High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides.

The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics.

Cognitive remediation improves memory and psychosocial functioning in first-episode psychiatric out-patients.

BACKGROUND: Cognitive remediation (CR) is an effective treatment for several psychiatric disorders. To date, there have been no published studies examining solely first-episode psychiatric cohorts, despite the merits demonstrated by early intervention CR studies. The current study aimed to assess the effectiveness of CR in patients with a first-episode of either major depression or psychosis. Method Fifty-five patients (mean age = 22.8 years, s.d. = 4.3) were randomly assigned to either CR (n = 28) or treatment as usual (TAU; n = 27). CR involved once-weekly 2-h sessions for a total of 10 weeks. Patients were comprehensively assessed before and after treatment. Thirty-six patients completed the study, and analyses were conducted using an intent-to-treat (ITT) approach with all available data. RESULTS: In comparison to TAU, CR was associated with improved immediate learning and memory controlling for diagnosis and baseline differences. Similarly, CR patients demonstrated greater improvements than TAU patients in psychosocial functioning irrespective of diagnosis. Delayed learning and memory improvements mediated the effect of treatment on psychosocial functioning at a marginal level. CONCLUSIONS: CR improves memory and psychosocial outcome in first-episode psychiatric out-patients for both depression and psychosis. Memory potentially mediated the functional gains observed. Future studies need to build on the current findings in larger samples using blinded allocation and should incorporate longitudinal follow-up and assessment of potential moderators (e.g. social cognition, self-efficacy) to examine sustainability and the precise mechanisms of CR effects respectively.

Factors affecting decision making about fertility preservation after cancer diagnosis: a qualitative study.

OBJECTIVE: To increase our understanding of factors underlying the decision to store gametes after the diagnosis of cancer. DESIGN: Qualitative interview study. SETTING: Andrology, Haematology, and Oncology Departments of a Scottish teaching hospital, and patients' own homes. POPULATION: Sixteen men and 18 women aged 17-49 years recently diagnosed with cancer; 15 health professionals concerned in cancer care. METHODS: Audio-recorded semi-structured interviews were transcribed verbatim and analysed thematically. Topics included perceptions of diagnosis; prognosis; future reproductive choices; priorities; quality of information received; communication and decisions made about future reproductive choices; and the role of partners, family, friends and healthcare professionals. Professional interviews examined their role in decision making and that of protocols and guidelines, together with information emerging from patient interview analysis. MAIN OUTCOME MEASURE: Themes identified following analysis of interview transcripts. RESULTS: The primary barriers to pursuing fertility preservation were the way in which information was provided and the 'urgent need for treatment' conveyed by staff. Survival was always viewed as paramount, with future fertility secondary. Sperm banking was viewed as 'part and parcel' of oncology care, and the majority of men quickly stored sperm as 'insurance' against future infertility. Few women were afforded the opportunity to discuss their options, reflecting clinicians' reservations about the experimental nature of egg and ovarian tissue cryopreservation, and the need for partner involvement in embryo storage. CONCLUSIONS: Significant gaps in the information provided to young women diagnosed with cancer suggest the need for an early appointment with a fertility expert.

Social approach in pre-school children with Williams syndrome: the role of the face.

BACKGROUND: Indiscriminate social approach behaviour is a salient aspect of the Williams syndrome (WS) behavioural phenotype. The present study examines approach behaviour in pre-schoolers with WS and evaluates the role of the face in WS social approach behaviour. METHOD: Ten pre-schoolers with WS (aged 3-6 years) and two groups of typically developing children, matched to the WS group on chronological or mental age, participated in an observed play session. The play session incorporated social and non-social components including two components that assessed approach behaviour towards strangers; one in which the stranger's face could be seen and one in which the stranger's face was covered. RESULTS: In response to the non-social aspects of the play session, the WS group behaved similarly to both control groups. In contrast, the pre-schoolers with WS were significantly more willing than either control group to engage with a stranger, even when the stranger's face could not be seen. CONCLUSION: The findings challenge the hypothesis that an unusual attraction to the face directly motivates social approach behaviour in individuals with WS.

Decision making about mode of delivery among pregnant women who have previously had a caesarean section: A qualitative study.

OBJECTIVE: To explore prospectively women's decision making regarding mode of delivery after a previous caesarean section. MAIN OUTCOME MEASURES: The evolution of decision making, women's participation in decision making, and factors affecting decision making. DESIGN AND METHODS: A qualitative study using diaries, observations and semi-structured interviews. Data were analysed thematically from both a longitudinal and a cross-sectional perspective. SETTING: An antenatal unit in a large teaching hospital in Scotland and participants' homes. SAMPLE: Twenty-six women who had previously had a caesarean section for a nonrecurrent cause. RESULTS: Women were influenced by their own previous experiences and expectations, and the final decision on mode of delivery often developed during the course of the pregnancy. Most acknowledged that any decision was provisional and might change if circumstances necessitated. Despite a universal desire to be involved in the process, many women did not participate actively and were uncomfortable with having responsibility for decision making. Feelings about the amount and quality of the information received regarding delivery options varied greatly, with many women wishing for information to be tailored to their individual clinical circumstances and needs. In contrast to the impression created in the media, there was no evidence of clear preferences or strong demands for elective caesarean section. CONCLUSION: Women who have had a previous caesarean section do not usually have firm ideas about mode of delivery. They look for targeted information and guidance from medical personnel based on their individual circumstances, and some are unhappy with the responsibility of deciding how to deliver in the current pregnancy.

No linkage or association of telomeric and centromeric T-cell receptor beta-chain markers with susceptibility to type 1 insulin-dependent diabetes in HLA-DR4 multiplex families.

The T-cell receptor beta locus (TCRB) on chromosome 7q35 was studied as a candidate region for genetic susceptibility to type 1 insulin-dependent diabetes (IDDM). A highly polymorphic microsatellite marker mapping to the TCRBV6.7 gene and a TCRB C-region RFLP were used to genotype the members of a total of 21 multiplex IDDM families from two different geographical areas. There was no evidence to support linkage to either of these markers with IDDM, and conventional two-point analysis excluded linkage to the telomeric end of the TCRB complex, in the region of the highly informative TCRBV6.7 marker. There was significant linkage of IDDM to the class II HLA-D locus with significant lod scores > 3.0 obtained for the HLA-DRB1 and HLA-DQB1 genes. Affected sib-pair (ASP) and transmission disequilibrium (TDT) association tests confirmed these findings.

Exploration of the function of a regulatory sulfhydryl of phosphoribulokinase from spinach.

Phosphoribulokinase from spinach is deactivated by reversible oxidation of Cys16 and Cys55 to an intrasubunit disulfide. Both residues have been assigned to the nucleotide-binding domain of the active site. Clearly, Cys16 does not play a significant role in catalysis, as complete methylation of this residue decreases kcat by only 50%. With methylated enzyme as the starting material, modification by 2-nitro-5-thiocyanobenzoate was used to probe the function of Cys55. The reagent rapidly inactivates methylated kinase, and activity is fully restored by dithiothreitol treatment. ATP, and ribulose 5-phosphate retard inactivation. The stoichiometry of incorporation indicates that only one site per subunit undergoes cyanylation. Mapping of tryptic digests demonstrates that Cys55 is selectively labeled by the reagent. The low level of activity observed after modification of Cys55 by the sterically unobtrusive cyano group suggests that Cys55 could play a facilitative role in catalysis; alternatively, slight reorientation of other catalytic groups as a consequence of cyanylation of Cys55 could account for the inactivation. In either event, major conformational changes need not be invoked to account for the loss of kinase activity concomitant with regulatory oxidation.

Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16.

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme, S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparent Kd of 3-4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [14C]reagent per mole of enzyme subunit. Amino acid analysis of the [14C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.

The aggregation states of spinach phosphoribulokinase.

Phosphoribulokinase (PRK; EC 2.1.7.19) is active in illuminated chloroplasts and inactive in darkened chloroplasts. This regulatory mechanism is mediated by thioredoxin-dependent reduction of a kinase disulfide in vivo. Extracts of spinach (Spinacia oleracea L.) leaves in the presence of 10 mM dithiothreitol contain a single 80-kDa form of PRK as judged by gel filtration. Gel filtration of thiol-free extracts of light-harvested tissue shows the presence of two inactive forms of PRK, the 80-kDa form and an aggregate (> 550 kDa) form, but treatment of both forms with dithiothreitol restores kinase activity. Gel filtration following extraction of dark-harvested tissue in the absence of dithiotreitol demonstrates the presence of only the heavier form. Inclusion of 400 mM (NH4)2SO4 in the homogenization buffer during extraction of light-harvested tissue suppresses the formation of the high-M r form of PRK, but does not eliminate the aggregate form observed in extracts of dark-harvested leaves. However, prolonged treatment of extracts from dark-harvested tissue with 400 mM (NH4)2SO4 results in conversion of the high-M r form of phosphoribulokinase to the low-M r form. The data are consistent with the heavier form of phosphoribulokinase being the normal in-vivo aggregation state in the dark, while the lighter form is the normal aggregation state in the light.This research was sponsored jointly by the science and education administration of the U.S. Department of Agriculture under Grant No. 88-37130-3722 from the Competitive Research Grants Office and by the Office of Health and Environmental Research, U.S. Department of Energy under Contract DE-AC05-84OR21400 with Martin Marietta Energy Systems Inc., Oak Ridge, Tenn., USA. The author is Postdoctoral Investigator supported by the U.S. Department of Agriculture through Subcontract No. 88-37130-3722 from the Biology Division of Oak Ridge National Laboratory to the University of Tennessee.

Catalytic nonessentiality of an active-site cysteinyl residue of phosphoribulokinase.

The activity of the Calvin cycle enzyme phosphoribulokinase is coupled to photosynthetic electron transport by reversible oxidation/reduction mediated by thioredoxin-f. Previous studies have shown that one of the regulatory sulfhydryl groups, that of Cys-16, is positioned at the nucleotide-binding domain of the active site. To determine if oxidative deactivation of the kinase reflects catalytic essentiality of Cys-16, the methylation of spinach phosphoribulokinase by methyl-4-nitrobenzenesulfonate has been examined. Methylation of the kinase results in a 50% loss of the initial activity relative to controls. The suppression of kcat is accompanied by a 6-fold increase in the Km for ATP, without change in the Km for ribulose 5-phosphate. The insensitivity of the modified enzyme, in contrast to the native, to iodoacetate and 5,5'-dithiobis(2-nitrobenzoate) indicates that Cys-16 is a site of methylation. This supposition is verified independently by peptide mapping and Edman degradation subsequent to S-carboxymethylation with [14C]iodoacetate of the methylated kinase. Retention of significant enzymatic activity after complete modification of Cys-16 with the small, uncharged methyl moiety demonstrates that this active-site residue is not essential for catalysis.

Fluorescence studies of phosphoribulokinase, a light-regulated, chloroplastic enzyme.

Recent characterization of spinach phosphoribulokinase has revealed that the homodimeric molecule contains only two tryptophans per 44-kDa subunit. We have performed steady-state and frequency domain studies of the intrinsic fluorescence of this protein. The fluorescence properties reflect contributions from both types of tryptophan residues. One of these appears to be relatively exposed to solvent and the quencher, acrylamide; fluoresce with a lambda max of 345 nm; decay with a fluorescence lifetime of 6.3 ns; have a relatively red-shifted absorption spectrum; and have a certain degree of independent motional freedom, with respect to the protein. The other tryptophan residue appears to be more buried; fluoresce with lambda max of 325 nm; have a lifetime of 1.7 ns; have a relatively blue-shifted absorption spectrum; and not to enjoy independent motional freedom. On comparison of phase-resolved spectral data and solute quenching data, we suggest that resonance energy transfer between the blue and red tryptophan residues may occur. We also describe the strategy of simultaneously fitting Stern-Volmer quenching data collected at two emission wavelengths.

Characterization of the regulatory thioredoxin site of phosphoribulokinase.

Phosphoribulokinase is light-regulated via thioredoxin by reversible oxidation/reduction of sulfhydryl/disulfide groups. To identify the cysteinyl residues that are involved in regulation, the S-carboxymethyl labeling patterns of the fully reduced (active) and oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced, [14C]carboxymethylated enzyme contained four labeled peptides, all of which were purified and sequenced by Edman degradation. If the enzyme was oxidized by 5,5'-dithiobis-(2-nitrobenzoic acid) prior to carboxymethylation and tryptic digestion, only two labeled peptides were observed, thereby revealing the identity of the regulatory cysteines as Cys-16 and Cys-55. The former was previously implicated as part of the nucleotide-binding domain of the active site (Porter, M.A., and Hartman, F.C. (1986) Biochemistry 25, 7314-7318), a conclusion reinforced by the present observation that the sequence around the Cys-16 is similar to a consensus sequence of ATP-binding sites from a number of proteins of diverse phylogenetic origin (Higgins, C.F., Hiles, I.D., Salmond, G.P.C., Gill, D.R., Downie, J.A., Evans, I.J., Holland, I.B., Gray, L., Buckel, S.D., Bell, A.W., and Hermondson, M. (1986) Nature 323, 448-450). The regulatory disulfide of phosphoribulokinase was found to be intrasubunit based on the stoichiometry of the oxidation and the failure to resolve oxidized and reduced enzyme by gel filtration under dissociation conditions.

Purification and characterization of ribulose-5-phosphate kinase from spinach.

An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44,000 Mr subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH2-terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation.

Evidence for a reactive cysteine at the nucleotide binding site of spinach ribulose-5-phosphate kinase.

Ribulose-5-phosphate kinase from spinach was rapidly inactivated by N-bromoacetylethanolamine phosphate in a bimolecular fashion with a k2 of 2.0 M-1 S-1 at 2 degrees C and pH 8.0. Ribulose 5-phosphate had little effect on the rate of inactivation, whereas complete protection was afforded by ADP or ATP. The extent of incorporation as determined with 14C-labeled reagent was about 1 molar equivalent per subunit in the presence of ATP with full retention of enzymatic activity, and about 2 molar equivalents per subunit in the completely inactivated enzyme. Amino acid analyses of enzyme derivatized with 14C-labeled reagent reveal that all of the covalently incorporated reagent was associated with cysteinyl residues. Hence two sulfhydryls are reactive, but the inactivation correlates with alkylation of one cysteinyl residue at or near the enzyme's nucleotide binding site. The kinase was also extremely sensitive to the sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethyl-maleimide. The reactive sulfhydryl groups are likely those generated by reduction of a disulfide during activation.

Acclimation to High CO(2) in Bean : Carbonic Anhydrase and Ribulose Bisphosphate Carboxylase.

Young bean plants (Phaseolus vulgaris L. cv Seafarer) grew faster in air enriched with CO(2) (1200 microliters per liter) than in ambient CO(2) (330 microliters per liter). However, by 7 days when increases in overall growth (dry weight, leaf area) were visible, there was a significant decline (about 25%) in the leaf mineral content (N, P, K, Ca, Mg) and a drop in the activity of two enzymes of carbon fixation, carbonic anhydrase and ribulose 1,5-bisphosphate (RuBP) carboxylase under high CO(2). Although the activity of neither enzyme was altered in young, expanding leaves during the acclimation period, in mature leaves the activity of carbonic anhydrase was reduced 95% compared with a decline of 50% in ambient CO(2). The drop in RuBP carboxylase was less extreme with 40% of the initial activity retained in the high CO(2) compared with 50% in the ambient atmosphere. While CO(2) enrichment might alter the flow of carbon into the glycolate pathway by modifying the activities of carbonic anhydrase or RuBP carboxylase, there is no early change in the ability of photosynthetic tissue to oxidize glycolate to CO(2).

Regulation of chloroplastic carbonic anhydrase : effect of magnesium.

It was previously reported that magnesium ion inhibited carbonic anhydrase (Bamberger and Avron 1975 Plant Physiol 56: 481-485). Studies with partially purified carbonic anhydrase from spinach (Spinacia oleracea L.) chloroplasts show that the effect was the result of the chloride counterion and not the magnesium ion. Enzyme activity was reduced 50% upon addition of 3 to 10 millimolar MgCl(2) or KCl while all additions of MgSO(4) between 0.3 and 10 millimolar were mildly stimulatory.