Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport

Recent 3-D structures of several intermediates in the photocycle of bacteriorhodopsin (bR) provide a detailed structural picture of this molecular proton pump in action. In this review, we describe the sequence of conformational changes of bR following the photoisomerization of its all-trans retinal chromophore, which is covalently bound via a protonated Schiff base to Lys216 in helix G, to a 13-cis configuration. The initial changes are localized near the protein's active site and a key water molecule is disordered. This water molecule serves as a keystone for the ground state of bR since, within the framework of the complex counter ion, it is important both for stabilizing the structure of the extracellular half of the protein, and for maintaining the high pK_a of the Schiff base (the primary proton donor) and the low pK_a of Asp85 (the primary proton acceptor). Subsequent structural rearrangements propagate out from the active site towards the extracellular half of the protein, with a local flex of helix C exaggerating an early movement of Asp85 towards the Schiff base, thereby facilitating proton transfer between these two groups. Other coupled rearrangements indicate the mechanism of proton release to the extracellular medium. On the cytoplasmic half of the protein, a local unwinding of helix G near the backbone of Lys216 provides sites for water molecules to order and define a pathway for the reprotonation of the Schiff base from Asp96 later in the photocycle. A steric clash of the photoisomerized retinal with Trp182 in helix F drives an outward tilt of the cytoplasmic half of this helix, opening the proton transport channel and enabling a proton to be taken up from the cytoplasm. Although bR is the first integral membrane protein to have its catalytic mechanism structurally characterized in detail, several key results were anticipated in advance of the structural model and the general framework for vectorial proton transport has, by and large, been preserved.     

Middle ear cell line that maintains vectorial electrolyte transport

The middle ear epithelium plays a major role in keeping the temporal bone cavities fluid-free and air-filled, which is a mandatory condition to allow optimum transmission of the sound vibrations from the tympanic membrane to the inner ear. Previous works have recently established the absorptive function of the middle ear epithelium, using primary cultures derived from Mongolian gerbil (Meriones unguiculatus). Because of the paucity of cells as obtained by enzymatic digestion, we developed a middle ear cell line (MESV) using wild-type SV40 infection of primary culture of Mongolian gerbil's middle ear epithelial cells. Transformation was attested by nuclear expression of SV40 large T antigen, prolonged in vitro passages (presently beyond 50 passages), and tumor-inducing ability when subcutaneously injected in athymic mice. Transport properties were evaluated after the fifteenth passage. MESV cells retained most cardinal properties of the original middle ear epithelial cells: cell polarization was evidenced by the presence of mature junctional complexes that separate the cell membrane in two distinct domains, with apical microvilli at the luminal side, and by vectorial sodium transport responsible for the transepithelial lumen-negative potential difference (?9.3 ± 0.14 mV in culture conditions (n=9), ?2.1 ± 0.25 mV after overnight growth factors and serum deprivation). Short-circuit current was, like in primary cultures, mainly related to a sodium transport occuring through amiloride-sensitive apical sodium channels, since apical addition of amiloride (10^?5 M) reduced I_sc from 7.0 = 1.4 to 0.6 ± 0.1 μA/cm² (P < 0.01, n = 6). Cellular cAMP content was increased by isoproterenol and prostaglandin E2 from 40.5 ± 5.6 to 258.5 ± 17.3 and 55.6 ± 6.2 pmol/mg protein per 5 min, respectively (P < 0.05, n = 10). Isoproterenol and prostaglandin E2 increased I_sc with very similar maximal effects: isoproterenol (10^?4 M) increased I_sc from 5.73 ± 0.31 to 12.77 ± 0.39 μA/cm², while prostaglandin E2 increased I_sc from 5.47 ± 0.21 to 12.87 ± 0.42 (n = 3). Since amiloride (10^?5 M) abolished this stimulation, this may be related to an increase of the electrogenic sodium transepithelial transport. The MESV cell line could provide an interesting tool as a model of middle ear epithelial cells for the study of pathophysiological modulations of ion transport. © 1993 Wiley-Liss, Inc.     

Cucurbits depicted in Byzantine mosaics from Israel, 350–600 ce

Background and Aims

Thousands of floor mosaics were produced in lands across the Roman and Byzantine empires. Some mosaics contain depictions of agricultural produce, potentially providing useful information concerning the contemporary presence and popularity of crop plants in a particular geographical region. Hundreds of floor mosaics produced in Israel during the Byzantine period have survived. The objective of the present work was to search these mosaics for Cucurbitaceae in order to obtain a more complete picture of cucurbit crop history in the eastern Mediterranean region.

Results and Conclusions

Twenty-three mosaics dating from 350–600 ce were found that had images positively identifiable as cucurbits. The morphological diversity of the cucurbit fruits in the mosaics of Israel is greater than that appearing in mosaics from any other Roman or Byzantine provincial area. The depicted fruits vary in shape from oblate to extremely long, and some are furrowed, others are striped and others lack definite markings. The cucurbit taxa depicted in the mosaics are Cucumis melo (melon), Citrullus lanatus (watermelon), Luffa aegyptiaca (sponge gourd) and Lagenaria siceraria (bottle gourd). Cucumis melo is the most frequently found taxon in the mosaics and is represented by round dessert melons and long snake melons. Fruits of at least two cultivars of snake melons and of watermelons are represented. To our knowledge, images of sponge gourds have not been found in Roman and Byzantine mosaics elsewhere. Indeed, the mosaics of Israel contain what are probably the oldest depictions of Luffa aegyptiaca in Mediterranean lands. Sponge gourds are depicted often, in 11 of the mosaics at eight localities, and the images include both mature fruits, which are useful for cleaning and washing, and immature fruits, which are edible. Only one mosaic has images positively identifiable as of bottle gourds, and these were round–pyriform and probably used as vessels.     

Foundations of vectorial metabolism and osmochemistry

Chemical transformations, like osmotic translocations, are transport processes when looked at in detail. In chemiosmotic systems, the pathways of specific ligand conduction are spatially orientated through osmoenzymes and porters in which the actions of chemical group, electron and solute transfer occur as vectorial (or higher tensorial order) diffusion processes down gradients of total potential energy that represent real spatially-directed fields of force. Thus, it has been possible to describe classical bag-of-enzymes biochemistry as well as membrane biochemistry in terms of transport. But it would not have been possible to explain biological transport in terms of classical transformational biochemistry or chemistry. The recognition of this conceptual asymmetry in favour of transport has seemed to be upsetting to some biochemists and chemists; and they have resisted the shift towards thinking primarily in terms of the vectorial forces and co-linear displacements of ligands in place of their much less informative scalar products that correspond to the conventional scalar energies. Nevertheless, considerable progress has been made in establishing vectorial metabolism and osmochemistry as acceptable biochemical disciplines embracing transport and metabolism, and bioenergetics has been fundamentally transformed as a result.     

Structures of the intermediates in the catalytic cycle of mitochondrial cytochrome c oxidase

Cytochrome c oxidase is the terminal complex of the respiratory chains in the mitochondria of nearly all eukaryotes. It catalyzes the reduction of molecular O₂ to water using electrons from the respiratory chain, delivered via cytochrome c on the external surface of the inner mitochondrial membrane. The protons required for water formation are taken from the matrix side of the membrane, making catalysis vectorial. This vectorial feature is further enhanced by the fact that the redox catalysis is coupled to the translocation of protons from the inside to the outside of the inner mitochondrial membrane. We are dealing with a molecular machine that converts redox free energy into a protonmotive force (pmf). Here, we review the current extensive knowledge of the structural changes in the active heme?copper site that accompany catalysis, based on a large variety of time-resolved spectroscopic experiments, X-ray and cryoEM structures, and advanced computational chemistry.     

Analysis of mosaic expression of the nptII gene in transgenic tobacco plant lines contrasting in mosaicism

Differences in the mosaic plants occurence frequency between the Nu5 and Nu6 lines of transgenic tobacco (Nicotiana tabacum) plants remained irrespective of the nptII marker gene allelic state. nptII gene transition from the hemizygous state (T₃) to the homozygous one (T₄) was accompanied by an increase in the frequency of mosaics in both lines. Transition from the homozygous state (T₄) into the hemizygous one (F₁) resulted in a further increase in the frequency of mosaic plants in the Nu5 line, whereas this parameter remained at a high level in the Nu6 line. pMAS promoter hypermethylation in plants of both lines, as well as differences in the 5??-part truncated nptII gene copy cytosin methylation level between the Nu5 and Nu6 lines, pointed to nptII gene mosaic expression epigenetic regulation.     

Interaction of angiotensin II type 1 receptor blockers with P-gp substrates in Caco-2 cells and hMDR1-expressing membranes

AimsThe inhibitory effect of angiotensin II type 1 receptor blockers (ARBs) on P-glycoprotein (P-gp) was examined to evaluate their clinical drug–drug interaction (DDI) potential.Main methodsWe performed an inhibition study on the vectorial transport of digoxin, a typical substrate for P-gp, using a human colonic adenocarcinoma cell line, Caco-2 cells, and verapamil-stimulated ATPase activity using human multidrug resistance 1 (hMDR1)-expressing membrane.Key findingsThe vectorial transport of digoxin was inhibited by candesartan cilexetil, irbesartan and telmisartan with the IC₅₀ values of 14.7, 34.0 and 2.19 µM, respectively. Those values were 7.4–426-fold higher than their theoretical clinical gastrointestinal concentration [I] at doses in clinical DDI studies. Other ARBs failed to show interaction with P-gp.SignificanceIt was demonstrated that candesartan cilexetil, irbesartan and telmisartan had the potential to inhibit the transport of various drugs via P-gp. Telmisartan, which caused an increase in the serum digoxin concentration in humans, had a sufficiently high [I]/IC₅₀ value, suggesting that DDI between digoxin and telmisartan was caused by the inhibition of digoxin efflux via intestinal P-gp.     

Inhibition of a cold-active alkaline phosphatase by imipenem revealed by in silico modeling of metallo-β-lactamase active sites

We demonstrate the inhibition of the native phosphatase activity of a cold active alkaline phosphatase from Vibrio (VAP) (IC₅₀ of 44 ± 4 (n = 4) μM at pH 7.0 after a 30 min preincubation) by a specific β-lactam compound (only by imipenem, and not by ertapenem, meropenem, ampicillin or penicillin G). The homologous scaffold was detected by an in silico analysis that established the spatial and electrostatic congruence of the active site of a Class B2 CphA metallo-β-lactamase from Aeromonas hydrophila to the active site of VAP. The tested β-lactam compounds did not inhibit Escherichia coli or shrimp alkaline phosphatase, which could be ascribed to the lower congruence indicated by CLASP. There was no discernible β-lactamase activity in the tested alkaline phosphatases. This is the first time a scaffold recognizing imipenem in an alkaline phosphatase (VAP) has been demonstrated.     

Thermoregulation in the semi-aquatic yellow anaconda, Eunectes notaeus

1.

Various factors affect a reptile's capacity for thermoregulation and most studies have focussed on terrestrial species.

2.

We investigated the thermoregulatory abilities of the yellow anaconda (Eunectes notaeus) in terms of selected body temperature (T_sel), set-point range (T_set) and body posture in terrestrial and aquatic thermal mosaics.

3.

Yellow anacondas selected higher body temperatures (T_b) and have a narrower T_set in a terrestrial environment than in an aquatic one.

4.

Coiled body postures were most frequently observed and were generally associated with higher T_b.

     

Development of selective and reversible pyrazoline based MAO-A inhibitors: Synthesis,biological evaluation and docking studies

3,5-Diaryl pyrazolines analogs were synthesized and evaluated for their monoamine oxidase (MAO) inhibitory activity. The compounds were found reversible and selective towards MAO-A with selectivity index in the magnitude of 10³–10⁵. The docking studies were carried out to gain further structural insights of the binding mode and possible interactions with the active site of MAO-A. Interestingly, the theoretical (K_i) values obtained by molecular docking studies were in congruence with their experimental (K_i) values.     

The plastoquinone pool as possible hydrogen pump in photosynthesis

The function of the plastoquinone pool as a possible pump for vectorial hydrogen (H⁺ + e^?) transport across the thylakoid membrane has been investigated in isolated spinach chloroplasts. Measurements of three different optical changes reflecting the redox reactions of the plastoquinone, the external H⁺ uptake and the internal H⁺ release led to the following conclusions:(1) A stoichiometric coupling of 1 : 1 : 1 between the external H⁺ uptake, the electron translocation through the plastoquinone pool and the internal H⁺ release (corrected for H⁺ release due to H₂O oxidation) is valid (pH_out = 8, excitation with repetitive flash groups). (2) The rate of electron release from the plastoquinone pool and the rate of proton release into the inner thylakoid space due to far-red illumination are identical over a range of a more than 10-fold variation.These results support the assumption that the protons taken up by the reduced plastoquinone pool are translocated together with the electrons through the pool from the outside to the inside of the membrane. Therefore, the plastoquinone pool might act as a pump for a vectorial hydrogen (H⁺ + e^?) transport. The molecular mechanism is discussed. The differences between this hydrogen pump of chloroplasts and the proton pump of Halobacteria are outlined.     

Characterization of a signal peptide sequence in the cell-free translation product of sheep elastin mRNA

In vitro explant cultures of near-term sheep nuchal ligament secrete tropoelastin of approximate M_r 70,000–72,000 while the elastin cell-free product of sheep nuchal ligament RNA is 2000 to 3000 M_r larger. Automated Edman degradation of immunoprecipitates of radiolabeled cell-free elastin precursor demonstrated the presence of a 26-residue signal sequence which was absent from sheep tropoelastin secreted from explant cultures. In addition, a 20-residue overlap was established between the cell-free product and the secreted protein. This overlap region, representing the N-terminal sequence of ovine tropoelastin, demonstrated complete homology with the N-terminal sequence of porcine tropoelastin and near complete homology with chick tropoelastin. These findings suggest that cotranslational removal of this hydrophobic peptide extension is likely a correlate of vectorial transport of elastin into the secretory apparatus.     

Evaluation of on-farm methods for testing the human–animal relationship in dairy herds with cubicle loose housing systems—test–retest and inter-observer reliability and consistency to familiarity of test person

The human–animal relationship is an important factor when considering animal welfare at herd level. In the present study, two behavioural tests for the on-farm assessment of the human–animal relationship at herd level of dairy cows housed in loose housing cubicle systems were evaluated with respect to inter-observer reliability, test–retest reliability, effect of familiarity of test person as well as inter-correlation of the two tests. In a voluntary animal approach (VAA) test, the number of cows and the latencies to approach and touch a stationary test person was measured. In an avoidance (AV) test, the cows’ avoidance reactions to an approaching test person were categorised. A first study was carried out in 12 commercial Danish and Austrian dairy farms. On each farm, both behaviour tests were carried out on the same day and repeated within 4–5 days. For each test, cows were tested by both an unfamiliar and a familiar test person (the stock-person) and two observers simultaneously registered the animals’ test responses. The inter-observer reliability of both behavioural tests was found to be high (VAA: 2.5-m approach r=0.98 (P<0.001) and touch r=0.97 (P<0.001); AV: Kappa coefficient_weighted=0.886 (prevalence index for flight distance≥2 m is 0.636)). The cows at herd level showed shortest latency for touching an unfamiliar test person on the first test day (P=0.006). Further, the AV test had a high test–retest reliability (Kappa coefficient_weighted=0.503 (prevalence index for flight distance≥2 m is −0.660)) and results indicated no effect of familiarity of test person (Kappa coefficient_weighted=0.463 (prevalence index for flight distance≥2 m is −0.677)). In a second study, the correlation between the two behavioural tests (similar measures) was evaluated. On each of 10 commercial Danish dairy farms with loose housing cubicle systems at four repeated sessions, both behaviour tests were carried out on the same day. For each test cows were tested by the stock-person. The VAA and AV tests at herd level were highly correlated (r_s=−0.84; P=0.002).The results suggest that the AV test is valid and applicable for on-farm assessment of the human–animal relationship at herd level. This accounts only partly for the VAA test, which seem to be more unclear regarding motivation for the animals’ approach behaviour.     

The Mathematics of Mosaic Analysis I: The Relationship between Sturts and Distance

In mosaic fate mapping the fraction of mosaics in which two structures are of different genotype is calculated. This frequency of separation has been called a "distance" and the units of this distance are called "sturts". The fundamental assumption of fate mapping is that the frequency of separation increases continuously with the actual distance between the anlage for these structures on the blastoderm. This paper shows that the frequency of separation does not increase beyond a certain value.—For the current theory to work as proposed, each mosaic animal must be half mutant and half normal. This is rarely the case in collections of mosaics. It has been thought that if some flies are less than half mutant and others more than half, these two types would introduce compensating errors in mapping distance. We show that this is not true and describe the nature of the errors introduced. It is probable that these errors are the main reason that mapping distances reported from different sets of mosaics have not been reproducible. This paper presents methods for the proper handling of data from mosaics with different amounts of mutant tissue.—We prove here that for mosaics with an arbitrary fraction of mutant tissue (m), the largest frequency of separation that can occur is 2m. We prove that sturts underestimate actual distance on the blastoderm by a factor of r/m, where r is the radius of the mutant patch, and that sturts give no information on distances greater than 2r. This, and not double crossing over, is the reason for the nonadditivity of sturts and the shrinking of large distances in sturt measures. Sturtoids overestimate distances by a factor of 1/(2r) and also give no information on distances over 2r. This paper gives formulae for correctly estimating distance when using a collection of mosaics with varying amounts of mutant tissue. We also describe the nature of the errors introduced by convoluted or elongate mosaic boundaries and by multiple mosaic patches.     

Formation of pure and mixed clones of c mutants in serratiaphage sigma after treatment of the free virion with UV or hydroxylamine

The clone composition of c mutants of phage σ induced by UV irradiation of the free virion was studied, using Hcr⁺, Hcr^? and UV-irradiated Hcr⁺ cells as hosts and 2, 3 and 4 different UV doses, respectively. Most of the c plaques contained only mutant phages, and the distribution of mosaics was asymmetrical, i.e. most mixed clones contained >80% mutant type. The frequency of mosaics decreased with increasing UV dose in all three host systems; however, the decrease was significant only with the UV-irradiated Hcr⁺ host. Propagation of UV-irradiated σ in Hcr⁺ and Hcr^? hosts, respectively, did not lead to a significant difference in the frequency of mosaics, but, using UV-irradiated Hcr⁺ host significantly increased the percentage of mixed clones.The composition of plaques containing c mutants, after UV irradiation and treatment with hydroxylamine, was also studied by picking and testing all plaques (mutant and wild-type) of the survivors of a single UV dose and a single incubation time, respectively. In both experiments, besides pure and nearly pure (visible) c mutant plaques, many cryptic mutants containing predominantly >20% mutant type were found. The distribution of mosaics was of an almost “inverse symmetrical” type, the class of clones with about 50% mutant and 50% wild-type being the rarest.From these results incomplete recombinational repair is suggested to be responsible for the formation of pure mutant clones in mutation induction.     

Action dichroism in perception of vectorial photo-excitation in the solar-tracking leaf ofLavatera cretica L.

The leaf lamina ofLavatera cretica L. exhibits a diaphototropic response that discriminates between two opposite, constant vectorial excitations by white light beams whose fluence rates differ by as little as 10% (50 versus 45 μmol·m⁻²·S⁻¹). The relationship between the response (angular velocity of laminar reorientation) and the fluence-rate ratio is linear. The lamina similarly discriminates between two such excitations by polarized light, one with the electrical vector transverse to the plane of the two beams (θ) and the opposite one with the vector parallel to that plane (⪙). When two such beams were of equal fluence rate, the lamina reoriented towards the ⪙ beam. When the fluence rate of the θ beam was maintained at 50 μmol·m⁻²·s⁻¹ and that of the ⪙ beam was reduced, the response to the latter (angular velocity of laminar reorientation) was reduced progressively. Further reduction in the fluence rate of the ⪙ polarized beam eventually resulted in reorientation in the opposite direction (towards the θ beam) and the response to the latter increased progressively with the reduction in fluence rate. The equilibrium was at a ⪙/θ ratio of 0.62. Measurements of reflectance of oblique beams of ⪙ and θ polarized light from the upper laminar surface, and of transmittance of such light ghrough the lamina, eliminated the possibility that optical dichroism of the lamina contributed significantly to these results. The implications of this action dichroism to the postulated mechanism of perception of vectorial excitation by these leaves is discussed. Dedicated to the 60thbirth day of Professor Hans Mohr     

Multivariate vectorial analysis of the visual evoked response

Principal component analysis of a set of V.E.R.s yields vectorial representations of the responses in a diagram of a reduced dimensionality. In a vectorial diagram the responses can be resolved into components, associated with the aspects of the stimulus modulation and the state of the visual system. The appearance of a scotopic and a photopic component is demonstrated. In another experiment small modulations of luminance and color evoked the responses. The distorting effect of a type of non-linearity of the visual system, that is latency variation of the responses and the components, is discussed and demonstrated.