Inhibition of Na+, K+-ATPase activates swelling-induced taurine efflux in a human neuroblastoma cell line

The Na⁺ pump (Na⁺, K⁺-ATPase) has been implicated in the regulation of many cellular functions, including cell volume regulation. The effects of inhibiting Na⁺ pump activity on cell volume and taurine efflux were evaluated in the human neuroblastoma cell line CHP-100. Cell volume changes monitored with the Coulter Multisizer technique and confocal microscopy showed that neuroblastoma cells exposed to ouabain swelled by 22 ± 4% (n = 5). The rapid cell swelling was followed by regulatory volume decrease (RVD). In cells treated with ouabain, ¹⁴C-taurine efflux increased by 183 ± 11% compared with controls. However, cells exposed simultaneously to ouabain and hypoosmotic solution resulted in a ¹⁴C-taurine efflux of 207 ± 18%. Western blot and immunofluorescence microscopy with specific monoclonal antibodies for the catalytic α isoforms of Na⁺, K⁺-ATPase demonstrated high levels of the ubiquitously expressed α1 and the neuronal-specific α3. Ouabain-binding data showed that CHP-100 cells express ∼3 × 10⁵ pump units/cell. The present data indicate that efflux of taurine may be involved during volume recovery subsequent to blockade of Na⁺, K⁺-ATPase in CHP-100 cells. J. Cell. Physiol. 174:145–153, 1998. © 1998 Wiley-Liss, Inc.     

5-Membered NH...N hydrogen bonded molecular scaffold in a model dipeptide containing 3-aminophenylacetic acid: Crystal and solution conformations

Crystallographic and spectroscopic studies of a model dipeptidecontaining unusual amino acid residues establish the presence ofan intramolecular, 5-membered NH...N hydrogen bond involvingan amide NH (from 3-amino phenyl acetic acid residue) and anamide N atom from an adjacent amino acid residue in solid stateand in solution. The dipeptide also forms an infinite -sheet ribbon structure in crystals.     

Genomic analysis of cyclic-di-GMP-related genes in rhizobial type strains and functional analysis in Rhizobium etli

Rhizobia are soil bacteria that can fix nitrogen in symbiosis with leguminous plants or exist free living in the rhizosphere. Crucial to their complex lifestyle is the ability to sense and respond to diverse environmental stimuli, requiring elaborate signaling pathways. In the majority of bacteria, the nucleotide-based second messenger cyclic diguanosine monophosphate (c-di-GMP) is involved in signal transduction. Surprisingly, little is known about the importance of c-di-GMP signaling in rhizobia. We have analyzed the genome sequences of six well-studied type species (Bradyrhizobium japonicum, Mesorhizobium loti, Rhizobium etli, Rhizobium leguminosarum, Sinorhizobium fredii, and Sinorhizobium meliloti) for proteins possibly involved in c-di-GMP signaling based on the presence of four domains: GGDEF (diguanylate cyclase), EAL and HD-GYP (phosphodiesterase), and PilZ (c-di-GMP sensor). We find that rhizobia possess a high number of these proteins. Conservation analysis suggests that c-di-GMP signaling proteins modulate species-specific pathways rather than ancient rhizobia-specific processes. Two hybrid GGDEF-EAL proteins were selected for functional analysis, R. etli RHE_PD00105 (CdgA) and RHE_PD00137 (CdgB). Expression of cdgA and cdgB is repressed by the alarmone (p)ppGpp. cdgB is significantly expressed on plant roots and free living. Mutation of cdgA, cdgB, or both does not affect plant root colonization, nitrogen fixation capacity, biofilm formation, motility, and exopolysaccharide production. However, heterologous expression of the individual GGDEF and EAL domains of each protein in Escherichia coli strongly suggests that CdgA and CdgB are bifunctional proteins, possessing both diguanylate cyclase and phosphodiesterase activities. Taken together, our results provide a platform for future studies of c-di-GMP signaling in rhizobia.     

Mitochondrial-targeted antioxidants represent a promising approach for prevention of cisplatin-induced nephropathy

Cisplatin is a widely used antineoplastic agent; however, its major limitation is the development of dose-dependent nephrotoxicity whose precise mechanisms are poorly understood. Here we show not only that mitochondrial dysfunction is a feature of cisplatin nephrotoxicity, but also that targeted delivery of superoxide dismutase mimetics to mitochondria largely prevents the renal effects of cisplatin. Cisplatin induced renal oxidative stress, deterioration of mitochondrial structure and function, an intense inflammatory response, histopathological injury, and renal dysfunction. A single systemic dose of mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently prevented cisplatin-induced renal dysfunction. Mito-CP also prevented mitochondrial injury and dysfunction, renal inflammation, and tubular injury and apoptosis. Despite being broadly renoprotective against cisplatin, Mito-CP did not diminish cisplatin's antineoplastic effect in a human bladder cancer cell line. Our results highlight the central role of mitochondrially generated oxidants in the pathogenesis of cisplatin nephrotoxicity. Because similar compounds seem to be safe in humans, mitochondrially targeted antioxidants may represent a novel therapeutic approach against cisplatin nephrotoxicity.     

Enantiopure ethyl 2,3-dibromopropionate: Enantioselective synthesis vs preparative HPLC enantioseparation of racemate on multigram scale

Racemic ethyl 2,3-dibromopropionate, commercially available at low price, is a key intermediate used in the synthesis of several heterocycle fragments, which are present in many biologically active compounds. Surprisingly, the enantiomers are not commercially available and have never been described in the literature. In this work, we undertook two different strategies to obtain these enantiomers, which are enantioselective synthesis and preparative HPLC enantioseparation of commercially available racemate on multigram scale. The first strategy has proved inadequate because racemization occurred during the synthesis (ee ≈ 9-50%). Conversely, the second strategy produced a very good enantioseparation of commercially available racemate (ee > 99.5% for both enantiomers) on multigram scale.     

Design,synthesis and molecular docking of novel diarylcyclohexenone and diarylindazole derivatives as tubulin polymerization inhibitors

New target compounds were designed as inhibitors of tubulin polymerization relying on using two types of ring B models (cyclohexenone and indazole) to replace the central ring in colchicine. Different functional groups (R¹) were attached to manipulate their physicochemical properties and/or their biological activity. The designed compounds were assessed for their antitumor activity on HCT-116 and MCF-7 cancer cell lines. Compounds 4b, 5e and 5f exhibited comparable or higher potency than colchicine against colon HCT-116 and MCF-7 tumor cells. The mechanism of the antitumor activity was investigated through evaluating the tubulin inhibition potential of the active compounds. Compounds 4b, 5e and 5f showed percentage inhibition of tubulin in both cell line homogenates ranging from 79.72% to 89.31%. Cell cycle analysis of compounds 4b, 5e and 5f revealed cell cycle arrest at G₂/M phase. Molecular docking revealed the binding mode of these new compounds into the colchicine binding site of tubulin.     

Exosomes surf on filopodia to enter cells at endocytic hot spots,traffic within endosomes,and are targeted to the ER

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.     

Predictors of Symptomatic Change and Adherence in Internet-Based Cognitive Behaviour Therapy for Social Anxiety Disorder in Routine Psychiatric Care

Objective

A central goal of health care is to improve patient outcomes. Although several studies have demonstrated the effectiveness of therapist guided internet-based cognitive behaviour therapy (ICBT) for social anxiety disorder (SAD), a significant proportion of patients do not respond to treatment. Consequently, the aim of this study was to identify individual characteristics and treatment program related factors that could help clinicians predict treatment outcomes and adherence for individuals with SAD.

Method

The sample comprised longitudinal data collected during a 4-year period of adult individuals (N = 764) treated for SAD at a public service psychiatric clinic. Weekly self-rated Liebowitz Social Anxiety Scale (LSAS-SR) scores were provided. Rates of symptomatic change during treatment and adherence levels were analysed using multilevel modelling. The following domains of prognostic variables were examined: (a) socio-demographic variables; (b) clinical characteristics; (c) family history of mental illness; and (d) treatment-related factors.

Results

Higher treatment credibility and adherence predicted a faster rate of improvement during treatment, whereas higher overall functioning level evidenced a slower rate of improvement. Treatment credibility was the strongest predictor of greater adherence. Having a family history of SAD-like symptoms was also associated with greater adherence, whereas Attention-Deficit/Hyperactivity Disorder (ADHD)-like symptoms, male gender, and family history of minor depression predicted lower adherence. Also, the amount of therapist time spent per treatment module was negatively associated with adherence.

Conclusions

Results from a large clinical sample indicate that the credibility of ICBT is the strongest prognostic factor explaining individual differences in both adherence level and symptomatic improvement. Early screening of ADHD-like symptoms may help clinicians identify patients who might need extra support or an adjusted treatment. Therapist behaviours that promote adherence may be important for treatment response, although more research is needed in order to determine what type of support would be most beneficial.