Formation of Meta III during the decay of activated rhodopsin proceeds via Meta I and not via Meta II

Thermal isomerization of the retinal Schiff base C=N double bond is known to trigger the decay of rhodopsin's Meta I/Meta II photoproduct equilibrium to the inactive Meta III state [Vogel, R., Siebert, F., Mathias, G., Tavan, P., Fan, G., and Sheves, M. (2003) Biochemistry 42, 9863-9874]. Previous studies have indicated that the transition to Meta III does not occur under conditions that strongly favor the active state Meta II but requires a residual amount of Meta I in the initial photoproduct equilibrium. In this study we show that the triggering event, the thermal isomerization of the protonated Schiff base, is independent of the presence of Meta II and occurs even under conditions where the transition to Meta II is completely prevented. We have examined two examples in which the transitions from Lumi to Meta I or from Meta I to Meta II are blocked. This was achieved using dry films of rhodopsin and rhodopsin reconstituted into rather rigid lipid bilayers. In both cases, the resulting fully inactive room temperature photoproducts decay specifically by thermal isomerization of the protonated Schiff base C=N double bond to an all-trans 15-syn chromophore isomer, corresponding to that of Meta III. This thermal isomerization becomes less efficient as the conformation of the respective photoproduct approaches that of Meta II and is fully absent in a pure Meta II state. These results indicate that the decay of the Meta I/Meta II photoproduct equilibrium to Meta III proceeds via Meta I and not via Meta II.     

<Emphasis Type="Italic">Babesia microti</Emphasis> Infection in Europe

The majority of babesia infections in Europe are life-threatening and caused by Babesia divergens and B. bovis. Although Babesia microti has been detected in ticks from Switzerland, few if any cases of babesiosis have been caused by B. microti. This first reported case, diagnosed by serology, DNA detection, and microscopy, is additionally interesting because there appears to be coinfection with the Lyme disease organism, Borrelia burgdorferi.     

Kinetics of Choline Uptake into Isolated Rat Forebrain Microvessels: Evidence of Endocrine Modulation

The active uptake of [methyl-3H]choline into isolated rat brain microvessel suspension was studied as a likely guide to the transport of choline across the blood-brain barrier. The method consisted primarily of incubation of the suspension with a fixed concentration of labeled choline in the presence of increasing concentrations of unlabeled choline or any other inhibitor (I) of active uptake, defined as the difference in uptake at 37 degrees and 0 degrees C. From the linear regression of (1/V) against [I], the following values of Vmax (nmol g-1 min-1) and Km (microM) were obtained for choline: 2-month-old males, 10.6 +/- 3.8 and 6.1 +/- 0.9; 3-month old random females, 28.4 +/- 5.9 and 12.6 +/- 4.0; females at metaestrus, 17.8 +/- 10.3 and 8.3 +/- 5.0; at diestrus, 31.1 +/- 9.3 and 13.0 +/- 2.6; at proestrus, 54.9 +/- 2.2 and 14.0 +/- 1.5; at estrus, 19.2 +/- 2.2 and 2.6 +/- 1.7. The differences between males and random females (p less than 0.018) and between females at proestrus and estrus (p less than 0.005) are significant. It is suggested that these inter- and intrasex variations in choline uptake reflect a dynamic adjustment of supply in accordance with brain demand for choline at the time of assay. Hemicholinium-3 was an effective inhibitor of choline uptake, Ki = 14.0 +/- 8.5 microM; dimethylaminoethanol was much less effective; and imipramine had no measurable effect.     

Adaptation to Short-Term Cataclysmic Events: Flooding in Premodern Riverine Societies

This article examines flooding and resilience in two riverine systems in the premodern Eastern Mediterranean. Flooding represents a distinct type of short-term cataclysmic events (SCEs) because of its frequency and long-term predictability which facilitates societal adaptation. We discuss the sources for premodern floods and their limitations before surveying Mesopotamia and Ancient Egypt as case studies. Both societies are compared with regard to their environment and how it shaped local flood management practices. We argue that although floods caused short-term societal disruption in these societies, they also stimulated the reorganization and regeneration of economic resources. Both Mesopotamian and Egyptian societies systematically managed and mitigated their risks and were, in general, resilient to flooding events.     

Nitroxide Radicals Prevent Metal-aggravated Reperfusion Injury in Isolated Rat Heart

The effects of Cu(II) and the stable nitroxide radical 4-OH-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TPL) on reperfusion injury following global myocardial ischemia have been studied using the isolated rat heart model in the Langendorff configuration. Hearts were equilibrated with Krebs-Henseleit buffer (KH-buffer) for 10 min and subjected to 18 min of normothermic global ischemia. After 20 min reperfusion, hemodynamic parameters recovered as follows: ventricular developed pressure (77%), dP/dt (71%) and -dP/dt (80%), heart rate (91%), and work index (70%). End-diastolic pressure was 16 mm Hg. When 10μM Cu-nitrilotriacetate or Cu-(histidine)₂ was included in the perfusate before, during, and following ischemia, the heart injury was more extensive and the work index only recovered to 17% of the preischemic value. The inclusion of 100μM TPL during reperfusion abolished the copper-induced sensi-tization. In the absence of copper, TPL did not provide any protection against ischemia-reperfusion damage to the heart. The inclusion of 100μM 1, 4-dihydroxy-2, 2, 6, 6-tetramethylpiperidine (TPL-H) during reperfusion, partially abolished the copper-induced sensitization. Since conversion between TPL and TPL-H takes place, the fact that both forms provide protection can increase their protective efficacy.     

Phosphatidylcholine Biosynthesis in the Neuroblastoma-Glioma Hybrid Cell Line NG 108-15: Stimulation by Phorbol Esters

Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High-affinity binding sites (KD = 2 nM for 1-h incubation at 20 degrees C) and low-affinity binding sites (KD = 40 nM) for 125I-labeled alpha-bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high-affinity binding of toxin is 0.56 nM (k1 = 6.3 X 10(-3) min-1 nM-1; k-1 = 3.5 X 10(-3) min-1) at 20 degrees C. Nicotine, d-tubocurarine, and acetylcholine are among the most effective inhibitors of high-affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulfhydryl residues. High-affinity toxin binding sites that have been subjected to reaction with the sulfhydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High-affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues.     

Structural changes in bacteriorhodopsin following retinal photoisomerization from the 13-cis form

Bacteriorhodopsin (BR), a light-driven proton pump in Halobacterium salinarum, accommodates two resting forms of the retinylidene chromophore, the all-trans form (AT-BR) and the 13-cis,15-syn form (13C-BR). Both isomers are present in thermal equilibrium in the dark, but only the all-trans form has proton-pump activity. In this study, we applied low-temperature Fourier-transform infrared (FTIR) spectroscopy to 13C-BR at 77 K and compared the local structure around the chromophore before and after photoisomerization with that in AT-BR. Strong hydrogen-out-of-plane (HOOP) vibrations were observed at 964 and 958 cm(-)(1) for the K state of 13C-BR (13C-BR(K)) versus a vibration at 957 cm(-)(1) for the K state of AT-BR (AT-BR(K)). In AT-BR(K), but not in 13C-BR(K), the HOOP modes exhibit isotope shifts upon deuteration of the retinylidene at C15 and at the Schiff base nitrogen. Whereas the HOOP modes of AT-BR(K) were significantly affected by the mutation of Thr89, this was not the case for the HOOP modes of 13C-BR(K). These observations imply that, while the chromophore distortion is localized near the Schiff base in AT-BR(K), it is located elsewhere in 13C-BR(K). By use of [zeta-(15)N]lysine-labeled BR, we identified the N-D stretching vibrations of the 13C-BR Schiff base (in D(2)O) at 2173 and 2056 cm(-)(1), close in frequency to those of AT-BR. These frequencies indicate strong hydrogen bonding of the Schiff base in 13C-BR, presumably with a water molecule as in AT-BR. In contrast, the N-D stretching vibration appears at 2332 and 2276 cm(-)(1) in 13C-BR(K) versus values of 2495 and 2468 cm(-)(1) for AT-BR(K), suggesting that the rupture of the Schiff base hydrogen bond that occurs in AT-BR(K) does not occur in 13C-BR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller in magnitude for 13C-BR than for AT-BR. These differences in the primary step are possibly related to the absence of light-driven proton pumping by 13C-BR.     

Agonists and partial agonists of rhodopsin: retinal polyene methylation affects receptor activation

Using Fourier transform infrared (FTIR) difference spectroscopy, we have studied the impact of sites and extent of methylation of the retinal polyene with respect to position and thermodynamic parameters of the conformational equilibrium between the Meta I and Meta II photoproducts of rhodopsin. Deletion of methyl groups to form 9-demethyl and 13-demethyl analogues, as well as addition of a methyl group at C10 or C12, shifted the Meta I/Meta II equilibrium toward Meta I, such that the retinal analogues behaved like partial agonists. This equilibrium shift resulted from an apparent reduction of the entropy gain of the transition of up to 65%, which was only partially offset by a concomitant reduction of the enthalpy increase. The analogues produced Meta II photoproducts with relatively small alterations, while their Meta I states were significantly altered, which accounted for the aberrant transitions to Meta II. Addition of a methyl group at C14 influenced the thermodynamic parameters but had little impact on the position of the Meta I/Meta II equilibrium. Neutralization of the residue 134 in the E134Q opsin mutant increased the Meta II content of the 13-demethyl analogue, but not of the 9-demethyl analogue, indicating a severe impairment of the allosteric coupling between the conserved cytoplasmic ERY motif involved in proton uptake and the Schiff base/Glu 113 microdomain in the 9-demethyl analogue. The 9-methyl group appears therefore essential for the correct positioning of retinal to link protonation of the cytoplasmic motif with protonation of Glu 113 during receptor activation.     

Use of FTIR spectroscopy to distinguish between capsular types and capsular quantities in Streptococcus pneumoniae

Fourier transform infrared (FTIR) spectroscopy has shown remarkable ability in distinguishing between bacterial species and identifying bacterial colony structures, when used in tandem with methods such as cluster analysis, principal component analysis, or linear discriminant analysis. The present work was aimed to evaluate the potential of FTIR-microscopy (FTIR-MSP) to distinguish between different serotypes and capsular quantities of Streptococcus pneumoniae. In general, the results obtained have consistently proven that the spectral information at the region 900-1,185 cm(-1) was sufficient to distinguish between various pneumococcal serotypes. Moreover, the method was able to differentiate between S. pneumoniae phase variants on the basis of their relative carbohydrate content. The unsupervised cluster analysis of the samples showed differences, not only in the carbohydrate content, but also in the region 1,350-1,480 cm(-1), which is dominated by absorptions due to lipids and phospholipids. This approach proved to be useful for the distinction between S. pneumoniae serotypes and between phase variants, which were shown to acquire different pathogenic capacity.