleotide of an exon and over 95% of them are predicted to result in missplicing. A novel human mutation at the last nucleotide of exon 4 in rhodopsin (RHO, c.936G>A) is shown to generate two misspliced transcripts in COS 7 cells and retinal explants. One of these transcripts skips exon 4 whereas the other activates a cryptic DS. Both are predicted to result in truncated RHO, explaining the pathogenic mechanism underlying the patient's RP phenotype. U1 snRNA-mediated DS recognition is a key step in the splicing process. As a therapeutic strategy, U1 snRNAs were adapted to the novel RHO mutation and tested for its potential to reverse missplicing. The rescue efficiency for misspliced transcripts of RHO was examined by quantitative RT-PCR. Using mutation-adapted U1 snRNA, we observed significantly reduced exon skipping that reached wild-type levels. Nevertheless, activation of the cryptic splice site (CS) was still detected. To test the feasibility of the strategy for mutations that only cause exon skipping, we inactivated the CS. Indeed, adapted U1 snRNA was able to rescue almost 95% [corrected] of misspliced transcripts. This study shows that modified U1 snRNAs constitute a promising therapeutic strategy to treat DS mutations. Our findings have implications for various diseases caused by similar mutations.

1. Magnesium-protoporphyrin chelatase activity, previously shown in whole cells of Rhodopseudomonas spheroides, could not be demonstrated in cell-free extracts prepared in different ways, although spheroplasts retained moderate activity. Slight activity was detected also in whole cells of Rhodospirillum rubrum. 2. The effects on the activity of the enzyme of inhibitors of electron and energy transfer were studied in whole cells of Rps. spheroides. Amytal, rotenone, azide and cyanide inhibited at low pO(2) in the dark but not under anaerobic conditions in the light. Antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, as well as uncouplers and oligomycin, inhibited under all environmental conditions. 3. The effects on magnesium chelatase activity of intermediates of the tricarboxylic acid cycle, of thenoyltrifluoroacetone, of a number of artificial electron donors or acceptors, of various quinones and of the oxidation-reduction indicator dyes Benzyl Viologen and Methyl Viologen are described. 4. It was concluded that electron transport between a b-type and a c-type cytochrome as well as associated energy conservation and transformation reactions were essential for activity. There was also a specific requirement for ATP. 5. Exogenous protoporphyrin and magnesium protoporphyrin monomethyl ester were incorporated into bacteriochlorophyll or late precursors by whole cells. 6. Evidence is presented that the insertion of magnesium was the only step inhibited by oxygen in the biosynthetic pathway between protoporphyrin and bacteriochlorophyll.

A pancreatic carcinoma cell line, AsPC-1, underwent apoptosis in vitro when heat-treated for 60 min at 43 degrees C. Apoptotic AsPC-1 cells liberated a monocyte chemotactic factor into the culture supernatant 24 to 30 h after the heat-treatment. This factor was immunologically identified as the cross-linked homodimer of S19 ribosomal protein (RP S19), since the majority of the chemotactic activity was absorbed by both anti--RP S19 rabbit antibodies and an anti--isopeptide bond monoclonal antibody immobilized on agarose beads. Intracellular transglutaminase activity increased during the apoptotic process, reaching the peak strength between 18 and 24 h after the heat-treatment. A recombinant RP S19 acquired the monocyte chemotactic capacity when incubated with the apoptotic cell extract obtained at the 18th hour. The chemotactic activity acquirement as well as the transglutaminase activity were blocked by treatment of the extract with anti--type II transglutaminase rabbit antibodies. When the recombinant RP S19 was treated with an authentic type II transglutaminase, the dimerization of RP S19 concomitant with the generation of the monocyte chemotactic activity was observed. Peptide-map analyses involving amino acid sequencing demonstrated that the inter-molecular isopeptide bond was heterogeneous: Gln12 or Gln137 and Lys29 or Lys122 were cross-linked. Site-directed mutagenic analysis indicated that the cross-linking of Gln137, but not other residues such as Gln12, Lys29, and Lys122, was essential for expression of the chemotactic activity.

The human immunodeficiency virus type 1 (HIV-1) negative factor, or Nef, has a variety of functions that are important in viral pathogenesis. Sequence analysis has identified nef mutations that are linked to the rate of disease progression in adults and children infected with HIV-1 subtype B. Here we have sequenced and analyzed HIV-1 subtype C nef sequences from 34 children with rapid (RP) or slow progressing (SP) disease and identified polymorphisms associated with disease stage including motifs involved in specific pathogenic functions. Unlike subtype B, insertions and deletions in the N-terminal variable region were observed exclusively in SP children (8 out of 25). Strong positive selection pressures were found in sites of known functional importance among SP sequences, whereas RP had strong negative selection across the gene. A lineage analysis of selection pressures indicated weaker pressure across the nef gene in SP sequences bearing a deletion in region 8-12, suggesting this deletion has functional importance in vivo. Together these results suggest a differential adaptation of certain Nef functions related to disease progression, some of which may be attributable to immune-imposed pressures. These data broadly reflect previous studies on subtype B, corroborate the decreased cytopathicity of SP viruses, but also highlight potential subtype differences that require further investigation.

Ors binding activity (OBA) represents a HeLa cell protein activity that binds in a sequence-specific manner to A3/4, a 36-bp mammalian replication origin sequence. OBA's DNA binding domain is identical to the 80-kDa subunit of Ku antigen. Ku antigen associates with mammalian origins of DNA replication in vivo, with maximum binding at the G1/S phase. Addition of an A3/4 double-stranded oligonucleotide inhibited in vitro DNA replication of p186, pors12, and pX24, plasmids containing the monkey replication origins of ors8, ors12, and the Chinese hamster DHFR oribeta, respectively. In contrast, in vitro SV40 DNA replication remained unaffected. The inhibitory effect of A3/4 oligonucleotide was fully reversed upon addition of affinity-purified Ku. Furthermore, depletion of Ku by inclusion of an antibody recognizing the Ku heterodimer, Ku70/Ku80, decreased mammalian replication to basal levels. By co-immunoprecipitation analyses, Ku was found to interact with DNA polymerases alpha, delta and epsilon, PCNA, topoisomerase II, RF-C, RP-A, DNA-PKcs, ORC-2, and Oct-1. These interactions were not inhibited by the presence of ethidium bromide in the immunoprecipitation reaction, suggesting DNA-independent protein associations. The data suggest an involvement of Ku in mammalian DNA replication as an origin-specific-binding protein with DNA helicase activity. Ku acts at the initiation step of replication and requires an A3/4-homologous sequence for origin binding. The physical association of Ku with replication proteins reveals a possible mechanism by which Ku is recruited to mammalian origins.

A reverse-phase HPLC System for isolation of the water insoluble alpha- and beta-polypeptides of the light-harvesting complex II (LH II) of Rhodopseudomonas (Rps.) palustris without employment of any detergent was developed. The material obtained was of high purity and suitable for direct microsequence analysis. Chromatographic analysis could resolve at least two major beta-polypeptides, beta a and beta b, two major alpha-polypeptides, alpha a and alpha b, and two additional minor polypeptides. N-terminal amino acid sequencing shows that the resolved peaks correspond to different polypeptide species and that the minor species have an N-terminal sequence identical to that of the alpha b polypeptide. An oligonucleotide derived from the amino terminal sequence of the alpha a polypeptide was utilized to screen a genomic library from Rps.palustris. Several independent clones have been characterized by Southern blot and nucleotide sequence analysis. We show that Rps.palustris contains at least four different clusters of beta and alpha genes. Two clones contain sequences potentially coding for beta a-alpha a and beta b-alpha b polypeptides; and two additional clones potentially coding for beta and alpha peptides which we named beta c-alpha c and beta d-alpha d, which did not correspond to the major purified polypeptides. In addition to the protein chemistry data, the conservation at the amino acid level and the presence of canonical ribosomal binding sites upstream of each of the identified genes strongly suggest that all four coding regions are expressed.

Several lines of evidence have shown a role for the nitric oxide/cyclic guanosine monophosphate signaling pathway in the development of spinal hyperalgesia. However, the roles of effectors for cyclic guanosine monophosphate are not fully understood in the processing of pain in the spinal cord. The present study showed that cyclic guanosine monophosphate-dependent protein kinase Ialpha but not Ibeta was localized in the neuronal bodies and processes, and was distributed primarily in the superficial laminae of the spinal cord. Intrathecal administration of a selective inhibitor of cyclic guanosine monophosphate-dependent protein kinase Ialpha, Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine, produced a significant antinociception demonstrated by the decrease in the number of flinches and shakes in the formalin test. This was accompanied by a marked reduction in formalin-induced c-fos expression in the spinal dorsal horn. Moreover, cyclic guanosine monophosphate-dependent protein kinase Ialpha protein expression was dramatically increased in the lumbar spinal cord 96 h after injection of formalin into a hindpaw, which occurred mainly in the superficial laminae on the ipsilateral side of a formalin-injected hindpaw. This up-regulation of cyclic guanosine monophosphate-dependent protein kinase Ialpha expression was completely blocked not only by a neuronal nitric oxide synthase inhibitor, 7-nitroindazole, and a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, but also by an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate (MK-801). The present results indicate that noxious stimulation not only initially activates but also later up-regulates cyclic guanosine monophosphate-dependent protein kinase Ialpha expression in the superficial laminae via an N-methyl-D-aspartate-nitric oxide-cyclic guanosine monophosphate signaling pathway, suggesting that cyclic guanosine monophosphate-dependent protein kinase Ialpha may play an important role in the central mechanism of formalin-induced inflammatory hyperalgesia in the spinal cord.

The 26S proteasome is a non-lysosomal protease in the cytosol and nucleus of eukaryotic cells. Its main function is to mediate ubiquitin-dependent proteolysis. The 26S proteasome is a multimeric complex composed by the 20S proteasome CP (core particle) and the 19S RPs (regulatory particles). Although the atomic structure of the 26S proteasome has not yet been determined, high-resolution structures are available for its CP. Studies on the complicated assembly pathway of the proteasome have revealed that it involves an unprecedented number of dedicated chaperones. Assembly of the CP alone involves three conserved proteasome-assembly chaperones [PACCCCCs have been implicated in the formation of rings of the seven distinct alpha subunits, UMP1 is important for the formation and dimerization of proteasome precursor complexes containing beta subunits. Dimerization coincides with the incorporation of the last beta subunit (beta7). Additional modules important for the assembly of precursor complexes and their dimerization reside in the beta subunits themselves, either as transient or as permanent extensions. Particularly important domains are the propeptide of beta5 and the C-terminal extensions of beta2 and beta7. Upon maturation of the active sites by autocatalytic processing, UMP1 is degraded by the native proteasome.

Retinitis pigmentosa (RP) is the most common inherited retinal dystrophy, with extensive allelic and nonallelic genetic heterogeneity. Autosomal recessive RP (arRP) is the most common form of RP worldwide, with at least nine loci known and accountable for approximately 10%-15% of all cases. Gamma-aminobutyric acid (GABA) is the major inhibitory transmitter in the CNS. Different GABA receptors are expressed in all retinal layers, and inhibition mediated by GABA receptors in the human retina could be related to RP. We have selected chromosomal regions containing genes that encode the different subunits of the GABA receptors, for homozygosity mapping in inbred families affected by arRP. We identify a new locus for arRP, on chromosome 6, between markers D6S257 and D6S1644. Our data suggest that 10%-20% of Spanish families affected by typical arRP could have linkage to this new locus. This region contains subunits GABRR1 and GABRR2 of the GABA-C receptor, which is the effector of lateral inhibition at the retina.

In LTK- cells stably transfected with rat D1A receptor cDNA, fenoldopam, a D1 agonist, increased phosphatidylinositol 4, 5-bisphosphate hydrolysis in a time-dependent manner. In the cytosol, phospholipase C (PLC) activity increased (50 +/- 7%) in 30 s, returned to basal level at 4 h, and decreased below basal values by 24 h; in the membrane, PLC activity also increased (36 +/- 13%) in 30 s, returned to basal level at 10 min, and decreased below basal value at 4 and 24 h. Fenoldopam also increased PLC-gamma protein in a time-dependent manner. The latter was blocked by the D1 antagonist SKF83742 and by a D1A antisense oligodeoxynucleotide, indicating involvement of the D1A receptor. The fenoldopam-induced increase in PLC-gamma and activity was mediated by protein kinase A (PKA) since it was blocked by the PKA antagonist Rp-8-CTP-adenosine cyclic 3':5'-monophosphorothioate (Rp-8-CTP-cAMP-S) and mimicked by direct stimulation of adenylyl cyclase with forskolin or by a PKA agonist, Sp-cAMP-S. Protein kinase C (PKC) was also involved, since the fenoldopam-induced increase in PLC-gamma protein was blocked by two different PKC inhibitors, calphostin C and chelerythrine; calphostin C also blocked the fenoldopam-induced increase in PLC activity. In addition, forskolin and a PKA agonist, Sp-8-CTP-cAMP-S, increased PKC activity, and direct stimulation of PKC with phorbol 12-myristate 13-acetate increased PLC-gamma protein and activity, effects that were blocked by calphostin C. We suggest that the D1A-mediated stimulation of PLC occurs as a result of PKA activation. PKA then stimulates PLC-gamma in cytosol and membrane via activation of PKC.

Memory consolidation is the process by which recently acquired information becomes stable and is modulated by different neurotransmitters depending on the structure involved and the nature of the memory. Here we evaluate the participation of both D1 and D5 dopamine receptors in the CA1 region of the hippocampus in the consolidation of the memory of two different tasks, object recognition (OR) and inhibitory avoidance (IA). For this, male rats with infusion cannulae stereotaxically implanted in the CA1 region of the dorsal hippocampus were trained in an OR task involving exposure to two different objects, or in a one-trial step-down IA task. At different times after the training, some of the animals received intrahippocampal infusions of the D1-family receptor antagonist SCH-23390. In a test session carried out 24h later, the animals that received infusions immediately or 60 min but not 180 min after the training showed impaired long-term memory. Since D1- and D5-subtypes engage different signaling pathways involving cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), respectively, we assessed whether they participate distinctively in consolidation. The animals that received intra-CA1 infusions of the PKA inhibitor, Rp-cAMP, or the PKC inhibitor, Gö6976, immediately after OR or IA training had a long-term memory impairment and the amnesic effect caused by SCH-23390 was reversed when co-infused with activators of PKA (8Br-cAMP) or PKC (PMA). These results indicate that both D1 and D5 dopamine receptors are required in the CA1 region of the hippocampus for consolidation of the two tasks. This supports the notion of a commonality of consolidation mechanisms across tasks.

We asked if the mechanisms of exocytosis and its regulation in epithelial cells share features with those in excitable cells. Cultured dog pancreatic duct epithelial cells were loaded with an oxidizable neurotransmitter, dopamine or serotonin, and the subsequent release of these exogenous molecules during exocytosis was detected by carbon-fiber amperometry. Loaded cells displayed spontaneous exocytosis that may represent constitutive membrane transport. The quantal amperometric events induced by fusion of single vesicles had a rapid onset and decay, resembling those in adrenal chromaffin cells and serotonin-secreting leech neurons. Quantal events were frequently preceded by a "foot," assumed to be leak of transmitters through a transient fusion pore, suggesting that those cell types share a common fusion mechanism. As in neurons and endocrine cells, exocytosis in the epithelial cells could be evoked by elevating cytoplasmic Ca(2+) using ionomycin. Unlike in neurons, hyperosmotic solutions decreased exocytosis in the epithelial cells, and giant amperometric events composed of many concurrent quantal events were observed occasionally. Agents known to increase intracellular cAMP in the cells, such as forskolin, epinephrine, vasoactive intestinal peptide, or 8-Br-cAMP, increased the rate of exocytosis. The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas the Sp-isomer, a specific agonist of PKA, evoked exocytosis. Thus, PKA is a downstream effector of cAMP. Finally, activation of protein kinase C by phorbol-12-myristate-13-acetate also increased exocytosis. The PMA effect was not mimicked by the inactive analogue, 4alpha-phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I. Elevation of intracellular Ca(2+) was not needed for the actions of forskolin or PMA. In summary, exocytosis in epithelial cells can be stimulated directly by Ca(2+), PKA, or PKC, and is mediated by physical mechanisms similar to those in neurons and endocrine cells.

Xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.1.3.22), a molybdenum-containing hydroxylase that produces superoxide and uric acid from purine substrates and molecular oxygen, is involved in the oxidative stress underlying several human pathologies including lung diseases. An enzymatic activity similar to xanthine oxidase was previously reported in bronchoalveolar lavage fluid of patients with chronic obstructive pulmonary disease (COPD-BAL), by fluorometric analysis of DNA unwinding and cytochrome c reduction kinetics. Here we report the detection of xanthine oxidase activity products by electron paramagnetic resonance (EPR) in presence of the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and reversed-phase high-performance liquid chromatography (RP-HPLC) in COPD-BAL (n = 14, average age of patients 65 years, range 38-81) and BAL from healthy nonsmoker controls (n = 6, average age 64 years, range 44-73). Superoxide DMPO adducts were detected in COPD-BAL and in an in vitro system containing xanthine and xanthine oxidase (XA/XO), but not in BAL controls and when superoxide dismutase (SOD, 1000 I.U./ml) was added to COPD-BAL. The HPLC analyses after addition of xanthine showed production of uric acid in COPD-BAL and in the XA/XO system but not in BAL controls. These results support the involvement of xanthine oxidase in the mechanisms of superoxide production by BAL supernatant, which increases oxidative stress in chronic obstructive pulmonary disease.

Pristionchus pacificus is a free-living nematode increasingly used as an organism for comparison to the more familiar model <em>C</em>aenorhabditis elegans. In this study, we examined the N-glycans of this organism isolated after serial release with peptide:N-glycosidases F and A; after fluorescent labelling with 2-aminopyridine, chromatographic fractionation by three types of <em>RP</em>-HPL<em>C</em> (with either classical <em>C</em>18, fused core <em>C</em>18 or alkylamide-bonded phases) followed by mass spectrometric analyses revealed key features of its N-glycome. In addition to paucimannosidic and oligomannosidic glycans typical of invertebrates, N-glycans with two core fucose residues were detected. Furthermore, a range of glycans carrying up to three phosphorylcholine residues was observed whereas, unlike <em>C</em>. elegans, no tetrafucosylated N-glycans were detected. Structures with three fucose residues, unusual methylation of core α1,3-fucose or with galactosylated fucose motifs were found in low amounts; these features may correlate with a different ensemble or expression of glycosyltransferase genes as compared to <em>C</em>. elegans. From an analytical perspective, both the alkylamide <em>RP</em>-amide and fused core <em>C</em>18 columns, as compared to a classical <em>C</em>18 material, offer advantages in terms of resolution and of elution properties, as some minor pyridylamino-labelled glycans (e.g. those carrying phosphorylcholine) appear in earlier fractions and so potential losses of such structures due to insufficient gradient length can be avoided.

The present study sets out to extend the utility of reversed-phase liquid chromatography (RP-HPLC) by demonstrating its ability to monitor dimerization and unfolding of de novo designed synthetic amphipathic alpha-helical peptides on stationary phases of varying hydrophobicity. Thus, we have compared the effect of temperature (5-80 degrees C) on the RP-HPLC (CRP-HPLC can monitor association of peptide molecules, either through oligomerization or aggregation, or monitor unfolding of alpha-helical peptides with increasing temperature. We believe that the conformation-dependent response of peptides to RP-HPLC under changing temperature has implications both for general analysis and purification of peptides but also for the de novo design of peptides and proteins.

OBJECTIVE

To evaluate the effectiveness of relapse prevention (RP) and brief intervention (BI) in reducing HIV risk-taking behaviours among injecting drug users (IDU) enrolled in methadone programmes. The hypotheses tested were: (1) that a six-session RP programme would be more effective in reducing HIV risk-taking behaviours than a one-session BI and a non-intervention control condition (C); and (2) that BI would be more effective in reducing HIV risk-taking behaviours than C.

METHODS

Clients of methadone programmes were randomly assigned to either RP, BI, or C. Follow-up occurred 6 months after pre-intervention assessment and was conducted by independent research assistants who were not aware of subjects' group allocations.

METHODS

Confidential assessment interviews and interventions generally took place at the methadone unit treating the subject.

METHODS

Ninety-five IDU enrolled in methadone programmes. Study entry criteria were: injection of any drug in the 6 months before the day of pre-intervention assessment; literacy in English; agreement to HIV-antibody testing for research purposes; and no known diagnosis of a serious mental illness. Eighty subjects were contacted successfully for a 6-month follow-up.

METHODS

The RP intervention was a six-session programme. Each 60-90 min session was conducted individually. The BI was a one-session motivational interview lasting 60-90 min, accompanied by a self-help booklet.

METHODS

All subjects were administered the Drug Use Scale and HIV Risk-Taking Behaviour Scale of the Opiate Treatment Index and consented to the collection of a capillary blood sample for HIV-antibody testing at pre-intervention assessment and follow-up. At follow-up, the Highest HIV Risk-Taking Behaviour Scale, collateral reports from subjects' sexual partners pertaining to the previous month and urinalysis results for the month before follow-up were collected.

RESULTS

Compliance with interventions was good. Correspondence of self-reports with urinalysis and collateral reports was satisfactory. There were no significant differences between groups in risk-taking behaviours during the month before follow-up. However, there was evidence of a lower rate of needle-risk behaviour (sharing and cleaning) during the heaviest risk-taking month since pre-intervention assessment in the group given RP. There were no indications that BI was of greater benefit than the usual methadone treatment and neither intervention appeared to reduce sexual risk behaviour.

CONCLUSIONS

The results are cautiously interpreted as showing that individual RP programmes decrease the level of needle-risk behaviour during relapse episodes, but further research is required to replicate this finding.

The aim of this study is to examine the effect of phosphorylation pathways on the electrically evoked fast motile response of isolated outer hair cells (OHCs). Transcellular electrical stimulation was applied in the microchamber to guinea pig OHCs and motility was measured before and after drug application. Forskolin (adenylate cyclase activator), phorbol 12-myristate 13-acetate (PMA, protein kinase C activator) and dibutyryl 3',5'-cyclic guanosine monophosphate (cGMP agonist) were studied. As controls, L15 medium and dimethyl-sulfoxide (DMSO) were used. In each group, 12 cells were measured. Forskolin and PMA were dissolved in 0.1% DMSO to render them membrane permeable. DMSO by itself caused a statistically significant electromotility magnitude decrease. Forskolin and PMA could not reverse the motility decrease due to DMSO, the effects seen in their presence were the same as observed with DMSO alone. Thus, neither 3',5'-cyclic AMP-dependent protein kinase nor calcium/phospholipid-dependent protein kinase appear to have modulatory effects on electromotility. Dibutyryl cGMP (DBcGMP), in concentrations of 200 microM, elicited a significant electromotility magnitude increase. The DBcGMP effect could be inhibited by co-application of 200 microM DBcGMP and 100 microM 8-Rp-pCPT-cGMPS (8-4-chlorophenylthio-guanosine 3',5'-cyclic monophosphothioate, Rp isomer, a cGMP antagonist). Our results suggest that OHC electromotility is modulated by a cGMP-dependent pathway.

BACKGROUND

Retinitis pigmentosa (RP) is one of the most common ophthalmic disorders affecting one in approximately 5000 people worldwide. A nuclear family was recruited from the Punjab province of Pakistan to study the genetic basis of autosomal recessive RP.

METHODS

All affected individuals underwent a thorough ophthalmic examination and the disease was characterised based upon results for fundus photographs and electroretinogram recordings. Genomic DNA was extracted from peripheral leucocytes. Exclusion studies were performed with short tandem repeat (STR) markers flanking reported autosomal recessive RP loci. Haplotypes were constructed and results were statistically evaluated.

RESULTS

The results of exclusion analyses suggested that family PKRPRP. Additional STR markers refined the critical interval and placed it in a 13.4 cM (17 Mb) region flanked by D2S293 proximally and D2S347 distally. Significant logarithm of odds (LOD) scores of 3.2, 3.25 and 3.18 at theta=0 were obtained with markers D2S1896, D2S2269 and D2S160. Sequencing of the coding exons of MERTK identified a mutation, c.718G->>T in exon 4, which results in a premature termination of p.E240X that segregates with the disease phenotype in the family.

CONCLUSIONS

Our results strongly suggest that the nonsense mutation in MERTK, leading to premature termination of the protein, is responsible for RP phenotype in the affected individuals of the Pakistani family.

Retinitis pigmentosa (RP) is an hereditary degenerative disease of the retina and a major cause of visual impairment, prevalence estimates ranging from 1 in 3000 to 1 in 7000. The condition may segregate as an autosomal dominant, autosomal recessive or an X-linked recessive trait and it may also occur on a sporadic basis in up to 50% of cases. In the autosomal dominant form, close linkage to the DNA marker CC----A transversion in codon 23, resulting in a proline----histidine substitution has now been identified in 17 of 148 unrelated ADRP patients in the United States (Dryja et al. 1990). This mutation is absent however in the original Irish pedigree (it is also absent in 21 other dominant Irish pedigrees, representing approximately 70% of the estimated ADRP population) indicating that another mutation, either in rhodopsin itself, or in a gene very closely linked to rhodopsin is responsible for the disease in that family. Analysis of other dominant pedigrees using the CCommunication), looser linkage, possibly indicative of a second locus on 3q (Olsson et al. 1990) or no linkage (Farrar et al. 1990, Blanton et al. 1990, Inglehearn et al. 1990). Extensive genetic heterogeneity thus exists in the autosomal dominant form of this disease, and in the light of these new observations, earlier tentative evidence for linkage of ADRP to the Rhesus locus on chromosome 1 will be re-evaluated. A locus for type II Usher syndrome (classical RP combined with congenital pedial deafness, and normal vestibular function) has now been established on the long arm of chromosome 1 (Kimberling et al. 1990). Type I Usher families, in which hearing loss is more profound and vestibular function absent, do not segregate with the same chromosome 1q markers, indicating the existence of another, as yet unlocated gene. In the X-linked form of the disease, two genes, XLRPRPCT TRUNCATED AT 400 WORDS)