Mathematical Modeling and Fluorescence Imaging to Study the Ca2+ Turnover in Skinned Muscle Fibers

AbstractA mathematical model was developed for the simulation of the spatial and temporal time course of Ca2+ ion movement in caffeine-induced calcium transients of chemically skinned muscle fiber preparations. Our model assumes cylindrical symmetry and quantifies the radial profile of Ca2+ ion concentration by solving the diffusion equations for Ca2+ ions and various mobile buffers, and the rate equations for Ca2+ buffering (mobile and immobile buffers) and for the release and reuptake of Ca2+ ions by the sarcoplasmic reticulum (SR), with a finite-difference algorithm. The results of the model are compared with caffeine-induced spatial Ca2+ transients obtained from saponin skinned murine fast-twitch fibers by fluorescence photometry and imaging measurements using the ratiometric dye Fura-2. The combination of mathematical modeling and digital image analysis provides a tool for the quantitative description of the total Ca2+ turnover and the different contributions of all interacting processes to the overall Ca2+ transient in skinned muscle fibers. It should thereby strongly improve the usage of skinned fibers as quantitative assay systems for many parameters of the SR and the contractile apparatus helping also to bridge the gap to the intact muscle fiber

Digestive enzyme activities and gastrointestinal fermentation in wood-eating catfishes

To determine what capabilities wood-eating and detritivorous catfishes have for the digestion of refractory polysaccharides with the aid of an endosymbiotic microbial community, the pH, redox potentials, concentrations of short-chain fatty acids (SCFAs), and the activity levels of 14 digestive enzymes were measured along the gastrointestinal (GI) tracts of three wood-eating taxa (Panaque cf. nigrolineatus “Marañon”, Panaque nocturnus, and Hypostomus pyrineusi) and one detritivorous species (Pterygoplichthys disjunctivus) from the family Loricariidae. Negative redox potentials (−600 mV) were observed in the intestinal fluids of the fish, suggesting that fermentative digestion was possible. However, SCFA concentrations were low (<3 mM in any intestinal region), indicating that little GI fermentation occurs in the fishes’ GI tracts. Cellulase and xylanase activities were low (<0.03 U g−1), and generally decreased distally in the intestine, whereas amylolytic and laminarinase activities were five and two orders of magnitude greater, respectively, than cellulase and xylanase activities, suggesting that the fish more readily digest soluble polysaccharides. Furthermore, the Michaelis–Menten constants (Km) of the fishes’ β-glucosidase and N-acetyl-β-d-glucosaminidase enzymes were significantly lower than the Km values of microbial enzymes ingested with their food, further suggesting that the fish efficiently digest soluble components of their detrital diet rather than refractory polysaccharides. Coupled with rapid gut transit and poor cellulose digestibility, the wood-eating catfishes appear to be detritivores reliant on endogenous digestive mechanisms, as are other loricariid catfishes. This stands in contrast to truly “xylivorous” taxa (e.g., beavers, termites), which are reliant on an endosymbiotic community of microorganisms to digest refractory polysaccharides

Pulsar-driven Jets in Supernovae, Gamma-Ray Bursts, and the Universe

The bipolarity of Supernova 1987A can be understood through its very early light curve observed from the CTIO 0.4-m telescope and IUE FES, and following speckle observations of the `Mystery Spot' by two groups. These indicate a highly directional beam/jet of light/particles, with initial collimation factors in excess of 10,000 and velocities in excess of 0.95 c, as an impulsive event of up to 1e-5 solar masses interacting with circumstellar material. These can be produced by a model proposed in 1972, by Bolotovskii and Ginzburg, which employs pulsar emission from polarization currents induced/(modulated faster than c) beyond the pulsar light cylinder by the periodic electromagnetic field (supraluminally induced polarization currents -- SLIP). SLIP accounts for the disruption of progenitors in supernova explosions and their anomalous dimming at cosmological distances, jets from Sco X-1 and SS 433, the lack/presence of intermittent pulsations from the high/low luminosity low mass X-ray binaries, long/short gamma-ray bursts and predicts that their afterglows are the pulsed optical/near infrared emission associated with these pulsars. SLIP may also account for the TeV e+/e- results from PAMELA and ATIC, the WMAP `Haze'/Fermi `Bubbles', and the r-process. SLIP jets from SNe of the first stars may allow galaxies to form without dark matter, and explain the peculiar, non-gravitational motions observed from pairs of distant galaxies by GALEX.Comment: This article has been published in the open source journal, Advances in Astronomy: http://www.hindawi.com/journals/aa/2012/898907 This arXiv version is out of date. arXiv admin note: substantial text overlap with arXiv:0909.2604 (Note: but less so by v2, Also Brook Sandford in Ackn. -JM

The astrophysics of energetic x-ray binaries

This thesis is a study of the X-ray binary Cygnus X-3, and related objects, using primarily observations at radio, sub-rom and infrared wavelengths. I find the emission mechanism to be synchrotron in the radio and sub-mm, while the infrared emission is thermal from the hot wind. The upper limit to the synchrotron emission is interpreted as being due to spectral ageing and implies a magnetic field strength of 7 T at a distance of 700 Rʘ from the centre of the system. The nature of the companion star in Cyg X-3 was investigated by midinfrared observations using the ISO satellite. I find that the spectrum shows a steady decrease and flattening at longer wavelengths which is consist ant with a standard wind emission. Thus the complete quiescent spectrum from Cyg X-3 shows two different emission mechanisms and covers a full 5 decades of frequency. High sensitivity and high time resolution radio photometry, taken during a minor flare period, reveal rapid (10 minute) increases and decreases in intensity which severely constrain the size of plasmons. Brightness temperatures of typically 1010K are found. I also consider models for the superluminal expansion and contraction of the Cyg X-3 source, observed on a milli-arcsecond scale by LIewell et al. (1998). The elliptical shape and the superluminal contraction are particularly hard to explain. Models involving photon beams illuminating shells, or propagating photon patterns are the most plausible. A thorough survey for maser emission from these sources has produced strong upper limits and improved our understanding of the circumstellar environment ofCyg X-3. This thesis has probed the radio-jet X-ray binaries, and made significant advances, opening up more questions about the nature of these sources and new areas of research

Gut microbiota mediates clearance of C. rodentium by phagocytes

Infections with enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli (E. coli) are a major cause of diarrhoea in the developing world. Asymptomatic EPEC-carriers are thought to be an important reservoir for these pathogens since they excrete pathogens unknowingly and thereby infect other people and spread disease. With C. rodentium we were able to mimic long-termcarrier situations in mice without gut microbiota. This enabled us to investigate how commensal bacteria initiate clearance of enteropathogens. During this work, we could show that a healthy gut microbiota influences the expression of inflammatory factors like IL-17A and consequently CXCL2 and ICAM- 1, thus mediating migration of neutrophils into the colon. Furthermore, we found that commensal bacteria enhance the phagocytic activity of neutrophils and in parallel elevate colonic IgG levels, subsequently leading to an efficient uptake and killing of C. rodentium. However, our findings demonstrate that in absence of gut microbiota these immune responses are impaired. As a consequence, this leads to a lifelong persistence of C. rodentium, which adapt a commensal-like phenotype at late time points of infection. Importantly, we here show that impaired immune responses can be restored by the transfer of gut microbiota, thus enabling clearance of the enteropathogen. Although many prior investigations have focused on infection with C. rodentium, it was not understood how gut microbiota induces clearance of the enteropathogen. The findings from this work might provide information for microbiota mediated preventive and therapeutic treatments of asymptomatic EPEC-carriers

Chemiluminescence detection in flow injection analysis and liquid chromatography

This thesis describes the development of chemiluminescence procedures for the detection of some organic compounds of interest to the oil industry.Water soluble tertiary amines were determined using flow-injection analysis with chemiluminescence detection. The chemiluminescence was generated by reacting the aqueous tertiary amine solution with aqueous sodium hypochlorite at pH 11.0, in the presence of rhodamine B, which sensitizes the emission. The simplex optimized flow-injection manifold was used to determine trimethylamine, triethylamine and tripropylamine in water and sea water. A standard-addition calibration procedure was used to determine trimethylamine in an industrial scrubbing medium.The chemiluminescence of tertiary amines was extended to the detection of the non-ionic surfactant, Nonidet AT 85, which contains a tertiary amine group. The flow-injection manifold was simplex optimised for this analyte in sea water. The surfactant was also determined in aqueous extracts of marine sediment. Calibration was by a standard addition procedure.Primary amines were determined by flow-injection analysis after fluorimetric derivatization with o-phthalaldehyde and 2-mercaptoethanol in non-aqueous media. The fluorescent derivative was detected by excitation using the peroxyoxalate chemi-excitation reaction.A pre-column fluorimetric derivatization procedure was developed for the determination of carboxylic acids in non-aqueous media. Straight chain acids over the range C2 to C20 and benzoic acid were derivatized with 9-anthracene methanol, using dicyclohexylcarbodiimide (DCC) as a coupling reagent to yield fluorescent esters. Separation was carried out by reversed-phase high-performance liquid chromatography and the peroxyoxalate chemi-excitation reaction was used to detect the fluorescent species.An automated flow-injection manifold was developed, including computerised data acquisition and automated sample uptake and injection. This enabled the system to operate whilst unattended. The precision of the data was higher than that obtained with the manual system.Finally, a flow-injection manifold was designed which incorporated a monochromator between the flow cell and the detector, thus enabling chemiluminescence spectra to be obtained

Selective targeting of immunoproteasome subunit LMP7 prevents colitis-associated carcinogenesis

Chronic inflammation is a well-known risk factor for the development of colonic tumorigenesis. In this study, we show that the immunoproteasome (iP) subunit LMP7 plays a crucial role in the progression of colitis-associated carcinogenesis (CAC). The activity and function of the iP complex has been extensively studied in the context of MHC class I-coupled antigen presentation, inflammation and infectious diseases. Here we show that the absence of LMP7 exerts a protective effect, since the LMP7-deficient mice fail to develop a full scale of carcinogenesis after CAC induction with AOM/DSS treatment. Our findings demonstrate that LMP7 deficiency results in reduced protein expression of pro-tumorigenic cytokines IL-6 and TNF-α in the colon after AOM/DSS treatment. Additionally, LMP7-deficient mice also exhibit significantly decreased mRNA levels of pro-tumorigenic chemokines CXCL1, CXCL2 and CXCL3, as well as cell adhesion molecule VCAM-1, thus highlighting the role of LMP7 in regulation of these pro-tumorigenic factors. The net result of the lack of pro-tumorigenic cytokines and chemokines is an impaired recruitment and subsequent activity of tumour-associated neutrophils (TANs) in the colonic lamina propria. Furthermore, we show that the absence or pharmacological inhibition of LMP7 and the consequent blockade of NF-κB, abrogated the production IL-17A, which possesses a potent carcinogenic activity in the gut. Moreover, in vivo administration of a selective LMP7 inhibitor „ONX-0914‟ during CAC induction reduced the tumour incidence in wild-type (WT) mice. Taken together, we identify the iP complex as a crucial mediator of inflammation-driven colon carcinogenesis and we also propose LMP7 as a potential therapeutic target for CAC to limit the ongoing tumorigenesis in inflammatory bowel disease (IBD) patients