Chloride transport in rabbit esophageal epithelial cells

We investigated Cl(-) transport pathways in the apical and basolateral membranes of rabbit esophageal epithelial cells (EEC) using conventional and ion-selective microelectrodes. Intact sections of esophageal epithelium were mounted serosal or luminal side up in a modified Ussing chamber, where transepithelial potential difference and transepithelial resistance could be determined. Microelectrodes were used to measure intracellular Cl(-) activity (a), basolateral or apical membrane potentials (V(mBL) or V(mC)), and the voltage divider ratio. When a basal cell was impaled, V(mBL) was -73 +/- 4.3 mV and a(i)(Cl) was 16.4 +/- 2.1 mM, which were similar in presence or absence of bicarbonate. Removal of serosal Cl(-) caused a transient depolarization of V(mBL) and a decrease in a(i)(Cl) of 6.5 +/- 0.9 mM. The depolarization and the rate of decrease of a(i)(Cl) were inhibited by approximately 60% in the presence of the Cl(-)-channel blocker flufenamate. Serosal bumetanide significantly decreased the rate of change of a(i)(Cl) on removal and readdition of serosal Cl(-). When a luminal cell was impaled, V(mC) was -65 +/- 3.6 mV and a was 16.3 +/- 2.2 mM. Removal of luminal Cl(-) depolarized V(mC) and decreased a by only 2.5 +/- 0.9 mM. Subsequent removal of Cl(-) from the serosal bath decreased a(i)(Cl) in the luminal cell by an additional 6.4 +/- 1.0 mM. A plot of V(mBL) measurements vs. log a(i)(Cl)/log a(o)(Cl) (a(o)(Cl) is the activity of Cl(-) in a luminal or serosal bath) yielded a straight line [slope (S) = 67.8 mV/decade of change in a(i)(Cl)/a(o)(Cl)]. In contrast, V(mC) correlated very poorly with log a/a (S = 18.9 mV/decade of change in a/a). These results indicate that 1) in rabbit EEC, a(i)(Cl) is higher than equilibrium across apical and basolateral membranes, and this process is independent of bicarbonate; 2) the basolateral cell membrane possesses a conductive Cl(-) pathway sensitive to flufenamate; and 3) the apical membrane has limited permeability to Cl(-), which is consistent with the limited capacity for transepithelial Cl(-) transport. Transport of Cl(-) at the basolateral membrane is likely the dominant pathway for regulation of intracellular Cl(-).     

Alpha activity decreases during the perception of Necker cube reversals: an application of wavelet transform

Since the first observation of perceptual reversal by Necker, many theoretical approaches have been proposed. In a previous study, we showed that a positive wave appeared approximately 250 ms prior to the button press of the subjects, indicating perceptual reversal during the observation of the Necker cube figure. A basic difficulty in this type of study is the possible jitter in the latency of the button press due to the variability of the subjects' reaction time during a recording session. To overcome this difficulty, a pattern selection method based on the wavelet transform was proposed in the previous study. A dominant positive wavelet coefficient in the delta band was found to represent the perceptual-reversal-related positivity. In the present study, we aim to analyze the changes in the alpha frequency band during perceptual reversal by using the Necker cube. The RMS values of the alpha frequency band were measured for two time periods: ±3 SD around the mean peak latency of the perceptual-reversal-related positivity and a time window of the same length before the positive wave. We found significantly increased delta power and decreased alpha power during the perceptual-reversal-related positivity. Received: 29 March 1999 / Accepted in revised form: 15 October 1999     

Synthesis and antioxidant properties of novel benzimidazoles containing substituted indole or 1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-naphthalene fragments

Some 6-fluoro-5-substituted-benzimidazole derivatives in which indole and 1,1,4,4-tetramethyl-1,2,3,4-tetrahydro-naphthalene groups were attached to the 2-position of the benzimidazole ring were synthesized and tested for antioxidant properties in vitro. Almost all the synthesized compounds at the 10(-3) M concentrations showed superoxide anion scavenging activity. Compounds 5, 3, 9, 4, 17 and 13 have strong inhibitory effects on superoxide anion formation (98%, 93%, 91%, 88%, 85% and 81%, respectively) at 10(-3) M concentration and these results are better than 30 IU of superoxide dismutase (SOD) (76%). Compound 11 is the most effective scavenger of 2,2-diphenyl-1-picrylhydrazyl (DPPH) stable free radical at 10(-3)M (61%) concentration.     

Plant growth promoting rhizobacteria enhanced leaf organic acids,FC-R activity and Fe nutrition of apple under lime soil conditions

Iron chlorosis in the calcareous soils is one most important stress factors worldwide that limits photosynthesis and decreases fruit yield and quality. Certain soil rhizobacteria produce organic compounds such as plant acids and they may reduce the soil rhizosphere pH and affect ferric chelate reductase (FC-R) activity in root. However, there is no knowledge regarding changes in organic acids content and FC-R activities of leaf due to rhizobacterial root inoculation. Therefore, the efficiency of six plant growth promoting rhizobacteria (PGPR) were tested on apple cv. Braeburn on M9 and MM106 rootstocks. The results of the experiment showed leaf organic acid contents, iron quantity of soil, root and leaf and root and leaf FC-R activity were significantly affected via rhizobacteria applications in apple plants. In MM106 and M9, there was a remarkable increase in Fe in M3 inoculated soil by 95 and 89%, respectively, compared to control. Average increases in citric, malic, malonic, butyric and lactic acid in the leaf were obtained from rhizobacterial root inoculations of 25.1, 21.8, 29.6, 18.0 and 18.2% in Braeburn/MM106, respectively. In Braeburn/M9, MFDCa1 application increased all organic acid concentrations compared to the control. MFDCa2 treatment caused the maximum leaf FC-R activity in Braeburn on M9 and MM106 (60.9 and 50.3 nmol Fe⁺² g^?1 FW h^?1, respectively) while the least values were determined in the control (33.5 and 29.9 nmol Fe⁺² g^?1 FW h^?1, respectively). This study showed the bacterial strains tested in our study may be used as a biofertilizer instead of Fe fertilizers.     

How Taxonomic Relations Affect the Physicochemical Properties of Chitin

Chitin specimens from 16 arthropod species (13 of Insecta and 3 of Arachnida) were isolated for the first time using the same method. Fourier Transform Infrared Spectrometry (FTIR), Thermogravimetric Analysis (TGA), X-ray diffraction (XRD), Scanning Electron Microscope (SEM) and elemental analysis have been applied to determine how physicochemical properties of chitin specimens are affected by taxonomic relationship. The characterisation studies revealed that physicochemical nature of the chitin specimens differed greatly and were found partially specific to taxa. Significant differences in the surface morphologies of chitin specimens were observed even in the same order. However, the chitin contents were recorded to be specific to the order in the class Insecta. The highest chitin content was observed in Coleoptera (18.2–25.2 %) followed by Hemiptera (10.6–14.5 %), Odonata (9.5–10.1 %), Hymenoptera (7.8–9.3 %), Diptera (8.1 %), Blattodea (4.7 %). In addition, the crystalline index (CrI) values of chitin specimens from Coleoptera were found to be higher than the other orders in Insecta. This study revealed that the chitin contents and CrI values can be related to taxonomical relationships.     

Development of a fast and low-cost qPCR assay for diagnosis of acute gas pharyngitis

Background

Group A streptococci (GAS) are the most common bacterial cause of acute pharyngitis and account for 15–30 % of cases of acute pharyngitis in children and 5–10 % of cases in adults. In this study, a real-time quantitative PCR (qPCR) based GAS detection assay in pharyngeal swab specimens was developed.

Methods

The qPCR assay was compared with the gold standard bacterial culture and a rapid antigen detection test (RADT) to evaluate its clinical performance in 687 patients. The analytical sensitivity of the assay was 240 cfu/swab. Forty-five different potential cross-reacting organisms did not react with the test. Four different laboratories for the reproducibility studies were in 100 % (60/60) agreement for the contrived GAS positive and negative swab samples.

Results

The relative sensitivities of the RADT and the qPCR test were 55.9 and 100 %; and the relative specificities were 100 and 96.3 %, respectively. Duration of the total assay for 24 samples including pre-analytical processing and analysis changed between 42 and 55 min depending on the type of qPCR instrument used. A simple DNA extraction method and a low qPCR volume made the developed assay an economical alternative for the GAS detection.

Conclusion

We showed that the developed qPCR test is rapid, cheap, sensitive and specific and therefore can be used to replace both antigen detection and culture for diagnosis of acute GAS pharyngitis.

     

Investigation of allosteric coupling in human β<Subscript>2</Subscript>-adrenergic receptor in the presence of intracellular loop 3

Background

This study investigates the allosteric coupling that exists between the intra- and extracellular parts of human β₂-adrenergic receptor (β₂-AR), in the presence of the intracellular loop 3 (ICL3), which is missing in all crystallographic experiments and most of the simulation studies reported so far. Our recent 1 μs long MD run has revealed a transition to the so-called very inactive state of the receptor, in which ICL3 packed under the G protein’s binding cavity and completely blocked its accessibility to G protein. Simultaneously, an outward tilt of transmembrane helix 5 (TM5) caused an expansion of the extracellular ligand-binding site. In the current study, we performed independent runs with a total duration of 4 μs to further investigate the very inactive state with packed ICL3 and the allosteric coupling event (three unrestrained runs and five runs with bond restraints at the ligand-binding site).

Results

In all three independent unrestrained runs (each 500 ns long), ICL3 preserved its initially packed/closed conformation within the studied time frame, suggesting an inhibition of the receptor’s activity. Specific bond restraints were later imposed between some key residues at the ligand-binding site, which have been experimentally determined to interact with the ligand. Restraining the binding site region to an open state facilitated ICL3 closure, whereas a relatively constrained/closed binding site hindered ICL3 packing. However, the reverse operation, i.e. opening of the packed ICL3, could not be realized by restraining the binding site region to a closed state. Thus, any attempt failed to free the ICL3 from its locked state due to the presence of persistent hydrogen bonds.

Conclusions

Overall, our simulations indicated that starting with very inactive states, the receptor stayed almost irreversibly inhibited, which in turn decreased the overall mobility of the receptor. Bond restraints which represented the geometric restrictions caused by ligands of various sizes when bound at the ligand-binding site, induced the expected conformational changes in TM5, TM6 and consequently, ICL3. Still, once ICL3 was packed, the allosteric coupling became ineffective due to strong hydrogen bonds connecting ICL3 to the core of the receptor.

     

GENDER INFLUENCES DIFFERENTIATION OF CHITIN AMONG BODY PARTS

Earlier reports have established that chitin isolates from each body part of an insect cuticle can exhibit diverse physicochemical properties. But it is still unknown if the gender of the insect can influence characteristics of chitin isolates from different body parts. The present study addresses this question. As a result, important physicochemical differences in the chitin samples from different body parts of Melolontha sp. were recorded on the basis of sex. The chitin samples were extracted from eight different body parts (antennae, head, eyes, thorax, abdomen, elytra, hindwings, and legs) of female and male. The most remarkable variations in the chitin isolates from female and male body parts were recorded in chitin content, crystallinity, thermal stability, and surface morphology. And also it was wondered these chitin isolates from different body parts of female and male could find different applications. To check this hypothesis, the chitin samples from female and male were interacted with bovine serum albumin (BSA) protein and important variations were observed.