haemolysin of Escherichia coli: Comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins

Abstract Lipid bilayer experiments were performed with chromosome-encoded haemolysin of Escherichia coli . The addition of the toxin to the aqueous phase bathing lipid bilayer membranes of asolectin resulted in the formation of transient ion-permeable channels with two states at small transmembrane voltages. One is prestate (single-channel conductance 40 pS in 0.15 M KCl) of the open state, which had a single-channel conductance of 420 pS in 0.15 M KCl and a mean lifetime of 30 s. Membranes formed of pure lipids were rather inactive targets for this haemolysin. Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH. The mobility sequence of the cations in the channel was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm. The pore-forming properties of chromosome-encoded haemolysin were compared with those of plasmid-encoded haemolysin. Both toxins share common features, oligomerize probably to form pores in lipid bilayer membranes. Both types of haemolysin channels have similar properties but different lifetimes.     

Glucan synthesis by intact cotton fibres fed with different precursors at the stages of primary and secondary wall formation

Seed clusters of individual locules from fruit capsules of Gossypium arboreum L. with adhering intact fibres were fed with radioactive uridinediphosphoglucose (UDPG), guanosinediphosphoglucose (GDPG), glucose and sucrose. The incorporation into high molecular weight glucans of the fibres was studied. For primary wall fibres, UDPG at 1 mM was by far the best precursor, whereas sucrose was the best precursor for secondary wall fibres. No competition was observed between the incorporation of glucose from UDPG and from sucrose when the two were fed simultaneously to secondary wall fibres, indicating that their metabolic pathways are well separated when they are fed from the apoplast. Inhibitors of respiratory ATP-formation strongly inhibited incorporation of sucrose but not that of UDPG. Sucrose incorporation was studied at five different stages of development of the cotton fibres. At the stage of most intense secondary wall formation the incorporation rate was about 300 times that during primary wall formation (24 days post anthesis (DPA)). Incorporation from 1 mM UDPG or GDPG by secondary wall fibres (35 DPA) was less than twice that of primary wall fibres (22 DPA), indicating that the two sugar nucleotides are not readily used as precursors for secondary wall cellulose when they are fed to the exterior of intact cells. The high molecular weight non-cellulosic glucans formed from UDPG and sucrose at 5 and 1,000 M were solubilized in strongly alkaline solutions or dimethyl-sulfoxide (DMSO) and were partially characterized by degradation with an exo--1,3-glucanase. After feeding for one hour, at most 1/3 of the radioactivity in high molecular weight material was found in cellulose and at least 2/3 in -1,3-glucan. The proportions varied little for fibres in the age range of 30 to 48 DPA when sucrose was the precursor although the total incorporation varied by a factor of about four. The fact that at all stages of secondary wall formation -1,3-glucan is synthesized at a very high rate, but that the total amount in the cell wall does not exceed 2% in the later stages of wall formation, can be interpreted in terms of a high turnover of this polysaccharide if it is assumed that wound effects are negligible in the system under study.Abbreviations UDPG uridinediphosphoglucose - GDPG guanosinediphosphoglucose - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulphonic acid - DMSO dimethyl-sulfoxide - DNP 2,4-dinitrophenol - DPA days post anthesis     

Fluorescent antibody method Conjugation Of Fluoresceinisothiocyanate With Immune ?-Globulin

ИЗyчaл0C044A; Bлияниe НeкOTOpыX ФaкTopoB (_pH, OTHoшeниe КpacиTeля к бeлкy, КOнпeнTpaция КOMпoнeнTOB, пpиcyTcTBиe opraничecкиX pacTBopиTeлeй B peaгиpyюшeй cмecи, TeMпepaTypa, Bpeмя peaкции) Ha эффeкTиBн0CTъ кOнъюгaции γ-глoбyлинa c флyopecцeинизOTиoциaнaTOM. КoличecTBO КpacиTeля, CBязaннoгo c γ-глoбyлинOM, знaч иTeлънO пoBышaлocъ, кoгдa пoBышaлcя B пpeдeлax 7,0–10,0_pH peaгиpyющeй cмeCи, Toгдa кaк ocTaлъныe фaкTopы нe oкaзыBaли нa мeчeныe бeлки B иccлeдyeмыx пpeдeлax cyщecTBeннoгo Bлияния. БылO ycTaнOBлeнo, чTO cпeцифичecкaя флyopecцeнпия cBязaннoгo кpacиTeля зaмeTнo пoнижaлacъ c пOBышeниeм мeчeннOcTи бeлкOB, a пoэTOмy нaибoлee цeлecooбpaзным пpeдcTaBляeTCя иcпoлъзOBaниe для мeTOдa флyopecцeнTныx aнTиTeл-кOнъюгaTOB c BecoBым COOTнOшeниeм бeлкOB и кpacиTeля, paBным пpиблизиTeльнO 60.     

Nocturnal masseter electromyographic activity of complete denture wearers

doi: 10.1111/j.1741‐2358.2011.00528.x Nocturnal masseter electromyographic activity of complete denture wearers Objective: Collection of normative data on activity patterns of the masseter during sleep in elderly denture wearers by portable electromyography (EMG) recorders. Background: Complete denture wearers might suffer from orofacial pain caused by myoarthropathies of the masticatory system. Indeed, denture instability may precipitate parafunctional habits and consequently muscle soreness and/or temporomandibular pain. Materials and methods: We collected normative masseter EMG data during sleep in 15 complete denture wearers (five women, 10 men, 56–88 years) by portable recorders in their natural environment. Activity periods (AP) were signal portions including subthreshold intervals ≤5 s. Signal amplitude was expressed in per cent of maximum voluntary contraction (%MVC). For this reason, maximum bite force was assessed. Twenty age‐matched dentate controls were also recorded for the maximum bite force. Results: We found 157.2 ± 86.5 AP per night, i.e. 24.0 ± 12.2 AP/h. Mean amplitude was 15.1 ± 4.3%MVC. AP lasted 6.8 ± 4.1 s, and their time integral was 126.3 ± 112.5%MVC?s. Maximum bite force was 116.8 ± 69.6 N in the edentulous, significantly lower than in controls (344.8 ± 150.4 N). Conclusions: Healthy complete denture wearers showed intermittent periods of nocturnal masseter activity of very low intensity and short duration.     

Systematic in vivo RNAi analysis identifies IAPs as NEDD8-E3 ligases

The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling.     

The RootChip: an integrated microfluidic chip for plant science

Studying development and physiology of growing roots is challenging due to limitations regarding cellular and subcellular analysis under controlled environmental conditions. We describe a microfluidic chip platform, called RootChip, that integrates live-cell imaging of growth and metabolism of Arabidopsis thaliana roots with rapid modulation of environmental conditions. The RootChip has separate chambers for individual regulation of the microenvironment of multiple roots from multiple seedlings in parallel. We demonstrate the utility of The RootChip by monitoring time-resolved growth and cytosolic sugar levels at subcellular resolution in plants by a genetically encoded fluorescence sensor for glucose and galactose. The RootChip can be modified for use with roots from other plant species by adapting the chamber geometry and facilitates the systematic analysis of root growth and metabolism from multiple seedlings, paving the way for large-scale phenotyping of root metabolism and signaling.     

Fully Automated Enhanced Tumor Compartmentalization: Man vs. Machine Reloaded

ObjectiveComparison of a fully-automated segmentation method that uses compartmental volume information to a semi-automatic user-guided and FDA-approved segmentation technique.MethodsNineteen patients with a recently diagnosed and histologically confirmed glioblastoma (GBM) were included and MR images were acquired with a 1.5 T MR scanner. Manual segmentation for volumetric analyses was performed using the open source software 3D Slicer version 4.2.2.3 (www.slicer.org). Semi-automatic segmentation was done by four independent neurosurgeons and neuroradiologists using the computer-assisted segmentation tool SmartBrush® (referred to as SB), a semi-automatic user-guided and FDA-approved tumor-outlining program that uses contour expansion. Fully automatic segmentations were performed with the Brain Tumor Image Analysis (BraTumIA, referred to as BT) software. We compared manual (ground truth, referred to as GT), computer-assisted (SB) and fully-automated (BT) segmentations with regard to: (1) products of two maximum diameters for 2D measurements, (2) the Dice coefficient, (3) the positive predictive value, (4) the sensitivity and (5) the volume error.ResultsSegmentations by the four expert raters resulted in a mean Dice coefficient between 0.72 and 0.77 using SB. BT achieved a mean Dice coefficient of 0.68. Significant differences were found for intermodal (BT vs. SB) and for intramodal (four SB expert raters) performances. The BT and SB segmentations of the contrast-enhancing volumes achieved a high correlation with the GT. Pearson correlation was 0.8 for BT; however, there were a few discrepancies between raters (BT and SB 1 only). Additional non-enhancing tumor tissue extending the SB volumes was found with BT in 16/19 cases. The clinically motivated sum of products of diameters measure (SPD) revealed neither significant intermodal nor intramodal variations. The analysis time for the four expert raters was faster (1 minute and 47 seconds to 3 minutes and 39 seconds) than with BT (5 minutes).ConclusionBT and SB provide comparable segmentation results in a clinical setting. SB provided similar SPD measures to BT and GT, but differed in the volume analysis in one of the four clinical raters. A major strength of BT may its independence from human interactions, it can thus be employed to handle large datasets and to associate tumor volumes with clinical and/or molecular datasets ("-omics") as well as for clinical analyses of brain tumor compartment volumes as baseline outcome parameters. Due to its multi-compartment segmentation it may provide information about GBM subcompartment compositions that may be subjected to clinical studies to investigate the delineation of the target volumes for adjuvant therapies in the future.