Photoinactivation of Acinetobacter baumannii and Escherichia coli B by a Cationic Hydrophilic Porphyrin at Various Light Wavelengths

Photodynamic treatment by the cationic TMPyP photosensitizer was undertaken on the multiple antibiotic-resistant bacteria Acinetobacter baumannii and Escherichia coli. Total eradication of the bacterial cultures was determined immediately after initiation of illumination when these bacteria were treated with 5, 10, 15, 20-tetra (4-N methylpyridyl)porphine (TMPyP) at a concentration of 29.4 μmol/L and illuminated by blue, green, or red light. Total eradication of both bacteria was obtained also after treatment of bacterial cultures with 3.7 μmol/L TMPyP and illumination with blue light (400–450 nm). On the other hand, an 8- or 16- to 20-fold higher light intensity, respectively, was required for total eradication upon illumination with green (480–550 nm) or red light (600–700 nm). A 407-nm blue light only 7 and 9 joules/cm², respectively, was needed for total eradication of both bacteria even at a concentration of 3.7 μmol/L TMPyP. X-ray-linked microanalysis demonstrated loss of potassium and a flood of sodium and chloride into the cells, indicating serious damage to the cytoplasmic membrane. Transmission electron microscopy (TEM) revealed structural changes and damage to the membrane of treated E. coli. In A. baumannii-treated cells, mesosomes and black dots that resemble aggregation of polyphosphate polymers could be seen. DNA breakage appeared only after a long period of illumination, when the bacterial cell was no longer viable. It can be concluded that cytoplasmic membrane damage and not DNA breakage is the major cause for bacterial death upon photosensitization. Received: 13 October 2000 / Accepted: 17 November 2000     

Electron crystallography of aquaporins

Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.     

Fluid shear stress and the vascular endothelium: for better and for worse

As blood flows, the vascular wall is constantly subjected to physical forces, which regulate important physiological blood vessel responses, as well as being implicated in the development of arterial wall pathologies. Changes in blood flow, thus generating altered hemodynamic forces are responsible for acute vessel tone regulation, the development of blood vessel structure during embryogenesis and early growth, as well as chronic remodeling and generation of adult blood vessels. The complex interaction of biomechanical forces, and more specifically shear stress, derived by the flow of blood and the vascular endothelium raise many yet to be answered questions:How are mechanical forces transduced by endothelial cells into a biological response, and is there a "shear stress receptor"?Are "mechanical receptors" and the final signaling pathways they evoke similar to other stimulus-response transduction systems?How do vascular endothelial cells differ in their response to physiological or pathological shear stresses?Can shear stress receptors or shear stress responsive genes serve as novel targets for the design of diagnostic and therapeutic modalities for cardiovascular pathologies?The current review attempts to bring together recent findings on the in vivo and in vitro responses of the vascular endothelium to shear stress and to address some of the questions raised above.     

Simple procedure for purification of diphtheria toxin and separation of its fragments by hydrophobic chromatography

Diphtheria toxin and fragment B bind to hydrocarbon-coated agaroses. Fragment A of the toxin is not adsorbed to such resins. Using Seph-C₄, the toxin and fragment B can be eluted from the column after adsorption by increasing the ionic strength of the eluent. The toxin is also eluted from the Seph-C₆ column, but fragment B is eluted only in the denatured form. Purification of the toxin can be achieved simply by passing the growth medium supernatant through a small size Seph-C₆ column and eluting the toxin by 0.1 m NaCl. The fragments of diphtheria toxin obtained after mild trypsin treatment can be separated purely on a Seph-C₄ column. The hydrophobic chromatography system may thus serve as a tool for purification of the toxin and its fragments: it may also be useful in large-scale preparations.     

Fragment-based phase extension for three-dimensional structure determination of membrane proteins by electron crystallography

In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.     

Dual role of SLP-76 in mediating T cell receptor-induced activation of phospholipase C-gamma1

Phospholipase C-gamma1 (PLC-gamma1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-gamma1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-gamma1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-gamma1 GEM recruitment, but were required for PLC-gamma1 phosphorylation at Tyr(783). Thus, GEM recruitment can be insufficient for full activation of PLC-gamma1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-gamma1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-gamma1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-gamma1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.     

Automatic noninvasive measurement of systolic blood pressure using photoplethysmography

Background

Systolic blood flow has been simulated in the abdominal aorta and the superior mesenteric artery. The simulations were carried out using two different computational hemodynamic methods: the finite element method to solve the Navier Stokes equations and the lattice Boltzmann method.

Results

We have validated the lattice Boltzmann method for systolic flows by comparing the veloCity and pressure profiles of simulated blood flow between methods. We have also analyzed flow-specific characteristics such as the formation of a vortex at curvatures and traces of flow.

Conclusion

The lattice Boltzmann Method is as accurate as a Navier Stokes solver for computing complex blood flows. As such it is a good alternative for computational hemodynamics, certainly in situation where coupling to other models is required.     

BMI may overestimate the prevalence of obesity among women of lower socioeconomic status

Objective: Our objective was to examine gender differences in height and weight associated with socioeconomic status (SES) and the consequent effect on body mass index in a multiethnic society. Research Methods and Procedures: A cross‐sectional study, the First Israeli National Health and Nutrition Survey, was performed on a representative population sample of 3246 adults 25 to 64 years of age, between the years 1999 to 2001. Height and weight were measured, and BMI and other weight‐height indices were calculated. SES was assessed by income and education. Results: Age‐adjusted height was significantly lower at lower levels of SES among both women and men (p < 0.001). As opposed to men, women of lower SES were heavier than those of higher SES, and the mean age‐adjusted weight was 4.6 kg higher among those of lower SES (p < 0.001). Thus, using the standard index of BMI, the prevalence of obesity was significantly higher among shorter women. Discussion: In this group of Israeli adults, the unfavorable effect of low SES on BMI was evident among women, partly due to their decreased height combined with increased weight common in this socioeconomic sector. Since BMI is only partly independent of height, it may overestimate the prevalence of obesity among women of lower SES. Alternative measures for classifying obesity in the lower SES groups that put less emphasis on height may be considered and studied.     

Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size

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Isolation and characterization of heat-modifiable proteins from the outer membrane of Porphyromonas asaccharolytica and Acinetobacter baumannii

Active porins were isolated and purified from the outer membranes of the gram-negative anaerobic rod Porphyromonas asaccharolytica and the aerobic coccobacillus Acinetobacter baumannii. The porins from both bacteria appear to be monomers when isolated and purified. Both porins exhibited decreased mobility on SDS-PAGE after boiling for 10 min in the sample buffer. After heating, their molecular weight is estimated at 43 kDa while without heating they run as proteins with a molecular weight of approximately 37 kDa. Due to their characteristic heat-modifiability, these proteins were named HMP (heat-modifiable protein)-P. asaccharolytica and HMP-A. baumannii. Amino acid analysis revealed both porins to be hydrophilic proteins. These proteins have been shown to be active in transporting sugars when incorporated into liposomes. The permeability of both porins for L-arabinose was less than that produced by the porin of Escherichia coli B. Permeability to high molecular weight disaccharides was lower than for small monosaccharides. Western blot analysis did not reveal any antigenic cross reaction between HMP-A. baumannii and the HMP-P. asaccharolytica. The results obtained in this study confirm that although these heat-modifiable proteins are pore forming proteins and have similar activity they differ in their antigenicity.