Synchronization and entrainment of cytoplasmic Ca2+ oscillations in cell clusters prepared from single or multiple mouse pancreatic islets

In contrast to pancreatic islets, isolated beta-cells stimulated by glucose display irregular and asynchronous increases in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)). Here, clusters of 5-30 cells were prepared from a single mouse islet or from pools of islets, loaded with fura-2, and studied with a camera-based system. [Ca(2+)](i) oscillations were compared in pairs of clusters by computing the difference in period and a synchronization index lambda. During perifusion with 12 mM glucose, the clusters exhibited regular [Ca(2+)](i) oscillations that were quasi-perfectly synchronized (Delta period of 1.4% and index lambda close to 1.0) between cells of each cluster. In contrast, separate clusters were not synchronized, even when prepared from one single islet. Pairs of clusters neighboring on the same coverslip were not better synchronized than pairs of clusters examined separately (distinct coverslips). We next attempted to synchronize clusters perifused with 12 mM glucose by applying external signals. A single pulse of 20 mM glucose, 10 mM amino acids, or 10 microM tolbutamide transiently altered [Ca(2+)](i) oscillations but did not reset the clusters to oscillate synchronously. On a background of 12 mM glucose, repetitive applications (1 min/5 min) of 10 microM tolbutamide, but not of 20 mM glucose, synchronized separate clusters. Our results identify a level of beta-cell heterogeneity intermediate between single beta-cells and the whole islet. They do not support the idea that substances released by islet cells serve as paracrine synchronizers. However, synchronization can be achieved by an external signal, if this signal has a sufficient strength to overwhelm the intrinsic rhythm of glucose-induced oscillations and is repetitively applied.     

Rye SCAR markers for male fertility restoration in the P cytoplasm are also applicable to marker-assisted selection in the C cytoplasm

The study aimed at testing the usefulness of recently developed SCAR markers on rye (Secale cereale L.) chromosome 4R in hybrid breeding based on the C source of male sterility-inducing cytoplasm. Of 10 markers studied, 4 revealed polymorphisms between 2 inbred lines (544cms-C and Ot0-20) crossed to develop F2 and BC1 mapping populations. Analyses performed on 94 F2 and 93 BC1 plants allowed to extend a formerly constructed genetic map of chromosome arm 4RL. Three SCAR markers (SCP14M55, SCP15M55 and SCP16M58) were mapped in the vicinity of gene Rfc1, which restores male fertility in the C cytoplasm. The 3 tested SCAR markers proved to be effective in marker-assisted selection (MAS) for male fertility/sterility.     

Oxytocin and its analogs, methyl-substituted in ortho-, meta- or para-position of aromatic ring of phenylalanine in position 2: NMR study and biological activities

Complete analyses of NMR data of oxytocin (OT) and 4 analogues, ([o-MePhe(2)]OT, [mMe-Phe(2)]OT, [m-OMePhe(2)]OT and [p-MePhe(2)]OT), are given. The same conformational behavior in solution on one hand and large differences in biological activities on the other hand indicate that the compounds adopt a "biologically active conformation" at the stage of interaction with the receptor when the character of the substituent and its position on the aromatic ring may play a role in hindering attaining the ideal complementarity of both interacting components.     

Functional analysis and comparative genomics of expressed sequence tags from the lycophyte Selaginella moellendorffii

Background

DNA microarrays are widely used in gene expression analyses. To increase throughput and minimize costs without reducing gene expression data obtained, we investigated whether four mRNA samples can be analyzed simultaneously by applying four different fluorescent dyes.

Results

Following tests for cross-talk of fluorescence signals, Alexa 488, Alexa 594, Cyanine 3 and Cyanine 5 were selected for hybridizations. For self-hybridizations, a single RNA sample was labelled with all dyes and hybridized on commercial cDNA arrays or on in-house spotted oligonucleotide arrays. Correlation coefficients for all combinations of dyes were above 0.9 on the cDNA array. On the oligonucleotide array they were above 0.8, except combinations with Alexa 488, which were approximately 0.5. Standard deviation of expression differences for replicate spots were similar on the cDNA array for all dye combinations, but on the oligonucleotide array combinations with Alexa 488 showed a higher variation.

Conclusion

In conclusion, the four dyes can be used simultaneously for gene expression experiments on the tested cDNA array, but only three dyes can be used on the tested oligonucleotide array. This was confirmed by hybridizations of control with test samples, as all combinations returned similar numbers of differentially expressed genes with comparable effects on gene expression.     

Soybean hull peroxidase immobilization on macroporous glycidyl methacrylates with different surface characteristics

Soybean hull peroxidase (SHP, E.C. 1.11.1.7) was immobilized by a glutaraldehyde and periodate method onto series of macroporous copolymers of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA), poly(GMA-co-EGDMA) with various surface characteristics and pore size diameters ranging from 44 to 200 nm. Glutaraldehyde immobilization method and poly(GMA-co-EGDMA) named SGE 20/12 with pore sizes of 120 nm gave immobilized enzyme with highest specific activity of 25 U/g. Deactivation studies showed that immobilization increased stability of SHP and that surface characteristics of the used copolymer had a major influence on a stability of immobilized enzyme at high temperatures and in an organic solvent. The highest thermostability was obtained using the copolymer SGE 20/12 with pore size of 120 nm, while the highest stability in dioxane had SHP immobilized onto copolymer SGE 10/4 with pore size of 44 nm. Immobilized SHP showed a wider pH optimum as compared to the native enzyme especially at alkaline pH values and 3.2 times increased K _m value for pyrogallol. After 6 cycles of repeated use in batch reactor, immobilized SHP retained 25 % of its original activity. Macroporous copolymers with different surface characteristics can be used for fine tuning of activity and stability of immobilized SHP to obtain a biocatalyst suitable for phenol oxidation or polymer synthesis in organic solvents.     

Proteomic Analysis of the Human Skin Proteome after In Vivo Treatment with Sodium Dodecyl Sulphate

Background

Skin has a variety of functions that are incompletely understood at the molecular level. As the most accessible tissue in the body it often reveals the first signs of inflammation or infection and also represents a potentially valuable source of biomarkers for several diseases. In this study we surveyed the skin proteome qualitatively using gel electrophoresis, liquid chromatography tandem mass spectrometry (GeLC-MS/MS) and quantitatively using an isobaric tagging strategy (iTRAQ) to characterise the response of human skin following exposure to sodium dodecyl sulphate (SDS).

Results

A total of 653 skin proteins were assigned, 159 of which were identified using GeLC-MS/MS and 616 using iTRAQ, representing the most comprehensive proteomic study in human skin tissue. Statistical analysis of the available iTRAQ data did not reveal any significant differences in the measured skin proteome after 4 hours exposure to the model irritant SDS.

Conclusions

This study represents the first step in defining the critical response to an irritant at the level of the proteome and provides a valuable resource for further studies at the later stages of irritant exposure.     

Targeting Complement C3a Receptor to Improve Outcome After Ischemic Brain Injury

Neurochemical Research - Ischemic stroke is a major cause of disability. No efficient therapy is currently available, except for the removal of the occluding blood clot during the first hours after...     

The preoperative activity of Th1 and Th17 cytokine axes in prediction of sepsis after radical cystectomy

The aim of the study was to correlate the preoperative activity of Th1 and Th17 cytokine axes with the development of sepsis after radical cystectomy. The study involved twenty patients with the infiltrative transitional cell carcinoma of the urinary bladder without previous radiotherapy/chemotherapy, who underwent open radical cystectomy with urinary diversion. Preoperative plasma concentrations of Th1 cytokines interleukin 12 (IL-12) and interferon gamma (IFN-γ), and Th17 cytokines IL-23 and IL-17, were measured using ELISA. Preoperative expression of mRNA for IL-12p35, IFN-γ, IL-23p19 and IL-17 was quantified by real-time RT-PCR using mRNA extracted from peripheral blood mononuclear cells. Eight patients developed postoperative sepsis, diagnosed within two weeks post-operation as systemic inflammatory response syndrome in the presence of local or systemic infection. The preoperative basal plasma concentrations of Th1 and Th17 cytokines were slightly above the detection limits, with a tendency toward lower concentrations in patients who developed sepsis, but the difference was not significant (p>0.05). The preoperative expression of mRNA encoding IL-12p35 and IL-17 was significantly lower in patients who developed sepsis (p=0.003 and p=0.028, respectively). The similar trend was observed for IL-23p19 and IFN-γ, but the differences did not reach the statistical significance (p=0.051 and p=0.172, respectively). These data suggest that determination of preoperative Th1 and Th17 cytokine mRNA levels might be useful in predicting sepsis development after radical cystectomy.