Correlation between Raman and X-ray crystallography data of (Pro-Pro-Gly)10

Model biopolymers are powerful tools to guide the interpretation of physical properties in complex systems. (Pro-Pro-Gly)(10), (PPG)(10), is a collagen-model peptide, whose structure is known at high resolution. Herein, Raman microscopy data of (PPG)(10) powders and single crystals are reported. The spectra interpretation leads to an accurate assignment of three well-resolved amide bands corresponding to the three peptide bonds (PP, PG and GP) present in the (PPG)(10) structure. These data together with the availability of torsional angles phi and psi derived from the high-resolution crystal structure, provide the opportunity to test the validity of theoretical equations for the calculation of non-canonical amide III bands and represent a reference for theoretical calculations of vibrational spectra and for polyproline II detection in complex proteins. Spectroscopic data do not support the indication of two distinct and equally populated up and down conformations of the pyrrolidine rings observed in the (PPG)(10) crystal structure.     

Imaging of calcium transients in skeletal muscle fibers.

Epifluorescence images of Ca2+ transients elicited by electrical stimulation of single skeletal muscle fibers were studied with fast imaging techniques that take advantage of the large fluorescence signals emitted at relatively long wavelengths by the dyes fluo-3 and rhod-2 in response to binding of Ca2+ ions, and of the suitable features of a commercially available CCD video camera. The localized release of Ca2+ in response to microinjection of InsP3 was also monitored to demonstrate the adequate space and time resolutions of the imaging system. The time resolution of the imager system, although limited to the standard video frequency response, still proved to be adequate to investigate the fast Ca2+ release process in skeletal muscle fibers at low temperatures.     

Cyanotoxins in small artificial dams in Kenya utilised for cage fish farming – a threat to local people?

Nine small artificial dams located in different climatic regions of Kenya were studied. The local communities use the stored water for various purposes, such as irrigation, domestic use, watering of livestock and cage fish farming. Such intense use is commonly accompanied by eutrophication, including fast growth of cyanobacteria, which at times produce cyanotoxins threatening human and animal life. We studied the pelagic community, analysed abiotic variables and identified microcystins by means of high performance liquid chromatography and ELISA kits at monthly intervals over a period of one year. Mass spectrometry (MALDI-TOF MS) was used to identify structural variants of microcystins by their protonated masses (M + H). Three dams contained microcystins, with the highly toxic Microcystin-LR being identified as the most prominent substance. Cell content of the toxin varied from 7.2 to 686.7 fg cell^?1. Basic limnological variables that indicate the probability of toxin presence were also recorded. Non-parametric Mann–Whitney U-test revealed significant differences in soluble reactive phosphorous, nitrate-N, water depth, total hardness and post-Nauplii stages sampled between toxin-producing and non-toxin-producing dams. Although most of the samples did not contain high amounts of cyanobacteria, the cyanotoxin-problem was evident, suggesting the need for regular cyanotoxin monitoring programs.     

Current issues in invertebrate phototransduction

Investigation of phototransduction in invertebrate photoreceptors has revealed many physiological and biochemical features of fundamental biological importance. Nonetheless, no complete picture of phototransduction has yet emerged. In most known cases, invertebrate phototransduction involves polyphosphoinositide and cyclic GMP (cGMP) intracellular biochemical signaling pathways leading to opening of plasma membrane ion channels. Excitation is Ca²⁺-dependent, as are adaptive feedback processes that regulate sensitivity to light. Transduction takes place in specialized subcellular regions, rich in microvilli and closely apposed to submicrovillar membrane systems. Thus, excitation is a highly localized process. This article focuses on the intracellular biochemical signaling pathways and the ion channels involved in invertebrate phototransduction. The coupling of signaling cascades with channel activation is not understood for any invertebrate species. Although photoreceptors have features that are common to most or all known invertebrate species, each species exhibits unique characteristics. Comparative electrophysiological, biochemical, morphological, and molecular biological approaches to studying phototransduction in these species lead to fundamental insights into cellular signaling. Several current controversies and proposed phototransduction models are evaluated.     

Influence of supplemental ultraviolet-B on indoleacetic acid and calmodulin in the leaves of rice (Oryza sativa L.)

IR68 and Dular rice cultivars were grown under ambient, 13.0 (simulating 20% ozone depletion) and 19.1 (simulating 40% ozone depletion) kJ m^-2 day^-1 of biologically effective ultraviolet-B (UV-B_BE) for 4 weeks. Plant height and leaf area were significantly reduced by supplemental UV-B_BE radiation. Greater reduction in leaf area than of plant height was observed. A decrease in indole-3-acetic acid (IAA) content and increase in peroxidase and IAA oxidase activities of UV-B treated plants in both cultivars were observed compared with ambient control. Calmodulin content also decreased after plants were treated with high supplemental UV-B for two weeks and medium UV-B treatment for four weeks. The results indicated that peroxidase and IAA oxidase activities in rice leaves were stimulated by supplemental UV-B, resulting in the destruction of IAA which in turn may cause inhibition of rice leaf growth. Although the mechanism is unclear, calmodulin is most likely involved in leaf growth.     

Mechanisms of inorganic carbon acquisition in Gracilaria gaditana nom. prov. (Rhodophyta)

The mechanisms for acquisition of dissolved inorganic carbon (DIC) in the red macroalga Gracilaria gaditana nom. prov. have been investigated. The capacity for HCO₃ ⁻ use by an extracellular carbonic anhydrase (CA; EC 4.2.1.1), and by an anion exchanger with similar properties to that of red blood cells (AE1), has been quantified. It was illustrated by comparing O₂ evolution rates with those theoretically supported by CO₂, as well as by photosynthesis-pH curves. Both external and internal CA, and a direct uptake were involved in HCO₃ ⁻ use, since photosynthesis and pH evolution were affected by acetazolamide, 6-ethoxyzolamide (inhibitors of external and total CA, respectively) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate, (DIDS; an inhibitor of HCO₃ ⁻ exchanger protein). The activity of the external CA was detected by a potentiometric method and by an alternative method based on the study of O₂ evolution after addition of CO₂ and acetazolamide. The latter method showed a residual photosynthetic rate due to direct HCO₃ ⁻ use. Inhibitors caused a reduction in the pH compensation points in pH-drift experiments. The CO₂ compensation points for photosynthesis increased when the inhibitors were applied, indicating a suppresion of the pathways involved in the carbon-concentrating mechanism. The net photosynthesis rates as a function of DIC concentration displayed a biphasic pattern that could be supported by the occurrence of the two mechanisms of HCO₃ ⁻ use. The potential contribution to HCO₃ ⁻ acquisition by the DIDS-sensitive mechanism was higher after culturing at a high pH. Our results suggest that the HCO₃ ⁻ use by Gracilaria gaditana is carried out by the two DIC uptake mechanisms. These operate simultaneously with different affinities for DIC, the indirect HCO₃ ⁻ use by an external CA activity being the main pathway. The presence of a carbon-concentrating mechanism confers eco-physiological advantages in a fluctuating ecosystem subjected daily to high pHs and low DIC concentrations. Received: 3 July 1998 / Accepted: 30 November 1998     

Localization of the K+ lock-In and the Ba2+ binding sites in a voltage-gated calcium-modulated channel. Implications for survival of K+ permeability.

Using Ba2+ as a probe, we performed a detailed characterization of an external K+ binding site located in the pore of a large conductance Ca2+-activated K+ (BKCa) channel from skeletal muscle incorporated into planar lipid bilayers. Internal Ba2+ blocks BKCa channels and decreasing external K+ using a K+ chelator, (+)-18-Crown-6-tetracarboxylic acid, dramatically reduces the duration of the Ba2+-blocked events. Average Ba2+ dwell time changes from 10 s at 10 mM external K+ to 100 ms in the limit of very low [K+]. Using a model where external K+ binds to a site hindering the exit of Ba2+ toward the external side (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-568), we calculated a dissociation constant of 2.7 mircoM for K) at this lock-in site. We also found that BK(Ca) channels enter into a long-lasting nonconductive state when the external [K+] is reduced below 4 microM using the crown ether. Channel activity can be recovered by adding K+, Rb+, Cs+, or NH4+ to the external solution. These results suggest that the BK(Ca) channel stability in solutions of very low [K+] is due to K+ binding to a site having a very high affinity. Occupancy of this site by K+ avoids the channel conductance collapse and the exit of Ba2+ toward the external side. External tetraethylammonium also reduced the Ba2+ off rate and impeded the channel from entering into the long-lasting nonconductive state. This effect requires the presence of external K+. It is explained in terms of a model in which the conduction pore contains Ba2+, K+, and tetraethylammonium simultaneously, with the K+ binding site located internal to the tetraethylammonium site. Altogether, these results and the known potassium channel structure (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69-77) imply that the lock-in site and the Ba2+ sites are the external and internal ion sites of the selectivity filter, respectively.