Building a biopsychosocial model of cancer-related fatigue: the BIOCARE FActory cohort study protocol.

BACKGROUND: Cancer-related fatigue (CRF) is the most common side effect of cancer and cancer treatment. CRF prevalence is up to 50% in breast cancer patients and can continue several years after cancer remission. This persistent subjective sense of exhaustion is multifactorial. Numerous parameters have been evidenced to be related to CRF across biological, physical, psychological, social and/or behavioral dimensions. Although CRF has been studied for many years, the majority of previous studies focused on only one dimension, i.e., physical function. Moreover, few studies investigated CRF longitudinally with repeated measures. These are the two main obstacles that limit the understanding of CRF mechanisms. The purpose of this study is to create a biopsychosocial model of CRF with simultaneous and longitudinal anthropometric, clinical, biological, physical, psychological and sociological parameters. METHODS: BIOCARE FActory is a multicentric prospective study that will consist of an 18-month follow-up of 200 women diagnosed with breast cancer. Four visits will be scheduled at diagnosis, after treatments, and 12 and 18 months after diagnosis. The same procedure will be followed for each visit. Each session will be composed of anthropometric data collection, a semi-structured interview, cognitive tests, postural control tests, neuromuscular fatigability tests and a cardiorespiratory fitness test. Clinical and biological data will be collected during medical follow-ups. Participants will also complete questionnaires to assess psychological aspects and quality of life and wear an actigraphy device. Using a structural equation modeling analysis (SEM), collected data will build a biopsychosocial model of CRF, including the physiological, biological, psychological, behavioral and social dimensions of CRF. DISCUSSION: This study aims to highlight the dynamics of CRF and its correlates from diagnosis to post treatment. SEM analysis could examine some relations between potential mechanisms and CRF. Thus, the biopsychosocial model will contribute to a better understanding of CRF and its underlying mechanisms from diagnosis to the aftermaths of cancer and its treatments. TRIAL REGISTRATION: This study is registered at ClinicalTrials.gov ( NCT04391543 ), May 2020.

Imaging host-Leishmania interactions: significance in visceral leishmaniasis.

Leishmaniasis is a neglected disease that is associated with a spectrum of clinical manifestations ranging from self-healing cutaneous lesions to fatal visceral infections, which primarily depends on the parasite species. In visceral leishmaniasis (VL), as opposed to cutaneous leishmaniasis (CL), parasites that infect host cells at the sand fly bite site have the striking ability to disseminate to visceral organs where they proliferate and persist for long periods of time. Imaging the dynamics of the host-Leishmania interaction in VL provides a powerful approach to understanding the mechanisms underlying host cell invasion, Leishmania dissemination and persistence within visceral organs and, to dissecting the immune responses to infection. Therefore, by allowing the visualization of the critical steps involved in the pathogenesis of VL, state-of-the-art microscopy technologies have the great potential to aid in the identification of better intervention strategies for this devastating disease. In this review, we emphasize the current knowledge and the potential significance of imaging technologies in understanding the infection process of visceralizing Leishmania species. Then, we discuss how application of innovative microscopy technologies to the study of VL will provide rich opportunities for investigating host-parasite interactions at a previously unexplored level and elucidating visceral disease-promoting mechanisms.

Activity of ethanol and daptomycin lock on biofilm generated by an in vitro dynamic model using real subcutaneous injection ports.

Vancomycin lock solution (LS) is recommended for the conservative treatment of subcutaneous injection port (SIP)-related infections, but may be associated with failure. We used an in vitro dynamic model of biofilm formation in an SIP, based on a continuous flow circulating via a real SIP, to assess the effectiveness of vancomycin (5 mg/ml), daptomycin (5 mg/ml) and ethanol 40 % LS in eradicating a pre-established Staphylococcus epidermidis biofilm. Heparin, Ringer's lactate and enoxaparin sodium LS were used as controls. The logarithmic reductions of colony-forming units (CFU) were compared by Student's t-test. After 24 h of exposure, the vancomycin LS did not exert a greater bactericidal effect than the heparin LS control (mean logarithmic reduction: 2.27 ± 0.58 vs. 1.34 ± 0.22, respectively, p = 0.3). The mean logarithmic reduction was greater with daptomycin LS (5.45 ± 0.14 vs. 0.39 ± 0.12, p < 0.01) and ethanol LS (6.79 ± 1.03 vs. 1.43 ± 0.54, p = 0.02). Bacterial revival after exposure to 24 h of LS was assessed. The mean viable bacteria count was significantly higher for vancomycin LS (9.36 ± 0.10 log(10)CFU) and daptomycin LS (9.16 ± 0.02 log(10)CFU) than for ethanol LS (2.95 ± 1.65 log(10)CFU). Ethanol appeared to be the most attractive option to treat SIP-related infection, but its poor ability to entirely disrupt the biofilm structure may require its use in association with a dispersal agent to avoid renewal of the biofilm.

Roles of capsule and lipopolysaccharide O antigen in interactions of human monocyte-derived dendritic cells and Klebsiella pneumoniae.

In humans, Klebsiella pneumoniae is a saprophytic bacterium of the nasopharyngeal and intestinal mucosae that is also frequently responsible for severe nosocomial infections. Two major factors of virulence, capsular polysaccharide (CPS) and lipopolysaccharide (LPS) O antigen, are involved in mucosal colonization and the development of infections. These bacterial surface structures are likely to play major roles in interactions with the mucosal immune system, which are orchestrated by a network of surveillance based on dendritic cells (DCs). To determine the roles of K. pneumoniae CPS and LPS in the DC response, we investigated the response of immature human monocyte-derived DCs to bacterial challenge with a wild-type strain and its isogenic mutants deficient in CPS or LPS O-antigen production. As observed by flow cytometry and confocal laser microscopy, the rate of phagocytosis was inversely proportional to the amount of CPS on the bacterial cell surface, with LPS playing little or no role. The K. pneumoniae wild-type strain induced DC maturation with upregulation of CD83, CD86, and TLR4 and downregulation of CD14 and DC-SIGN. With CPS mutants, we observed a greater decrease in DC-SIGN, suggesting a superior maturation of DCs. In addition, incubation of DCs with CPS mutants, and to a lesser extent with LPS mutants, resulted in significantly higher Th1 cytokine production. Combined, our findings suggest that K. pneumoniae CPS, by hampering bacterial binding and internalization, induces a defective immunological host response, including maturation of DCs and pro-Th1 cytokine production, whereas the LPS O antigen seems to be involved essentially in DC activation.

Biofilms in hospital effluents as a potential crossroads for carbapenemase-encoding strains.

Bacterial resistance to carbapenem, which is mainly due to the successful dissemination of carbapenemase-encoding genes, has become a major health problem. Few studies have aimed to characterize the level of resistance in the environment, notably in hospital wastewater, which is a likely hotspot for exchange of antibiotic resistance genes. In this work, we looked for the presence of imipenem-resistant bacteria and imipenem in the effluent of the teaching hospital of Clermont-Ferrand, France. Selective growth of bacteria from 14-day old biofilms formed in the pipe sewer showed that 22.1% of the isolates were imipenem-resistant and identified as Aeromonas (n = 23), Pseudomonas (n = 10), Stenotrophomonas (n = 4) and Acinetobacter (n = 1). Fifteen of these strains harbored acquired carbapenemase-encoding genes blaVIM (n = 11), blaOXA-48 (n = 2), blaGES (n = 1), blaNDM (n = 1). All isolates also harbored associated resistances to aminoglycosides, fluoroquinolones and/or tetracyclin. S1-nuclease pulsed-field gel electrophoresis analysis of eight selected isolates showed that four of them harbored one to two plasmids of molecular weight between 48.5 Kb and 194 Kb. In vitro transformation assays evidenced the presence of blaVIM and blaNDM on plasmids with the blaVIM harboring 80 Kb plasmid having conjugative capacity. The predicted environmental concentration of imipenem in the hospital effluent was 3.16 µg/L, suggesting that biofilm bacteria are subjected to sub-MICs of imipenem within the effluent. However, no imipenem molecule was detected in the hospital effluent, probably owing to its instability: in vitro assays indicated that imipenem's biological activity was no longer detectable after 45 h of storage. However, the predictive value of the hazard quotient relative to the development of resistance was >1.0 (HQr = 28.9 ±â€¯1.9), which indicates a possible risk. The presence of carbapenemase-encoding genes in hospital effluent biofilm strains and their ability to transfer are therefore a potential hazard that should not be neglected and points to the need for monitoring antibiotic resistance in hospital wastewater.

Depression Severity as a Risk Factor of Sarcopenic Obesity in Morbidly Obese Patients.

SETTING: Etiopathogenic factors of physical disability in obesity are numerous, underestimated and not sought in the non-geriatric population. Amongst these factors, depression may favor the development of sarcopenic obesity by reducing strength and physical performance even in the absence of overt muscle loss. Objectives and participants: To study the link between depression status and muscle functional disorders (dynapenia) in a population of adult subjects with severe and morbid obesity. MEASUREMENTS: Patients were assessed for body composition, grip strength, the Short Physical Performances Battery test (SPPB), for depression according to the Beck II score as well as for metabolic parameters through biological tests. RESULTS: In 373 obese subjects (mean age 44 ± 13y and BMI 43 ± 6 kg/m²), the prevalence of depression was 53% with 18% having mild depression, 18% moderate depression and 16% severe depression. Depression was significantly related to dynapenia: 62% of dynapenic (D) patients suffered from depression compared to 50% of non-dynapenic (ND) patients (p = 0.036). The Beck questionnaire score for D patients was 20 ± 13 and 15 ± 10 for ND patients (p = 0.001). The depression intensity was significantly correlated with dynapenia with D patients having a higher severe depression degree than ND patients (30% versus 11%; p < 0.0001). Fat-free to fat mass ratio was also significantly correlated with dynapenia (p = 0.01). In multivariate analysis, the presence of depression was twice as likely to be associated with dynapenia. CONCLUSIONS: Depression is associated with a reduction of muscle function in severe obesity in relation to its severity and to changes in fat to fat-free mass, suggesting that screening and prevention of sarcopenic obesity should be considered in adult obese patients with depression.

Extended-spectrum beta-lactamase production is associated with an increase in cell invasion and expression of fimbrial adhesins in Klebsiella pneumoniae.

Extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae strains are suggested to possess higher pathogenic potential than non-ESBL producers. Microbial adherence to and invasion of host cells are critical steps in the infection process, so we examined the expression of type 1 and 3 fimbrial adhesins by 58 ESBL-producing and 152 nonproducing isolates of K. pneumoniae and their abilities to invade ileocecal and bladder epithelial cells. Mannose-sensitive hemagglutination of guinea pig erythrocytes and mannose-resistant hemagglutination of ox erythrocytes were evaluated to determine the strains' abilities to express type 1 and type 3 fimbriae, respectively. Bacterial adhesion to and invasion of epithelial cells were tested by enzyme-linked immunosorbent assay and imipenem killing assay, respectively. The adherence of ESBL- and non-ESBL-producing strains to epithelial cells did not differ significantly (P > 0.05). In contrast, the proportion of strains capable of invading (>5% relative invasion) ileocecal and bladder epithelial cells was significantly higher among ESBL producers (81%, n = 47/58, and 27.6%, n = 16/58, respectively) than among non-ESBL producers (61%, n = 93/152, and 10%, n = 15/152, respectively) (P = 0.0084, odds ratio [OR] = 2.711, 95% confidence interval [CI] = 1.302 to 5.643 and P = 0.0021, OR = 4.79, 95% CI = 1.587 to 7.627). The mean invasion by ESBL producers (5.5% +/- 2.8% and 3.3% +/- 2.7%, respectively) was significantly higher than that by non-ESBL producers (2.9% +/- 2.6% and 1.8% +/- 2%, respectively) (P < 0.0001). Likewise, the proportion of ESBL producers coexpressing both fimbrial adhesins was significantly higher (79.3%; n = 46/58) than that of non-ESBL producers (61.8%; n = 94/152) (P = 0.0214; OR = 2,365; 95% CI = 1.157 to 4.834). Upon acquisition of SHV-12-encoding plasmids, two transconjugants switched on to produce type 3 fimbriae while expression of type 1 fimbriae was not affected. The acquisition of an ESBL plasmid appeared to upregulate the phenotypic expression of one or more genes, resulting in greater invasion ability.

[Pathophysiology of post-operative cognitive dysfunction: current hypotheses]. / Physiopathologie de la dysfonction cognitive postopératoire du sujet âgé: hypothèses actuelles.

Post-operative cognitive dysfunction (POCD) has been reported after a variety of surgical procedures. POCD is associated with a decline in performance of activities of daily living of elderly patients and can cause substantial damage to family and/or to social support systems. The incidence of POCD in the first week after surgery is 23% in patients between 60 and 69 years of age and 29% in patients older than 70. Cognitive dysfunction was still present in 14% of patients over 70 at three month after surgery. The risk of POCD increases with age, and the type of surgery is also important since there is very low incidence of POCD after minor surgery. For many years, it has been known that general anaesthesia is associated with persistent changes in gene expression in the brain for at least 72 hours. These observed modifications suggest an interesting hypothesis to explain the side effects of anaesthetic agents on cognitive dysfunction, particularly in the elderly. The inflammatory response to surgery is consistent with the hypothesis that inflammation contributes to cognitive decline in the elderly. Most of the drugs administered during anaesthesia interact with the cerebral cholinergic system, which seems to be impaired with ageing. One can hypothesize that this cholinergic dysfunction is a potent factor in the pathogenesis of POCD. These findings have implications for the information provided before obtaining consent from elderly patients prior to surgery; a careful evaluation of mental status is mandatory for all elderly patients undergoing general anaesthesia. Perioperative physicians should be familiar with the prevention, diagnosis, and management of postoperative cognitive dysfunction.

Opposing effect of Lactobacillus on in vitro Klebsiella pneumoniae in biofilm and in an in vivo intestinal colonisation model.

Beneficial bacteria represent potential sources of therapy, particularly in the battle against antibiotic-resistant pathogens. The Gram-negative bacillus Klebsiella pneumoniae is not only a paradigm of multi-resistant opportunistic pathogen, but it is also able to colonise the human intestine and displays a high capacity to form biofilm. In this study, the anti-biofilm activity of 140 neutralised Lactobacillus supernatants was assessed against K. pneumoniae. Among the 13 strains whose supernatant significantly impaired biofilm formation, Lactobacillus plantarum CIRM653 was selected because it was also able to impair K. pneumoniae preformed biofilm, independently of a bactericidal effect. Mixed K. pneumoniae/L. plantarum CIRM653 biofilms had reduced tridimensional structures associated with a significant decrease in K. pneumoniae biomass. Further investigation showed that L. plantarum CIRM653 supernatant induced transcriptional modifications of K. pneumoniae biofilm-related genes, including down-regulation of the quorum sensing-related lsr operons and over-expression of type 3 pili structure genes. Increased production of type 3 pili was validated by Western-blot, hemagglutination and adhesion assays. L. plantarum CIRM653 activity against K. pneumoniae was also assessed in a murine intestinal colonisation model: a constant faecal pathogen burden was observed, as against a gradual decrease in the control group. These results reveal that an in vitro a priori attracting anti-biofilm activity of Lactobacillus might be counterbalanced by an in vivo behaviour in a complex microbiota environment with potential deleterious dispersal of highly adherent K. pneumoniae cells, raising the question of the accuracy of in vitro assays in screening of beneficial microbes.

ATP binding cassette modulators control abscisic acid-regulated slow anion channels in guard cells

In animal cells, ATP binding cassette (ABC) proteins are a large family of transporters that includes the sulfonylurea receptor and the cystic fibrosis transmembrane conductance regulator (CFTR). These two ABC proteins possess an ion channel activity and bind specific sulfonylureas, such as glibenclamide, but homologs have not been identified in plant cells. We recently have shown that there is an ABC protein in guard cells that is involved in the control of stomatal movements and guard cell outward K+ current. Because the CFTR, a chloride channel, is sensitive to glibenclamide and able to interact with K+ channels, we investigated its presence in guard cells. Potent CFTR inhibitors, such as glibenclamide and diphenylamine-2-carboxylic acid, triggered stomatal opening in darkness. The guard cell protoplast slow anion current that was recorded using the whole-cell patch-clamp technique was inhibited rapidly by glibenclamide in a dose-dependent manner; the concentration producing half-maximum inhibition was at 3 &mgr;M. Potassium channel openers, which bind to and act through the sulfonylurea receptor in animal cells, completely suppressed the stomatal opening induced by glibenclamide and recovered the glibenclamide-inhibited slow anion current. Abscisic acid is known to regulate slow anion channels and in our study was able to relieve glibenclamide inhibition of slow anion current. Moreover, in epidermal strip bioassays, the stomatal closure triggered by Ca2+ or abscisic acid was reversed by glibenclamide. These results suggest that the slow anion channel is an ABC protein or is tightly controlled by such a protein that interacts with the abscisic acid signal transduction pathway in guard cells.

Metal(loid)s and radionuclides cytotoxicity in Saccharomyces cerevisiae. Role of YCF1, glutathione and effect of buthionine sulfoximine.

The presence of heavy metal(loid)s in soils and waters is an important issue with regards to human health. Taking into account speciation problems, in the first part of this report, we investigated under identical growth conditions, yeast tolerance to a set of 15 cytotoxic metal(loid)s and radionuclides. The yeast cadmium factor 1 (YCF1) is an ATP-Binding Cassette transporter mediating the glutathione detoxification of heavy metals. In the second part, metal(loid)s that could be handled by YCF1 and a possible re-localisation of the transporter after heavy metal exposure were evaluated. YCF1 and a C-terminal GFP fusion, YCF1-GFP, were overexpressed in wild-type and Deltaycf1 strains. Both forms were functional, conferring a tolerance to Cd, Sb, As, Pb, Hg but not to Ni, Zn, Cu, Ag, Se, Te, Cr, Sr, Tc, U. Confocal experiments demonstrated that during exposure to cytotoxic metals, the localisation of YCF1-GFP was restricted to the yeast vacuolar membrane. In the last part, the role of glutathione in this resistance mechanism to metal(loid)s was studied. In the presence of heavy metals, application of buthionine sulfoximine (BSO), a well-known inhibitor of gamma-glutamylcysteine synthetase, led to a decrease in the cytosolic pool of GSH and to a limitation of yeast growth. Surprisingly, BSO was able to phenocopy the deletion of gamma-glutamylcysteine synthetase after exposure to Cd but not to Sb or As. In the genetic context of gsh1 and gsh2 yeast mutants, the critical role of GSH for Cd, As, Sb and Hg tolerance was compared to that of wild-type and Deltaycf1.

Chloroplast targeting of phytochelatin synthase in Arabidopsis: effects on heavy metal tolerance and accumulation.

The enzymatically synthesized thiol peptide phytochelatin (PC) plays a central role in heavy metal tolerance and detoxification in plants. In response to heavy metal exposure, the constitutively expressed phytochelatin synthase enzyme (PCS) is activated leading to synthesis of PCs in the cytosol. Recent attempts to increase plant metal accumulation and tolerance reported that PCS over-expression in transgenic plants paradoxically induced cadmium hypersensitivity. In the present paper, we investigate the possibility of synthesizing PCs in plastids by over-expressing a plastid targeted phytochelatin synthase (PCS). Plastids represent a relatively important cellular volume and offer the advantage of containing glutathione, the precursor of PC synthesis. Using a constitutive CaMV 35S promoter and a RbcS transit peptide, we successfully addressed AtPCS1 to chloroplasts, significant PCS activity being measured in this compartment in two independent transgenic lines. A substantial increase in the PC content and a decrease in the glutathione pool were observed in response to cadmium exposure, when compared to wild-type plants. While over-expressing AtPCS1 in the cytosol importantly decreased cadmium tolerance, both cadmium tolerance and accumulation of plants expressing plastidial AtPCS1 were not significantly affected compared to wild-type. Interestingly, targeting AtPCS1 to chloroplasts induced a marked sensitivity to arsenic while plants over-expressing AtPCS1 in the cytoplasm were more tolerant to this metalloid. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.

Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and shoots.

Transcriptional regulation in response to cadmium treatment was investigated in both roots and leaves of Arabidopsis, using the whole genome CATMA microarray containing at least 24,576 independent probe sets. Arabidopsis plants were hydroponically treated with low (5 microM) or high (50 microM) cadmium concentrations during 2, 6, and 30 hours. At each time point, Cd level was determined using ICP-AES showing that both plant tissues are able to accumulate the heavy metal. RT-PCR of eight randomly selected genes confirmed the reliability of our microarray results. Analyses of response profiles demonstrate the existence of a regulatory network that differentially modulates gene expression in a tissue- and kinetic-specific manner in response to cadmium. One of the main response observed in roots was the induction of genes involved in sulfur assimilation-reduction and glutathione (GSH) metabolism. In addition, HPLC analysis of GSH and phytochelatin (PC) content shows a transient decrease of GSH after 2 and 6 h of metal treatment in roots correlated with an increase of PC contents. Altogether, our results suggest that to cope with cadmium, plants activate the sulfur assimilation pathway by increasing transcription of related genes to provide an enhanced supply of GSH for PC biosynthesis. Interestingly, in leaves an early induction of several genes encoding enzymes involved in the biosynthesis of phenylpropanoids was observed. Finally, our results provide new insights to understand the molecular mechanisms involved in transcriptional regulation in response to cadmium exposure in plants.

Cloning of AtMRP1, an Arabidopsis thaliana cDNA encoding a homologue of the mammalian multidrug resistance-associated protein.

A cDNA encoding a putative ATP-binding cassette (ABC) transporter from Arabidopsis was cloned and sequenced based on an EST clone homologous to ABC sequences in other species. The cDNA is 5.5 kb long and contains an ORF encoding a 1623 amino acids protein. This sequence is the first MRP-like protein found in plants.

Mechanism of action of K channel openers on skeletal muscle KATP channels. Interactions with nucleotides and protons.

The molecular mechanisms underlying the actions of K channel openers (KCOs) on KATP channels were studied with the patch clamp technique in excised inside-out patches from frog skeletal muscle fibers. Benzopyran KCOs (levcromakalim and SR 47063) opened channels partially blocked by ATP, ADP, or ATP gamma s, with and without Mg2+, but they had no effects in the absence of internal nucleotides, even after channel activity had significantly declined because of rundown. The effects of KCOs could therefore be attributed solely to a competitive interaction between KCOs and nucleotides, as confirmed by observations that ATP decreased the apparent affinity for KCOs and that, conversely, KCOs decreased ATP or ADP sensitivity. Protons antagonized the action of the non-benzopyran KCOs, pinacidil and aprikalim, by enhancing their dissociation rate. This effect resembled the effect of acidification on benzopyran KCOs (Forestier, C., Y. Depresle, and M. Vivaudou. FEBS Lett. 325:276-280, 1993), suggesting that, in spite of their structural diversity, KCOs could act through the same binding sites. Detailed analysis of the inhibitory effects of protons on channel activity induced by levcromakalim or SR 47063 revealed that, in the presence of 100 microM ATP, this effect developed steeply between pH 7 and 6 and was half maximal at pH 6.6. These results are in quantitative agreement with an allosteric model of the KATP channel possessing four protonation sites, two nucleotidic sites accessible preferentially to Mg(2+)-free nucleotides, and one benzopyran KCO site. The structural implications of this model are discussed.

LPS-induced immune response in Drosophila.

The study of the regulation of the inducible synthesis of antimicrobial peptides in Drosophila melanogaster has established this insect as a powerful model in which to study innate immunity. In particular, the molecular characterization of the regulatory pathway controlling the antifungal peptide drosomycin has revealed the importance of Toll receptors in innate immunity. We report here that injection of LPS into flies induces an immune response, suggesting that LPS receptors are used in Drosophila to detect Gram-negative bacteria infection. We have identified in the recently sequenced genome of Drosophila eight genes coding for Toll-like receptors in addition to Toll, which may function as LPS receptors. However, overexpression of a selection of these genes in tissue-culture cells does not result in up-regulation of the antibacterial peptide genes. These results are discussed in light of the recent data from genetic screens aimed at identifying the genes controlling the antibacterial response in Drosophila.

Intracellular protons control the affinity of skeletal muscle ATP-sensitive K+ channels for potassium-channel-openers.

Levcromakalim, a potential antihypertensive agent, is known to activate potassium channels dependent on intracellular ATP (K-ATP channels). In inside-out patches excised from frog skeletal muscle, levcromakalim or a related compound, SR 47063, caused a strong and persistent activation of K-ATP channels at a cytoplasmic pH of 7.1. However, at pH 6, these activators could no longer affect the K-ATP currents. Conversely, in the continuous presence of activator, lowering pH from 7.1 to 6 returned channel activity to its level in pH 6 alone. After wash-out of the activator, recovery from activation took minutes at pH 7.1 but only seconds at pH 6, thus ruling out an effect of protons on the activators in solution. These experiments suggest that K-channel-activators are unable to bind to their receptor when it is protonated, and more generally, they provide evidence at the microscopic level for proton-induced allosteric modulation of drug-receptor interaction.

Antibodies to the CFTR modulate the turgor pressure of guard cell protoplasts via slow anion channels.

The plasma membrane guard cell slow anion channel is a key element at the basis of water loss control in plants allowing prolonged osmolite efflux necessary for stomatal closure. This channel has been extensively studied by electrophysiological approaches but its molecular identification is still lacking. Recently, we described that this channel was sharing some similarities with the mammalian ATP-binding cassette protein, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel [Leonhardt, N. et al. (1999) Plant Cell 11, 1141-1151]. Here, using the patch-clamp technique and a bioassay, consisting in the observation of the change in guard cell protoplasts volume, we demonstrated that a functional antibody raised against the mammalian CFTR prevented ABA-induced guard cell protoplasts shrinking and partially inhibited the slow anion current. Moreover, this antibody immunoprecipitated a polypeptide from guard cell protein extracts and immunolabeled stomata in Vicia faba leaf sections. These results indicate that the guard cell slow anion channel is, or is closely controlled by a polypeptide, exhibiting one epitope shared with the mammalian CFTR.

Effects of irradiation on human platelets stored at 20 degrees C.

The morphologic and functional properties of platelets after irradiation with 2500 rads and storage, in first-generation containers, for 48 h in a liquid phase at 20 degrees C with continuous horizontal agitation have been analyzed and compared with a control group of the same platelets which were not irradiated. The preservation technique induced changes in the morphology and aggregation stimulated by ADP and collagen. However, no significant differences were found between the irradiated and non-irradiated groups. Irradiation is not a conditioning factor to add to the hazards of preserving platelets in a liquid phase.

Skeletal muscle regeneration and the mitotic clock.

Regeneration of muscle fibers following damage requires activation of quiescent satellite cells, their proliferation and finally their differentiation and fusion into multinucleated myotubes, which after maturation will replace the damaged fiber. The regenerative potential of human skeletal muscle will be determined, at least partly, by the proliferative capacity of the satellite cells. In this study, we have measured the proliferative life span of human satellite cells until they reach senescence. These analyses were performed on cell populations isolated from old and young donors as well as from one child suffering from Duchenne muscular dystrophy, where extensive regeneration had occurred. In order to see if there are any age-related changes in the myogenic program we have also compared the program of myogenic differentiation expressed by satellite cells from these subjects at different stages of their proliferative lifespan.