Search

Purpose: To explore the effects of the Personalized Citizen Assistance for Social Participation (APIC), an intervention adapted here for visual impairment, involving weekly stimulation sessions over six to twelve months, provided by trained and supervised attendants, on seven outcomes (social participation, leisure, independence, mobility, quality of life, health-related quality of life, and empowerment) in older adults with visual impairment, and to document its facilitators and barriers., Methods: A mixed-method design, which included a pre-experimental and an exploratory qualitative clinical research component, was used on 8 older adults (7 women) with visual impairment aged 70-86, and 8 attendants (5 women) aged 20-74. Before the intervention, directly after, and four months later, older adults completed questionnaires on the 7 outcomes. During the intervention, attendants completed diaries and participated in monthly meetings. Semi-structured interviews were administered to all participants after the intervention., Results: Social participation, leisure, mobility, quality of life and empowerment had increased immediately after the APIC. These improvements were still generally observed four months later. Participants reported that the APIC improved older adults' capabilities, social participation, and social environment., Conclusions: The APIC is a promising intervention which helps older adults with visual impairment to deal with social restrictions.

Purpose: This exploratory study aimed to evaluate the preferences, expectations, and sense of safety of blind or visually impaired persons regarding three types of pedestrian phasing with audible pedestrian signals configurations that exist in Quebec City (Canada). These include: 1) exclusive phasing with non-directional audible pedestrian signals; 2) exclusive phasing with directional audible pedestrian signals; and 3) concurrent phasing with directional audible pedestrian signals., Methods: Thirty-two blind or visually impaired persons were asked to complete a survey. Their preferences and expectations regarding audible pedestrian signals were documented through a series of simulations. Their sense of safety regarding the three existing configurations were also documented. Subsequently, semi-directed, individual interviews with 11 of the individuals who had completed the survey were conducted to build off the collected information., Results: No formal consensus regarding many of the issues discussed were established as participants' responses varied too significantly. However, findings suggest that the exclusive phasing with directional audible pedestrian signals configuration is perceived to be the safest option by the participants., Conclusion: This study may have practical implications on the design of intersections (e.g., selection of a type of pedestrian phasing with audible pedestrian signal) and the training of blind or visually impaired pedestrians.IMPLICATIONS FOR REHABILITATIONThe addition of audible pedestrian signals to pedestrian signals heightens the sense of safety of blind or visually impaired persons.This study may have practical implications on the design of intersections with audible pedestrian signals and the selection of a type of audible pedestrian signals based on intersection characteristics.Since many participants reported a lower sense of safety when faced with concurrent phasing, it is recommended that more intensive orientation and mobility interventions be provided to blind or visually impaired pedestrians who use this type of traffic signals.Educating road users about blind or visually impaired pedestrians appears also essential.

The cessation of driving is a difficult transition for the elderly, but it can be facilitated through interventions. The purpose of this study was to explore the satisfaction, usefulness and applicability of the CarFreeMe intervention in the French-Canadian context. A qualitative clinical research device was used on ten older adults aged between 61 and 90 years. The participants had stopped driving within the last twelve months or were planning to stop driving in the near future and did not have cognitive impairments. After the intervention, the participants were generally satisfied and reported on its usefulness and applicability in a French-Canadian context. In addition, they identified the positive impacts related to their social involvement as they re-engaged in or pursued their significant activities. Further research is required to assess the intervention's effects and the practicability of implementing it in Canada.

Background: Exclusive use of Del Nido cardioplegia administration in all adult patients undergoing cardiac surgery has been studied for operative, postoperative and myocardial protection outcomes., Methods: From November 2016 to October 2017, Del Nido cardioplegia was used in 131 consecutive patients (DN group). Using a propensity score, DN group was compared to 251 patients having received intermittent cold blood cardioplegia (CB group)., Results: Preoperative characteristics were similar in DN and CB groups. Operative outcomes were statistically different (p < 0.0001): cardiopulmonary bypass (CPB) time (DN 105.9 ± 46.5, CB 131.2 ± 38.8); aortic cross-clamp time (DN 80.8 ± 35.5, CB 102.2 ± 31.3); operative time (DN 203.1 ± 65.0, CB 241.5 ± 54.7); total cardioplegia volume (DN 1328 ± 879, CB 3773 ± 1226); and peak glycemia on CPB (DN 8.2 ± 2.3, CB 9.0 ± 1.8). No statistical differences were noted in intensive care unit stay, hospital stay and hospital death. Myocardial protection outcomes were similar: discharge left ventricular ejection fraction (DN 52 ± 11, CB 51 ± 10); Troponin levels at the end of the surgery (DN 871 ± 1623, CB 1958 ± 854), day 1 (DN 853 ± 1139, CB 993 ± 8234) and day 4 (DN 442 ± 540, CB 463 ± 317)., Conclusion: Del Nido cardioplegia use in all adult cardiac surgeries is associated with improved surgical efficiency. The design of larger trials including adults combined cardiac procedures and emergencies is needed.

Background: Although walking without vision seems to carry a high cognitive cost, few studies have measured the cognitive load involved in this activity in blind people. The aim of this study was to assess the cognitive load of walking in blind people, using gait analysis, a dual task paradigm and a subjective assessment of cognitive load., Methods: In a quantitative quasi-experimental design, 25 blind adults walked 40 meters. In one trial, participants walked normally (control condition). In another, they walked while performing an auditory simple reaction time task, and in the third trial they walked, performed the simple reaction time task and avoided obstacles. In addition to the simple reaction time task performance, walking speed was recorded, and participants provided a subjective assessment of cognitive load after each trial. Performance of participants aged less than 60 years were compared with those aged over than 60 years., Results: Walking significantly reduced performance of the simple reaction time task; carrying out the simple reaction time task while walking significantly reduced walking performance and increased the subjective assessment of cognitive load; and simple reaction time task performance decreased and subjective assessment increased when obstacles were present. Few significant age effects were found., Significance: Walking without vision involves a cognitive load that increases when the environment becomes complex. Each of the three methods used is relevant when assessing the cognitive load involved in walking in blind people, and could be useful in rehabilitation intervention. The results obtained allowed recommendations to be suggested for the design of technological mobility devices., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: Adaptation to blindness can lead to the enhancement of the attentional capacities and working memory in young people. However, although the effects of ageing on the cognition of sighted people and people with age-related visual impairments are well-documented, no study seems to have investigated the age-related changes of these cognitive processes in people who are blind. The aim of this study was to assess the effects of age on the attentional processes and working memory in blind people., Method: A cross-sectional study was conducted on 43 blind participants and 42 sighted participants. The participants performed auditory computerized tests assessing selective, sustained and divided attention, attentional switching, and working memory., Results: Two-way analysis of variance revealed significant visual status effect and age effect on most of the variables studied. No interaction was found between visual status and age effects., Conclusions: These results suggest that the trajectories of cognitive age-related change are similar in blind people and in sighted people. This study has implications for rehabilitation, such as cognitive intervention. Implications for Rehabilitation Blind people show improved attentional capacities compared to sighted people, even in old blind people. Old blind people have lower performances than younger blind people in tests assessing selective, sustained and divided attention, and working memory. Cognitive approaches to rehabilitation may help people who are blind to deal with age-related cognitive decline and its effects on everyday functioning. A high level of cognitive stimulation, provided by a cognitive training or a developed social participation, might reduce the age-related effects in people who are blind.

Although attentional processes and working memory seem to be significantly involved in the daily activities (particularly during navigating) of persons who are blind and who use these abilities to compensate for their lack of vision, few studies have investigated these mechanisms in this population. The aim of this study is to evaluate the selective, sustained and divided attention, attentional inhibition and switching and working memory of blind persons. Early blind, late blind and sighted participants completed neuropsychological tests that were designed or adapted to be achievable in the absence of vision. The results revealed that the early blind participants outperformed the sighted ones in selective, sustained and divided attention and working memory tests, and the late blind participants outperformed the sighted participants in selective, sustained and divided attention. However, no differences were found between the blind groups and the sighted group in the attentional inhibition and switching tests. Furthermore, no differences were found between the early and late blind participants in this set of tests. These results suggest that early and late blind persons can compensate for the lack of vision by an enhancement of the attentional and working memory capacities., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: According to international treatment guidelines, inhaled rapid-acting β2 agonists should be used for the control of symptoms in patients with asthma. We compared the efficacy and safety of an extrafine combination inhaler containing a corticosteroid (beclometasone) plus a rapid-onset, long-acting β2 agonist (formoterol) with a short-acting β2 agonist (salbutamol) as reliever strategies in patients taking beclometasone-formoterol combination as maintenance treatment., Methods: In a double-blind trial undertaken in 183 centres in 14 European countries over 48 weeks, patients (aged ≥18 years) with asthma that was not fully controlled, with a forced expiratory volume in 1 s (FEV1) of at least 60% predicted, had a 2-week run in. During this period, patients were treated with a combination of beclometasone 100 μg and formoterol 6 μg per one inhalation twice daily plus salbutamol 100 μg as required delivered by use of a pressurised metered-dose inhaler. They were then randomly assigned in a 1:1 ratio with a computer-generated randomisation list to receive beclometasone 100 μg plus formoterol 6 μg or salbutamol 100 μg as reliever in addition to maintenance with beclometasone 100 μg plus formoterol 6 μg twice daily. Primary outcome was the time to first severe exacerbation (admission to hospital or visit to emergency department, or use of systemic steroids for ≥3 consecutive days). Secondary outcomes were number of severe exacerbations (events per 100 patients per year), time to and number of mild exacerbations, additional exacerbation variables, lung function, symptom scores, and asthma control. Analysis was by intention to treat. The study is registered with ClinicalTrials.gov, number NCT00861926., Findings: 1714 patients were randomly assigned to the as-needed beclometasone-formoterol (n=857) and as-needed salbutamol groups (n=857), and 1701 were analysed (852 and 849, respectively). 326 severe exacerbations were reported by 251 patients during the study, and 99 versus 152 patients had at least one exacerbation during the 48 weeks, respectively. Compared with beclometasone-formoterol plus salbutamol as needed, beclometasone-formoterol for both maintenance and reliever treatment significantly increased the time to first exacerbation (209 days vs 134 days) by 75 days, with a 36% reduction in risk (hazard ratio 0·64 [95% CI 0·49 to 0·82]; p=0·0005), and the estimated probability was 12% and 18%, respectively (p=0·0003). The number of days with mild asthma exacerbations was also lower with as-needed beclometasone-formoterol than with as-needed salbutamol (56·04 days per patient per year vs 65·11 days per patient per year; 0·86 [0·76 to 0·98]; p=0·021). From the run-in period to week 48, both treatments improved symptoms (mean change -1·59 [-1·94 to -1·25] in the as-needed beclometasone-formoterol group vs -1·44 [-1·78 to -1·10] in the as-needed salbutamol group, difference -0·15 [-0·60 to 0·30]; p=0·507), percentage of asthma control days (9·5% [7·3 to 11·8] vs 10·9% [8·7 to 13·1], respectively, -1·4 [-4·3 to 1·6]; p=0·359), use of reliever (-0·29 [-0·38 to -0·20] vs -0·27 [-0·36 to -0·19], respectively, -0·02 [-0·13 to 0·10]; p=0·794), and lung function (FEV1, 0·090 [0·060 to 0·120] vs 0·090 [0·060-0·120], respectively, 0·001 [-0·040 to 0·040]; p=0·969), and were well tolerated (patients with serious adverse events, 32 [4%] and 41 [5%], respectively)., Interpretation: Our results lend support to the use of the combination of a single inhaled corticosteroid plus a rapid-onset, long-acting β2 agonist for maintenance and relief in patients with moderate to severe asthma and provide encouraging data for the formulation of beclometasone-formoterol for this use., Funding: Chiesi Farmaceutici., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In contrast to the human genome, the mouse genome contains two HEPC genes encoding hepcidin, a key regulator of iron homeostasis. Here we report a comparative analysis of sequence, genomic structure, expression and iron regulation of mouse HEPC genes. The predicted processed 25 amino acid hepcidin 2 peptide share 68% identity with hepcidin 1 with perfect conservation of eight cysteine residues. Both HEPC1 and HEPC2 genes have similar genomic organization and have probably arisen from a recent duplication of chromosome 7 region, including the HEPC ancestral gene and a part of the adjacent USF2 gene. Insertion of a retroviral intracisternal A-particle element was found upstream of the HEPC1 gene. Both genes are highly expressed in the liver and to a much lesser extent in the heart. In contrast to HEPC1, a high amount of HEPC2 transcripts was detected in the pancreas. Expression of both genes was increased in the liver during carbonyl-iron and iron-dextran overload. Overall our data suggest that both HEPC1 and HEPC2 genes are involved in iron metabolism regulation but could exhibit different activities and/or play distinct roles.

Originally identified as a gene up-regulated by iron overload in mouse liver, the HEPC gene encodes hepcidin, the first mammalian liver-specific antimicrobial peptide and potential key regulator of iron metabolism. Here we demonstrate that during rat liver development, amounts of HEPC transcripts were very low in fetal liver, strongly and transiently increased shortly after birth, and reappeared in adult liver. To gain insight into mechanisms that regulate hepatic expression of hepcidin, 5'-flanking regions of human and mouse HEPC genes were isolated and analyzed by functional and DNA binding assays. Human and mouse HEPC promoter-luciferase reporter vectors exhibited strong basal activity in hepatoma HuH-7 and mouse hepatocytes, respectively, but not in non-hepatic U-2OS cells. We found that CCAAT/enhancer-binding protein alpha (C/EBPalpha) and C/EBPbeta were respectively very potent and weak activators of both human and mouse promoters. In contrast, co-expression of hepatocyte nuclear factor 4alpha (HNF4alpha) failed to induce HEPC promoter activity. By electrophoretic mobility shift assay we demonstrated that one putative C/EBP element found in the human HEPC promoter (-250/-230) predominantly bound C/EBPalpha from rat liver nuclear extracts. Hepatic deletion of the C/EBPalpha gene resulted in reduced expression of HEPC transcripts in mouse liver. In contrast, amounts of HEPC transcripts increased in liver-specific HNF4alpha-null mice. Decrease of hepcidin mRNA in mice lacking hepatic C/EBPalpha was accompanied by iron accumulation in periportal hepatocytes. Finally, iron overload led to a significant increase of C/EBPalpha protein and HEPC transcripts in mouse liver. Taken together, these data demonstrate that C/EBPalpha is likely to be a key regulator of HEPC gene transcription and provide a novel mechanism for cross-talk between the C/EBP pathway and iron metabolism.

F-box proteins, a critical component of the evolutionary conserved ubiquitin-protein ligase complex SCF (Skp1/Cdc53-Cullin1/F-box), recruit substrates for ubiquitination and consequent degradation through their specific protein-protein interaction domains. Here, we report the identification of full-length cDNAs encoding three novel human F-box proteins named FBG3, FBG4 and FBG5 which display similarity with previously identified NFB42 (FBX2) and FBG2 (FBX6) proteins. All five proteins are characterized by an approximately 180-amino-acid (aa) conserved C-terminal domain and thus constitute a third subfamily of mammalian F-box proteins. Analysis of genomic organization of the five FBG genes revealed that all of them consist of six exons and five introns. FBG1, FBG2 and FBG3 genes are located in tandem on chromosome 1p36, and FBG4 and FBG5 are mapped to chromosome 19q13. FBG genes are expressed in a limited number of human tissues including kidney, liver, brain and muscle tissues. Expression of rat FBG2 gene was found related to differentiation/proliferation status of hepatocytes. Specifically, FBG2 mRNA was expressed in foetal liver, decreased after birth and re-accumulated in adult liver. Expression of FBG2 was strongly inhibited in hepatoma cells by okadaic acid.

Aceruloplasminemia is an autosomal recessive disease of iron overload associated with mutation(s) in the ceruloplasmin gene. We report here a new case of aceruloplasminemia in a woman who is a compound heterozygote for two new mutations. Besides this novel genotypic profile, this observation provides new insights on: (i) iron metabolism with normal erythroid repartition, in the absence of serum non-transferrin-bound iron and with an increase of 59Fe plasma clearance; (ii) hepatic abnormalities associated with the presence of iron-free foci; (iii) the therapeutic management of the disease, chronic subcutaneous infusion of deferrioxamine being remarkably effective at reducing hepatic iron overload.

We have investigated the effect of iron overload on the expression of mouse GSTA1, A4, M1, and P1 in liver, the main iron storage site during iron overload, and in kidney. In iron-overloaded animals, mRNA and protein levels of GSTA1, A4, and M1 were increased in liver. In kidney, GSTA4 protein level was also increased while, unexpectedly, GSTA1 and M1 expression was strongly decreased. We showed, by immunohistochemistry, that GSTA4 was more abundant in hepatocytes of periportal areas and in convoluted proximal tubular cells in normal liver and kidney, respectively. In iron-overloaded mice, GSTA4 staining was more intense in cells that preferentially accumulated iron, and conjugation of 4-hydroxynonenal, a specific substrate of GSTA4, was enhanced in both organs. Moreover an acute exposure of primary cultures of mouse hepatocytes to iron-citrate strongly induced oxidative stress and cellular injury and resulted in an increase in GSTA4 expression, while cotreatment with iron-citrate and either desferrioxamine or vitamin E prevented both toxicity and GSTA4 induction. These data demonstrate that GSTA1 and M1 are differentially regulated in liver and kidney while GSTA4 is induced in both organs during iron overload. Moreover, they support the view that iron-induction of GSTA4 is related to an overproduction of free radicals.

In humans, hepatic iron overload can lead to hepatocellular carcinoma development. Iron related dysregulation of hepatic genes could play a role in this phenomenon. We previously found that the carbonyl-iron overloaded mouse was a useful model to study the mechanisms involved in the development of hepatic lesions related to iron excess. The aim of the present study was to identify hepatic genes overexpressed in conditions of iron overload by using this model. A suppressive subtractive hybridization was performed between hepatic mRNAs extracted from control and 3% carbonyl-iron overloaded mice during 8 months. This methodology allowed us to identify stearoyl coenzyme A desaturase 1 (SCD1) mRNA overexpression in the liver of iron loaded mice. The corresponding enzymatic activity was also found to be significantly increased. In addition, we demonstrated that both SCD1 mRNA expression and activity were increased in another iron overload model in mice obtained by a single iron-dextran subcutaneous injection. Moreover, we found, in both models, that SCD1 mRNA was not only influenced by the quantity of iron in the liver but also by the duration of iron overload since SCD1 mRNA upregulation was not detected in earlier stages of iron overload. In addition, we found that cellular repartition likely influenced SCD1 mRNA expression. In conclusion, we demonstrated that iron excess in the liver induced both the expression of SCD1 mRNA and its corresponding enzymatic activity. The level and duration of iron overload, as well as cellular repartition of iron excess in the liver likely play a role in this induction. The fact that the expression and activity of SCD1, an enzyme adding a double bound into saturated fatty acids, are induced in two models of iron overload in mice leads to the conclusion that iron excess in the liver may enhance the biosynthesis of unsaturated fatty acids.

Considering that the development of hepatic lesions related to iron overload diseases might be a result of abnormally expressed hepatic genes, we searched for new genes up-regulated under the condition of iron excess. By suppressive subtractive hybridization performed between livers from carbonyl iron-overloaded and control mice, we isolated a 225-base pair cDNA. By Northern blot analysis, the corresponding mRNA was confirmed to be overexpressed in livers of experimentally (carbonyl iron and iron-dextran-treated mice) and spontaneously (beta(2)-microglobulin knockout mice) iron-overloaded mice. In addition, beta(2)-microglobulin knockout mice fed with a low iron content diet exhibited a decrease of hepatic mRNA expression. The murine full-length cDNA was isolated and was found to encode an 83-amino acid protein presenting a strong homology in its C-terminal region to the human antimicrobial peptide hepcidin. In addition, we cloned the corresponding rat and human orthologue cDNAs. Both mouse and human genes named HEPC are constituted of 3 exons and 2 introns and are located on chromosome 7 and 19, respectively, in close proximity to USF2 gene. In mouse and human, HEPC mRNA was predominantly expressed in the liver. During both in vivo and in vitro studies, HEPC mRNA expression was enhanced in mouse hepatocytes under the effect of lipopolysaccharide. Finally, to analyze the intracellular localization of the predicted protein, we used the green fluorescent protein chimera expression vectors. The murine green fluorescent protein-prohepcidin protein was exclusively localized in the nucleus. When the putative nuclear localization signal was deleted, the resulting protein was addressed to the cytoplasm. Taken together, our data strongly suggest that the product of the new liver-specific gene HEPC might play a specific role during iron overload and exhibit additional functions distinct from its antimicrobial activity.

F-box proteins are critical components of the SCF ubiquitin-protein ligase complex and are involved in substrate recognition and recruitment for ubiquitination and consequent degradation by the proteasome. We have isolated cDNAs encoding a further 10 mammalian F-box proteins. Five of them (FBL3 to FBL7) share structural similarities with Skp2 and contain C-terminal leucine-rich repeats. The other 5 proteins have different putative protein-protein interaction motifs. Specifically, FBS and FBWD4 proteins contain Sec7 and WD40-repeat domains, respectively. The C-terminal region of FBA shares similarity with bacterial protein ApaG while FBG2 shows homology with the F-box protein NFB42. The marked differences in F-box gene expression in human tissues suggest their distinct role in ubiquitin-dependent protein degradation.

Iron is required for cellular life. However, abnormalities of its metabolism may lead to iron deficiency or iron overload, both conditions which are deleterious. Therefore, stock and distribution of iron in the body must be very stable. Classically, four major proteins are involved in iron metabolism: (a) transferrin which is implicated in its plasmatic transport, (b) transferrin receptor which regulates iron-transferrin uptake, (c) ferritin, the major iron storage protein, and (d) IRP (Iron Regulatory Protein) which regulates both the entry and storage of iron by linking to the IRE (Iron Responsive Element), a nucleotidic sequence found on transferrin receptor and ferritin mRNA. Thus, IRP adapts gene expression to the iron cellular status. Recent data give informations about new proteins involved in iron metabolism: HFE whose gene is mutated in genetic hemochromatosis, ceruloplasmin which permits cellular iron egress and frataxin which is implicated in the exit of iron from mitochondria.

Background/aims: In humans, chronic iron excess may induce hepatic fibrosis and/or hepatocellular carcinoma. This work was undertaken to investigate hepatic iron overload outcome in iron-overloaded mice., Methods: BALB/cJ male mice received supplements of 0, 0.5, 1.5 and 3% carbonyl-iron for 2, 4, 8 and 12 months. Histological staining, immunohistochemistry using ferritin antibodies and electron microscopic studies were performed on liver. Liver iron concentration was measured biochemically. Mitotic index and hepatocyte nuclear size were evaluated on Feulgen-stained slides., Results: Liver iron concentration was increased, reaching 13 times control value after 12 months in 3% iron-overloaded mice, and iron was found predominantly in hepatocytes, with a porto-centrolobular decreasing gradient. Neither hepatic fibrosis nor hepatocellular carcinoma was found. Perls' stain positive inclusions containing ferritin were found within hepatocyte nuclei in 3%-overloaded mice. Electron microscopy disclosed that inclusions consisted of ferritin particle aggregates without a limiting membrane. Mice overloaded with 3% iron for 12 months showed larger hepatocyte nuclei than control mice and a mitotic index increase with presence of abnormal tripolar mitotic figures. In addition, some iron-free hepatocytes were observed., Conclusions: Carbonyl-iron supplementation produces significant iron overload in mice but does not result in liver fibrosis or hepatocellular carcinoma after 12 months. However, nuclear changes were produced in hepatocytes, and occasional iron-free hepatocytes were observed: these may represent preneoplastic changes caused by iron overload.

Iron is an essential element in cellular metabolism and the growth of all living species, and is involved in DNA replication. The risk of hepatocellular carcinoma development is associated with an increase in iron availability. The aim of the present work was to investigate the effect of an oral iron chelator, deferiprone (CP20), on HepG2 cell-line proliferation in culture. HepG2 cell cultures were maintained in the absence of fetal calf serum (FCS) and in the presence or not (control cultures) of CP20 at the concentrations of 50 or 100 microM; deferoxamine (DFO) was used as an iron chelator reference. Cell proliferation was investigated by the analysis of DNA synthesis using [3H] methyl-thymidine incorporation and of the cell cycle by flow cytometry. Iron chelation efficiency in the culture model was studied by analyzing the effect of CP20 on radioactive iron uptake, intracellular ferritin level, and transferrin receptor expression. CP20, at the concentration of 50 or 100 microM, inhibited DNA synthesis after 48 hr of incubation and induced an accumulation of the cells in the S phase of the cell cycle. Iron chelators inhibited cellular iron uptake, decreased intracellular ferritin level, and increased transferrin receptor protein and mRNA levels. Our results show that CP20 as well as deferoxamine inhibit HepG2 cell proliferation and block cell cycle in the S phase.

Bombesin stimulation of inositol 1,4,5-trisphosphate (Ins P3) formation in rat sonicated pancreatic acinar cells was inhibited by an antibody directed against the pertussis toxin (PTX)-sensitive GTP-binding G alpha i3 protein but not by an anti-G alpha q-11 antibody. After solubilization and gel filtration, [125I-Tyr4]bombesin binding sites were recovered in a peak of protein of 67 approximately 90 kDa with a maximal enrichment corresponding to a molecular mass of 83-kDa. Results obtained from the non-hydrolysable GTP analog guanosine-5'-[gamma-thio]triphosphate (GTP gamma S) binding, PTX-stimulated ADP-ribosylation and immunoblotting showed that the 83-kDa fraction contained the G alpha i3 protein but not the G alpha q-11 protein. Furthermore, GTP gamma S increased the bombesin binding dissociation constant (KD) from 0.32 to 0.60 nM, while the anti-G alpha i3 antibody decreased the maximal binding capacity (Bmax) from 50 to 25 fmol/mg protein without affecting the KD. Mixing solubilized bombesin binding sites with a phospholipase C (PLC) preparation from rat pancreas reconstituted a bombesin-stimulated PLC activity which was markedly inhibited by the anti-G alpha i3 antibody but unaffected by the anti-G alpha q-11 antibody. In addition, this stimulation was inhibited by an anti-PLC beta 1 antibody. This result supports the involvement of the PLC beta 1 isoform in bombesin receptor activation.

The effect of somatostatin analogue, lanreotide, and bombesin/GRP antagonist, BIM 26226, on the growth of colon cancer peritoneal carcinomatosis in the rat was studied. BDIX rats were i.p. injected with DHD/K12 rat colon cancer cells at day 0 and received from day 3 either lanreotide, BIM 26226, combination of treatments or peptide solvents. At sacrifice, an day 45, no significant difference between groups was observed for peritoneal tumor growth, hepatic metastases, ascite volume and labeling indices in normal colonic mucosa and tumoral tissues. Survival times were similar in other lanreotide-treated and control groups. However, BIM 26226 decreased plasma gastrin level, consistently with a physiological effect of this peptide. Ln all groups, somatostatin and bombesin receptors were found on mucosal and tumoral tissues. Interestingly, bombesin receptor number was higher in severe than in minor cancer stages, contrarily to that of somatostatin receptors. Moreover, an up-regulation of somatostatin and bombesin receptors was observed in BIM 26226- and lanreotide-treated group tumors, respectively, Despite the presence of these specific receptors, lanreotide and BIM 26226 were inactive on tumor growth in this model.

Objectives and Method: The beta 3 adrenergic receptor stimulates lipolysis and colonic relaxation in the rat, and, suggestively, in man. Several human forms generated by different mRNA splicings can occur: the A form of 396 amino acids and the B and C forms extended by 12 and 6 amino acids respectively, in the C-terminus region. In order to characterize these different forms as expressed in CHO cells, we studied adenylyl cyclase stimulation by the beta 3 agonists, SR58611A and BRL37344 and its inhibition by the beta 3 antagonist SR59230., Results: This antagonist totally inhibited SR58611-adenylyl cyclase stimulation with the following hierarchy of potency: C form >> B > A. In rat, a unique form is expressed which is close to the human B form. This form was the less sensitive to beta 1 and beta 2 antagonists., Conclusion: These findings constitute a molecular pharmacological basis for the design of beta 3 agonists of therapeutic value.

We previously reported the immunopurification of a somatostatin receptor from the human tumoral gastric cell HGT1 using the monoclonal antibody 30F3 (Reyl-Desmars, F., Le Roux, S., Linard, C., Benkouka, F., and Lewin, M. J. M. (1989) J. Biol. Chem. 264, 18789-18795). Screening of a lambda gt11 HGT1-cDNA library with 30F3 led us to isolate a cDNA encoding an 86-kDa polypeptide displaying 100% structural identity with the 86-kDa subunit (p86-Ku) of the Ku autoantigen. Recombinant p86 expressed in Escherichia coli cross-reacted with 30F3 and specifically bound [125I-Tyr11]somatstatin-14. Binding was totally displaced by somatostatin-14, somatostatin-28, and SMS 201-995, with IC50 values of 0.7, 1.0, and 1.2 nM, respectively. In a search for a biological effect associated with binding, we purified a 36-kDa, okadaic acid-sensitive phosphatase (protein phosphatase-2A (PP2A)) from rat gastric cytosol. PP2A catalyzed 32P release from p34cdc2-phosphorylated histone H1. However, PP2A-induced 32P release was concentration dependently inhibited by recombinant p86-Ku, with a decrease in maximal velocity without a change in Km. Steric exclusion high pressure chromatography indicated that the inhibition resulted from direct interaction of the enzyme with p86-Ku. Furthermore, it was antagonized by increased concentrations of somatostatin-14 and prevented by preincubating p86-Ku with 30F3. Given the key role played by PP2A in cell cycle regulation, the current findings suggest that p86-Ku could be a physiological target of somatostatin antiproliferative action.

The histamine H3 receptor agonist (R)alpha-methylhistamine (MeHA) inhibited, in a nanomolar range, basal and carbachol-stimulated inositol phosphate formation in the human gastric tumoral cell line HGT1-clone 6. The inhibition was reversed by micromolar concentrations of the histamine H3 receptor antagonist thioperamide and was sensitive to cholera or pertussis toxin treatment. Using [3H]N alpha-MeHA as specific tracer, high affinity binding sites were demonstrated with a Bmax of 54 +/- 3 fmol/mg of protein and a KD of either 0.61 +/- 0.04 or 2.2 +/- 0.4 nM, in the absence or presence of 50 microM GTP[gamma]S, respectively. The binding sites were solubilized by Triton X-100 and prepurified by gel chromatography. They were separated from the histamine H2 receptor sites by filtration through Sepharose-famotidine and finally retained on Sepharose-thioperamide. The purified sites concentrated in one single silver-stained protein band of 70 kDa in SDS-polyacrylamide gel electrophoresis. They specifically bound [3H]N alpha-MeHA with a KD of 1.6 +/- 0.1 nM and a Bmax of 12,000 +/- 750 pmol/mg of protein. This corresponds to a 90,225-fold purification over cell lysate and a purity degree of 84%. Binding was competitively displaced by N alpha-MeHA (IC50 = 5.8 +/- 0.7 nM), (R) alpha-MeHA (IC50 = 9 +/- 1 nM), and thioperamide (IC50 = 85 +/- 10 nM), but not by famotidine (H2 antagonist) or by mepyramine (H1 antagonist). These findings provide the first evidence for solubilization, purification, and molecular mass characterization of the histamine H3 receptor protein and for the negative coupling of this receptor phosphatidylinositol turnover through a so far unidentified G protein.

This communication reports the solubilization, the purification and the molecular characterization of the H2-histamine receptor from the cell line HGT-1 derived from a human gastric cancer. The receptor has been solubilized by Triton X100 and purified by gel filtration onto Sephacryl, affinity-chromatography (Sepharose-famotidine) and high performance liquid chromatography (HPLC). The purified receptor specifically bound the H2 selective ligand 3H-methyltiotidine with a kD of 160 nM (vs 50 nM for the intact HGT-1 cell) and a maximal binding capacity of 14,000 pmol/mg protein which represents a 12,170-fold enrichment and a degree of purity of 98%. It is a glycoprotein of 70 kDa molecular mass containing N-acetylglucosamine residues.