Purification of Lactobacillus secreted proteins

Summary We describe a rapid and reliable one-step method for purification of Lactobacillus secreted proteins. With electroendosmotic preparative electrophoresis and a modified synthetic medium the L. plantarum aggregation promoting factor, a 32 kDa secreted protein, and the thermostable -amylase of Bacillus stearothermophilus cloned in L. reuteri were purified. Although the growth rate was reduced, the production of secreted proteins was not affected.     

Formation of benzodiazepine-like molecules in rat brain

The possible biosynthetic origin of benzodiazepine-like molecules was investigated in mammalian tissue. Rat brain homogenates or cortical slices incubated under physiological conditions showed a 4 to 7 fold increase in the content of BZD-like compounds as compared with control non incubated or boiled tissue. The quantitative analysis was performed by a radioimmunoassay with a specific monoclonal antibody. The active fraction eluting just before diazepam exhibited a Mr lower than 1300 and inhibited the [3H]flunitrazepam binding to the central benzodiazepine receptor. No activity was measured in the absence of tissue. These data suggest that under our experimental conditions, low molecular weight substances similar to benzodiazepines are formed in rat brain.     

Simplified construction and characterization of yeast artificial chromosome libraries.

Three yeast artificial chromosome (YAC) libraries were constructed using two human cell lines and the pYAC-RC vector. The main differences from the previously described methods were: i) genomic DNA was digested in low melting point (LMP) agarose blocks with the rare cutting enzyme ClaI; ii) DNA was ligated in melted LMP agarose after agarase treatment; iii) spheroplast regeneration plating was done in calcium alginate thin layer. In addition, a panel of PCR primers was used to identify quickly the presence in the libraries of repetitive and single copy human DNA sequences.     

p140Cap protein suppresses tumour cell properties, regulating Csk and Src kinase activity

We recently identified p140Cap as a novel adaptor protein, expressed in epithelial-rich tissues and phosphorylated upon cell matrix adhesion and growth factor treatment. Here, we characterise p140Cap as a novel Src-binding protein, which regulates Src activation via C-terminal Src kinase (Csk). p140Cap silencing increases cell spreading, migration rate and Src kinase activity. Accordingly, increased expression of p140Cap activates Csk, leading to inhibition of Src and downstream signalling as well as of cell motility and invasion. Moreover, cell proliferation and "in vivo" breast cancer cell growth are strongly impaired by high levels of p140Cap, providing the first evidence that p140Cap is a novel negative regulator of tumour growth.     

Human leukemic K562 cells: suppression of hemoglobin accumulation by a monoclonal antibody to human transferrin receptor

The receptor for transferrin plays an important role both in tumor cell growth and in hemoglobin synthesis. In this paper, we demonstrate that the monoclonal antibody 42/6 to human transferrin receptor inhibits iron uptake in the human leukemic K562 cell line and suppresses hemoglobin accumulation in K562 cells induced to erythroid differentiation by butyric acid. In contrast, only slight inhibitory effects were observed on cell proliferation of both uninduced and erythroid-induced K562 cells treated with the 42/6 monoclonal antibody. In addition, the 42/6 monoclonal antibody to human transferrin receptor does not inhibit butyric acid-induced accumulation of gamma-globin mRNA. The effect of the 42/6 monoclonal antibody on hemoglobin synthesis appears to be restricted to human cell lines, as murine Friend erythroleukemic cells undergo erythroid differentiation when cultured in the presence of hexamethylenebisacetamide plus the 42/6 monoclonal antibody. The findings reported in this paper suggest (a) a dissociation of iron transport and accumulation of heme molecules from the expression of globin genes and (b) a different requirement of iron uptake by different iron-dependent functions such as cell proliferation and hemoglobin expression.     

Reassessment of the Psychometric Characteristics and Factor Structure of the ‘Perceived Stress Questionnaire’ (PSQ): Analysis in a Sample of Dental Students

Background

The training to become a dentist can create psychological distress. The present study evaluates the structure of the ‘Perceived Stress Questionnaire’ (PSQ), its internal consistency model and interrelatedness with burnout, anxiety, depression and resilience among dental students.

Methods

The study employed a cross-sectional design. A sample of Spanish dental students (n = 314) completed the PSQ, the ‘Goldberg Anxiety and Depression Scale’ (GADS), ‘Connor-Davidson Resilience Scale’ (10-item CD-RISC) and ‘Maslach Burnout Inventory-Student Survey’ (MBI-SS). The structure was estimated using Parallel Analysis from polychoric correlations. Unweighted Least Squares was the method for factor extraction, using the Item Response Theory to evaluate the discriminative power of items. Internal consistency was assessed by squaring the correlation between the latent true variable and the observed variable. The relationships between the PSQ and the other constructs were analysed using Spearman’s coefficient.

Results

The results showed a PSQ structure through two sub-factors (‘frustration’ and ‘tenseness’) with regard to one general factor (‘perceived stress’). Items that did not satisfy discriminative capacity were rejected. The model fit were acceptable (GFI = 0.98; RSMR = 0.06; AGFI = 0.98; NFI = 0.98; RFI = 0.98). All the factors showed adequate internal consistency as measured by the congeneric model (≥0.91). High and significant associations were observed between perceived stress and burnout, anxiety, depression and resilience.

Conclusions

The PSQ showed a hierarchical bi-factor structure among Spanish dental students. Using the questionnaire as a uni-dimensional scale may be useful in perceived stress level discrimination, while the sub-factors could help us to refine perceived stress analysis and improve therapeutic processes.     

P2X receptors in mouse Leydig cells

ATP-activated currents were studied in Leydig cells of mice with the patch-clamp technique. Whole cell currents were rapidly activating and slowly desensitizing (55% decrement from the peak value on exposure to 100 µM ATP for 60 s), requiring 3 min of washout to recover 100% of the response. The concentration-response relationships for ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPS), and 2-methylthio-ATP (2-MeS-ATP) were described by the Hill equation with a concentration evoking 50% of maximal ATP response (K_d) of 44, 110, and 637 µM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP ATPS > 2-MeS-ATP > 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). -Methylene-ATP (-MeATP), GTP, UTP, cAMP, and adenosine were ineffective. Suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) blocked the responses in a concentration-dependent manner. The ATP-activated currents were dependent on extracellular pH, being maximal at pH 6.5 and decreasing with both acidification and alkalinization (apparent dissociation constant (pK_a) of 5.9 and 7.4, respectively). The whole cell current-voltage relationship showed inward rectification and reversed near 0 mV. Experiments performed in bi-ionic conditions for measurement of reversal potentials showed that this channel is highly permeable to calcium [permeability (P)_Ca/P_Na = 5.32], but not to chloride (P_Cl/P_Na = 0.03) or N-methyl-D-glucamine (NMDG) (P_NMDG/P_Na = 0.09). Unitary currents recorded in outside-out patches had a chord conductance of 27 pS (between –90 and –50 mV) and were inward rectifying. The average current passing through the excised patch decreased with time [time constant () = 13 s], resembling desensitization of the macroscopic current. These findings indicate that the ATP receptor present in Leydig cells shows properties most similar to those of cloned homomeric P2X₂. patch clamp; single channels; ATP; desensitization     

Ketamine Self-Administration Elevates αCaMKII Autophosphorylation in Mood and Reward-Related Brain Regions in Rats

Modulation of αCaMKII expression and phosphorylation is a feature shared by drugs of abuse with different mechanisms of action. Accordingly, we investigated whether αCaMKII expression and activation could be altered by self-administration of ketamine, a non-competitive antagonist of the NMDA glutamate receptor, with antidepressant and psychotomimetic as well as reinforcing properties. Rats self-administered ketamine at a sub-anesthetic dose for 43 days and were sacrificed 24 h after the last drug exposure; reward-related brain regions, such as medial prefrontal cortex (PFC), ventral striatum (vS), and hippocampus (Hip), were used for the measurement of αCaMKII-mediated signaling. αCaMKII phosphorylation was increased in these brain regions suggesting that ketamine, similarly to other reinforcers, activates this kinase. We next measured the two main targets of αCaMKII, i.e., GluN2B (S1303) and GluA1 (S831), and found increased activation of GluN2B (S1303) together with reduced phosphorylation of GluA1 (S831). Since GluN2B, via inhibition of ERK, regulates the membrane expression of GluA1, we measured ERK2 phosphorylation in the crude synaptosomal fraction of these brain regions, which was significantly reduced suggesting that ketamine-induced phosphorylation of αCaMKII promotes GluN2B (S1303) phosphorylation that, in turn, inhibits ERK 2 signaling, an effect that results in reduced membrane expression and phosphorylation of GluA1. Taken together, our findings point to αCaMKII autophosphorylation as a critical signature of ketamine self-administration providing an intracellular mechanism to explain the different effects caused by αCaMKII autophosphorylation on the post-synaptic GluN2B- and GluA1-mediated functions. These data add ketamine to the list of drugs of abuse converging on αCaMKII to sustain their addictive properties.