Alterations in expression of myosin and myosin light chain kinases in response to vascular injury

Histochemical analysis ofballoon-injured rat carotid arteries revealed a coordinated expressionof nonmuscle myosin heavy chain-A and -B (NM-A and NM-B) in response toinjury. Expression of these nonmuscle myosin forms shifts from themedia to the adventitia and intima. In contrast, expression of smoothmuscle myosin heavy chain-1 (SM-1) within the media is not altered,whereas smooth muscle myosin heavy chain-2 (SM-2) expression declines.Western blotting shows a statistically significant increase inexpression of NM-A that occurs within 6 h in response to carotidinjury, suggesting this myosin form may be an appropriate experimental marker for proliferating, migrating cells in injured vessels. Nooverall change in the relative expression level of NM-B was detected,suggesting that compensatory declines in media expression are balancedby increases in the intima and adventitia. Expression of SM-1 did notchange in response to injury, whereas the expression of SM-2significantly declined between 24 h and 7 days. Expression ofmyosin light chain kinase is also negatively regulated, and the declinein its expression parallels downregulation of SM-2.

     

Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins.

The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid. High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top. Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions. The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones. It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight. By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.     

Molecular Mechanisms of Polymyxin B-Membrane Interactions: Direct Correlation Between Surface Charge Density and Self-Promoted Transport

We have studied the interaction of the polycationic peptide antibiotic polymyxin B (PMB) with asymmetric planar bilayer membranes via electrical measurements. The bilayers were of different compositions, including those of the lipid matrices of the outer membranes of various species of Gram-negative bacteria. One leaflet, representing the bacterial inner leaflet, consisted of a phospholipid mixture (PL; phosphatidylethanolamine, -glycerol, and diphosphatidylglycerol in a molar ratio of 81:17:2). The other (outer) leaflet consisted either of lipopolysaccharide (LPS) from deep rough mutants of PMB-sensitive (Escherichia coli F515) or -resistant strains (Proteus mirabilis R45), glycosphingolipid (GSL-1) from Sphingomonas paucimobilis IAM 12576, or phospholipids (phosphatidylglycerol, diphytanoylphosphatidylcholine). In all membrane systems, the addition of PMB to the outer leaflet led to the induction of current fluctuations due to transient membrane lesions. The minimal PMB concentration required for the induction of the lesions and their size correlated with the charge of the lipid molecules. In the membrane system resembling the lipid matrix of a PMB-sensitive strain (F515 LPS/PL), the diameters of the lesions were large enough (d= 2.4 nm ± 8%) to allow PMB molecules to permeate (self-promoted transport), but in all other systems they were too small. A comparison of these phenomena with membrane effects induced by detergents (dodecyltriphenylphosphonium bromide, dodecyltrimethylammonium bromide, sodiumdodecylsulfate) revealed a detergent-like mechanism of the PMB-membrane interaction. Received: 16 September 1997/Revised: 25 November 1997     

The Extracellular Domains of IgG1 and T Cell-Derived IL-4/IL-13 Are Critical for the Polyclonal Memory IgE Response In Vivo

IgE-mediated activation of mast cells and basophils contributes to protective immunity against helminths but also causes allergic responses. The development and persistence of IgE responses are poorly understood, which is in part due to the low number of IgE-producing cells. Here, we used next generation sequencing to uncover a striking overlap between the IgE and IgG1 repertoires in helminth-infected or OVA/alum-immunized wild-type BALB/c mice. The memory IgE response after secondary infection induced a strong increase of IgE⁺ plasma cells in spleen and lymph nodes. In contrast, germinal center B cells did not increase during secondary infection. Unexpectedly, the memory IgE response was lost in mice where the extracellular part of IgG1 had been replaced with IgE sequences. Adoptive transfer studies revealed that IgG1⁺ B cells were required and sufficient to constitute the memory IgE response in recipient mice. T cell-derived IL-4/IL-13 was required for the memory IgE response but not for expansion of B cells from memory mice. Together, our results reveal a close relationship between the IgE and IgG1 repertoires in vivo and demonstrate that the memory IgE response is mainly conserved at the level of memory IgG1⁺ B cells. Therefore, targeting the generation and survival of allergen-specific IgG1⁺ B cells could lead to development of new therapeutic strategies to treat chronic allergic disorders.     

Cg2091 encodes a polyphosphate/ATP-dependent glucokinase of Corynebacterium glutamicum

The Corynebacterium glutamicum gene cg2091 is encoding a polyphosphate (PolyP)/ATP-dependent glucokinase (PPGK). Previous work demonstrated the association of PPGK to PolyP granules. The deduced amino acid sequence of PPGK shows 45% sequence identity to PolyP/ATP glucomannokinase of Arthrobacter sp. strain KM and 50% sequence identity to PolyP glucokinase of Mycobacterium tuberculosis H37Rv. PPGK from C. glutamicum was purified from recombinant Escherichia coli. PolyP was highly preferred over ATP and other NTPs as substrate and with respect to the tested PolyPs differing in chain length; the protein was most active with PolyP₇₅. Gel filtration analysis revealed that PolyP supported the formation of homodimers of PPGK and that PPGK was active as a homodimer. A ppgK deletion mutant (ΔppgK) showed slowed growth in minimal medium with maltose as sole carbon source. Moreover, in minimal medium containing 2 to 4% (w/v) glucose as carbon source, ΔppgK grew to lower final biomass concentrations than the wild type. Under phosphate starvation conditions, growth of ΔppgK was reduced, and growth of a ppgK overexpressing strain was increased as compared to wild type and empty vector control, respectively. Thus, under conditions of glucose excess, the presence of PPGK entailed a growth advantage.     

Coupling GIS and LCA for biodiversity assessments of land use

Purpose  

Geospatial details about land use are necessary to assess its potential impacts on biodiversity. Geographic information systems (GIS) are adept at modeling land use in a spatially explicit manner, while life cycle assessment (LCA) does not conventionally utilize geospatial information. This study presents a proof-of-concept approach for coupling GIS and LCA for biodiversity assessments of land use and applies it to a case study of ethanol production from agricultural crops in California.     

A review of non-invasive optical-based image analysis systems for continuous bioprocess monitoring

To observe and control cultivation processes, optical sensors are used increasingly. Important variables for controlling such processes are cell count, cell size distribution and the morphology of cells. Among turbidity measurement methods, imaging procedures are applied for determining these process values. A disadvantage of most previously developed imaging procedures is that they are only available offline, which requires sampling. On the other hand, available imaging inline probes can only deliver a limited number of process values so far. This contribution gives an overview of optical procedures for the inline determination of cell count, cell size distribution and other variables. In particular, by in situ microscopy, an imaging procedure will be described, which allows the determination of direct and non-direct cell variables in real time without sampling.     

Multispectral imaging of ion transport in neutral carrier-based cation-selective membranes.

BACKGROUND: High-resolution spectroscopic imaging of the cross section of ion-selective membranes during real-time electrochemical measurements is termed spectroelectrochemical microscopy (SpECM). SpECM is aimed for optimizing the experimental conditions in mass transport controlled ion-selective electrode (ISE) membranes for improved detection limit. METHODS: The SpECM measurements are performed in a thin layer electrochemical cell. The key element of the cell is a membrane strip spacer ring assembly which forms a two compartment electrochemical cell. The cell is placed onto the stage of a microscope and the membrane strip is positioned in the center of the field of view. A slice of the image is focused onto the entrance slit of the imaging spectrometer. RESULTS: SpECM has been used for the determination of the diffusion coefficients of different membrane ingredients and for the quantitative assessment of the charged site concentrations in ISE membranes and membrane plasticizers. In addition, changes in the concentration profiles of the ionophore (free and complexed) and charged mobile sites inside the ISE membranes are documented upon the application of large external voltages. CONCLUSIONS: This account demonstrates the power and advantages of SpECM, a multispectral imaging method for investigations of mass transport processes in ISE membranes during electrochemical measurements.