Multiple Proteases to Localize Oxidation Sites

Proteins present in cellular environments with high levels of reactive oxygen and nitrogen species and/or low levels of antioxidants are highly susceptible to oxidative post-translational modification (PTM). Irreversible oxidative PTMs can generate a complex distribution of modified protein molecules, recently termed as proteoforms. Using ubiquitin as a model system, we mapped oxidative modification sites using trypsin, Lys-C, and Glu-C peptides. Several M+16 Da proteoforms were detected as well as proteoforms that include other previously unidentified oxidative modifications. This work highlights the use of multiple protease digestions to give insights to the complexity of oxidative modifications possible in bottom-up analyses.     

Gas Exchange in Renal Failure: II—Pulmonary Gas Exchange during Peritoneal Dialysis

Blood gas analysis studies have been made in patients undergoing peritoneal dialysis. It has been shown that oxygen tensions are reduced when fluid has been run into the peritoneal cavity and that this fall in Pao₂ is reversed after running out the dialysate. The change in Pao₂ is greater with 2-litre than with 1-litre cycles.     

The Steady-State Properties of Ion Exchange Membranes with Fixed Sites

The properties of the steady states of a system composed of two solutions separated by a quite general type of ion exchange membrane having fixed sites are derived as functions of the compositions of the solutions and of the difference of electric potential between the two solutions. These properties are evaluated with the restraints that the membrane is solely permeable to cations or anions, no flow of solvent occurs, and the solutions contain no more than two permeant ionic species, which are monovalent. Under the assumptions that the difference of standard chemical potentials of the permeant species and the ratio of their mobilities are constant throughout the membrane, even when the spacing of sites is variable, explicit expressions are derived for the electric current, individual fluxes, and concentration profiles. An unexpectedly simple dependence of these expressions upon distribution of sites is found.     

Importance of Multiple Methylation Sites in Escherichia coli Chemotaxis

Bacteria navigate within inhomogeneous environments by temporally comparing concentrations of chemoeffectors over the course of a few seconds and biasing their rate of reorientations accordingly, thereby drifting towards more favorable conditions. This navigation requires a short-term memory achieved through the sequential methylations and demethylations of several specific glutamate residues on the chemotaxis receptors, which progressively adjusts the receptors’ activity to track the levels of stimulation encountered by the cell with a delay. Such adaptation also tunes the receptors’ sensitivity according to the background ligand concentration, enabling the cells to respond to fractional rather than absolute concentration changes, i.e. to perform logarithmic sensing. Despite the adaptation system being principally well understood, the need for a specific number of methylation sites remains relatively unclear. Here we systematically substituted the four glutamate residues of the Tar receptor of Escherichia coli by non-methylated alanine, creating a set of 16 modified receptors with a varying number of available methylation sites and explored the effect of these substitutions on the performance of the chemotaxis system. Alanine substitutions were found to desensitize the receptors, similarly but to a lesser extent than glutamate methylation, and to affect the methylation and demethylation rates of the remaining sites in a site-specific manner. Each substitution reduces the dynamic range of chemotaxis, by one order of magnitude on average. The substitution of up to two sites could be partly compensated by the adaptation system, but the full set of methylation sites was necessary to achieve efficient logarithmic sensing.     

真核基因转录激活的多位点协同调控

真核基因的转录激活具有协同性的特征,表现为多个转录调控位点的共同作用效果大于每个位点单独作用之和,受多个位点调控的基因转录呈S型曲线.多个激活蛋白之间的相互作用、激活蛋白与各DNA位点的协同结合以及激活蛋白与转录机器的协同作用,三种途径都对协同性转录激活行为产生影响.协同性转录激活的本质是多个结合在调控位点上的激活蛋白之间直接或间接的相互作用.多位点的协同转录调控机制有助于理解生物的多种调控过程和建立基因调控网络.     

Microvascular Regulation of Cutaneous Gas Exchange in Amphibians

SYNOPSIS. Gas exchange across amphibian skin is regulated bythe cutaneous microcirculation. Parameters involved in regulatinggas exchange are capillary density, radius and blood flow. Changesin capillary density and radius should affect gas exchange byaltering the cutaneous diffusing capacity (D₂) while changesin capillary blood flow affect the perfusive conductance ofthe skin. A simple model predicts that the effect of capillary densitychanges on D₂ will become more pronounced as capillary densityand epidermal thickness decrease. Changes in capillary radiusshould have only a minor effect on D₂ Previous analyses havesuggested that cutaneous gas exchange is not significantly affectedby the perfusive conductance of the skin. Consequently, it hasbeen thought that changes in total capillary blood flow havelittle impact on cutaneous gas exchange. Earlier analyses, however,may have underestimated the importance of perfusive conductancein amphibian skin, primarily because functional heterogeneitiesin the microcirculation were not considered. The density of perfused capillaries is regulated in the footweb of Rana esculenta by environmental Po₂ and PCO₂, and alsoby lung ventilation. In Rana catesbeiana, capillary densityin the web decreases during air exposure. Chronic exposure toenvironmental hypoxia increases total capillary density in bullfrogtadpole skin. In Rana pipiens, regulation of cutaneous gas exchangeby environmental and pulmonary O₂ probably involves changesin total capillary blood flow.     

Two Steps of Gas Exchange in Leaf Photosynthesis

Photosynthesis and transpiration of excised leaves of Taraxacum officinale L. and a few other species of plants were measured, using an open gas analysis system. The rates of CO₂ uptake and transpiration increased in two steps upon illumination of stomata-bearing epidermis of these leaves at a light intensity of 50 mW × cm⁻². Abscisic acid inhibited only the second step of gas exchange. Illumination of the astomatous epidermis of hypostomatous leaves caused only the first step of gas exchange. These data indicate that the first and second steps arise from cuticular and stomatal gas exchange, respectively. The rate of the cuticular photosynthesis in a Taraxacum leaf reached saturation at a light intensity of 5 mW × cm⁻², and the rates of the stomatal photosynthesis and transpiration reached saturation at a higher intensity of 35 mW × cm⁻². The cuticular photosynthesis of a Taraxacum leaf was 18% of the stomatal photosynthesis at 50 mW × cm⁻² and 270% at 5 mW × cm⁻². The other species of leaves showed the same trend. The importance of cuticular CO₂ uptake in leaf photosynthesis, especially under low light intensity was stressed from these data.     

Models for Cutaneous Gas Exchange and Transport

SYNOPSIS. Models suited for quantitative analysis of cutaneousgas exchange and transport with particular emphasis on diffusionlimitation are discussed. I. The diffusive transfer of gasesbetween body and environment can be compared to diffusion acrossa homogeneous flat tissue sheet characterized by a diffusionconductance or diffusing capacity (D). II. A flat barrier separatingthe medium from blood flow is a widely useful model. The characteristic parameter is theratio , where rß is the effective solubility of the gas in blood. A high value means predominant perfusionlimitation; low signifies diffusion limitation. III. In some conditions, as in still wateror at body-soil contact, a considerable diffusion resistancein the medium may become important. IV. Unequal distributionof D to may strongly reduce gas exchange efficiency. V. Diffusive arteriovenous shuntingof gases may lead to additional reduction of gas exchange efficiency.VI. The parallel arrangement of skin circulation with respectto tissue circulation (in contrast to the arrangement in seriespresent in fish gills and in tetrapod lungs) leads to mixingof arterialized with venous blood and reduced efficiency ofblood flow in gas transport.     

Multiple Human Taste Receptor Sites: A Molecular Modeling Approach

Numerous experimental data on the human peripheral taste systemsuggest the existence of multiple low-affinity and low-specificityreceptor sites which are responsible for the detection and thecomplete discrimination of a very large number of organic molecules.According to this hypothesis, a given molecule interacts withnumerous taste receptors and vice versa. Statistical analysisof taste intensities estimated by 58 human subjects for variousmolecules enables the calculation of taste intermolecular distances.For the present modeling study, we hypothesized that a shorttaste distance (i.e. taste similarity) between two distinctmolecules indicates that they bind with similar distributionsof affinities to the taste receptors, and hence display similarbinding motifs. In order to find common molecular binding motifsamong 14 selected organic tastants, hydrogen-bonding and hydrophobicinteraction properties were mapped onto their molecular surfaces.The 14 surfaces were then cut in 240 fragments, most of whichwere made up of 2–4 potentially interacting zones. A correspondenceindex was defined to measure the analogy between two optimallysuperimposed fragments. The 75 most representative fragmentswere all matched pairwise. Twelve distinct clusters of fragmentswere isolated from the 2775 calculated comparisons. These 12fragment types were used to calculate structural similaritydistances. We then performed a combinatorial analysis to identifywhich fragment combination best reconciled structural and tastedistances. We finally identified an optimal subset of sevenfragment types out of the 12, which significantly and best accountedfor the 91 pairwise taste distances between all 14 modeled tastants.These seven validated fragment types are therefore presentedas good candidates to be recognized by the same number of distincttaste receptor sites. Potential applications of these identifiedbinding motifs to tastant design are suggested. Chem. Senses21: 425–445, 1996.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).     

Exchange of the Coronavirus Replicase Polyprotein Cleavage Sites Alters Protease Specificity and Processing

Coronavirus nonstructural proteins 1 to 3 are processed by one or two papain-like proteases (PLP1 and PLP2) at specific cleavage sites (CS1 to -3). Murine hepatitis virus (MHV) PLP2 and orthologs recognize and cleave at a position following a p4-Leu-X-Gly-Gly-p1 tetrapeptide, but it is unknown whether these residues are sufficient to result in processing by PLP2 at sites normally cleaved by PLP1. We demonstrate that exchange of CS1 and/or CS2 with the CS3 p4-p1 amino acids in engineered MHV mutants switches specificity from PLP1 to PLP2 at CS2, but not at CS1, and results in altered protein processing and virus replication. Thus, the p4-p1 residues are necessary for PLP2 processing but require a specific protein or cleavage site context for optimal PLP recognition and cleavage.Coronaviruses are positive-strand RNA viruses that translate their first open reading frames (ORF1a and ORF1b) into polyproteins that are processed by viral proteases into intermediate and mature nonstructural proteins (nsp1 to -16) (Fig. ​(Fig.11 A) (4, 7, 17, 20). nsp1, -2, and -3 are liberated at cleavage sites (CSs) between nsp1-2 (CS1), nsp2-3 (CS2), and nsp3-4 (CS3) by one or two papain-like protease (PLP) activities encoded within nsp3 (1, 2, 12, 13, 15) (Fig. ​(Fig.1B).1B). Murine hepatitis virus (MHV) and human coronavirus 229E (HCoV-229E) use two PLPs (PLP1 and PLP2) to process at CS1 to -3, while severe acute respiratory syndrome coronavirus (SARS-CoV) and avian infectious bronchitis virus (IBV) use a single PLP each (PLpro and PLP2, respectively) (10, 20, 25, 26). The factors determining the evolution and use of one versus two PLPs by different coronaviruses for processing of nsp1, -2, and -3 are unknown. Mutations at MHV CSs or within PLP1 alter replication and protein processing in surprising ways (8, 13). Loss of processing at MHV CS1 and CS2 by CS deletion or mutation results in changes in the timing and extent of virus replication. Inactivation of MHV PLP1 is more detrimental for virus replication than deletion of CS1 and CS2 or than inactivation of PLP1 combined with the CS deletions, even though not all of the mutant viruses process at CS1 or CS2 or display similar protein processing phenotypes. In contrast to MHV results, the HCoV-229E PLP1 and PLP2 have both been shown to process at CS1 and CS2, albeit at different efficiencies (Fig. ​(Fig.1B)1B) (24). Finally, the single SARS-CoV PLP2 homolog (PLpro) mediates efficient processing at CS1 to -3, each of which has an upstream position 4-Leu-X-Gly-Gly-position 1 (p4-LXGG-p1) amino acid motif implicated in PLpro processing (10, 16, 18). MHV possesses a p4-LXGG-p1 sequence only at CS3 and is cleaved by PLP2. These results suggest that p4-LXGG-p1 may be the critical determinant of recognition by PLP2/PLpro, but this hypothesis has not been tested in studies of replicating virus. Thus, it remains unknown whether the differences in PLP/CS recognition and processing are determined by the proximal p4-p1 residues (22).Open in a separate windowFIG. 1.MHV replicase organization, coronavirus PLP-mediated processing, and experimental design of cleavage site replacement viruses. (A) ORF1 of MHV genome RNA is shown, with overlapping ORF1a and ORF1b. The ORF1ab polyprotein is shown with nonstructural proteins (nsp1 to -16) indicated by vertical lines and numbers. Viral papain-like protease domains in nsp3 are shown as a white box containing black letters (PLP1) and a black box containing white letters (PLP2), and the nsp5 protease (3CLpro) is indicated as a gray box with a white number. Cleavage sites for PLP1 (CS1 and CS2 [shown as white arrowheads]), PLP2 (CS3 [shown as a black arrowhead]), and nsp5 (CS4 to -14 [shown as gray arrowheads]) are indicated. (B) The organization of nsp1 to nsp4 is shown for representative coronaviruses. PLPs are indicated, with the hatched box in IBV indicating a probable catalytically inactive remnant of PLP1. Processing events that were confirmed as occurring in vitro or during infection are shown by arrows with solid lines and large arrowheads, indicating single or dominant protease activity. The dashed lines and small arrowheads indicate minor or secondary cleavage activities. The CS amino acid sequences from position 4 (p4) to p1′ are shown for each CS, with a space and arrow representing the site of proteolytic processing. (C) The CS substitution viruses were engineered to replace the original CS amino acid sequences at CS1 and/or CS2 with that of the CS3 amino acid sequence p4-LKGG-p1. Both CS substitutions were also engineered into a catalytically inactive PLP1 (P1ko) background. PLPs are shown as numbers in boxes within nsp3. Engineered catalytically inactivated PLP1 is shown as a hatched box. Arrowheads indicate cleavage events of the WT virus and are linked to the enzyme predicted to mediate processing at the CS, as indicated by white boxes containing black characters (PLP1) or black boxes containing white characters (PLP2). The p4 through p1 amino acid residues for each CS are shown below each diagram. White and black vertical bars show the respective predicted PLP1 and PLP2 cleavage sites. Engineered substitutions are indicated in bold characters. Asterisks indicate engineered mutant genomes that could not be recovered as infectious virus.In this study, we used MHV as a model to test whether PLP/CS specificities could be switched by an exchange of CS amino acid sequences and to determine the impact of CS exchange on protein processing and virus replication. Replacement of the CS3 p4-LKGG-p1 at CS2, but not at CS1, was sufficient for a switch in protease specificity from PLP1 to PLP2. Some combinations of CS exchange could not be recovered with inactive PLP1, and recovered mutant viruses had altered protein processing and/or impaired growth compared to the wild type (WT). The results confirm that p4-LXGG-p1 amino acid sequences are necessary determinants of cleavage by PLP2 but also indicate that a larger cleavage site or a different protein context is required for efficient recognition and processing. Finally, the results support the conclusion that complex relationships with respect to the timing and extent of PLP/CS interactions are essential for successful replication and, likely, for virus fitness.     

Gas Exchange between Equisetum limosum and its Environment

Equisetum limosum grows in standing water. Its perennating rhizomesramify in the bottom mud, which is a markedly oxygen-deficientmedium. During the summer months aerial shoots are present andextend above the surface of the water to a height of about 1m. The concentration of oxygen in the internal atmosphere ofplants in the field decreases progressively from the stem tothe perennating rhizomes. There is a reverse gradient of carbondioxide concentrations. The structure of the nodal diaphragms of the aerial stem isdescribed and the progressive occlusion of the pores of thediaphragms between May and November is demonstrated. This occlusionof the diaphragms has been correlated with a reduction in thecase of gaseous diffusion along lengths of aerial stems. Oxygen is present in the rhizomes throughout the winter, despitethe fact that the dead aerial stems have broken off and thestumps have been submerged by the rise in water level, thusisolating the rhizome from atmospheric oxygen. The results are discussed in relation to the ability of theplant to grow in an oxygen-deficient environment.     

Gas Exchange and Flowering in Verticillium-wilted Pepper Plants

In Navarra, Northern Spain, Verticillium dahliae Kleb. is one of the pathogens that causes drastic reductions in pepper production. The aim of this study therefore was to describe how infection by V. dahliae affects gas exchange during the flowering of pepper in order to determine some possible factors contributing to the significant decrease of plant yield. Verticillium was inoculated when plants had started flowering. The first leaf wilting symptoms appeared on day 18 after inoculation, but leaf water potential rapidly decreased after infection. The inoculated plants produced more flowers than the controls between 15 and 33 days after inoculation, but flower production declined after day 33. Inoculated plants also suffered more defoliation and chlorophyll degradation. Leaf conductance and photosynthesis clearly decreased in both groups of plants as a consequence of senescence, but the values in those inoculated were significantly lower. Results suggest that the decrease in photosynthesis was in part due to defoliation and chlorophyll degradation, as well as premature flower fall. These factors contributed to the negative effects of Verticillium infection on pepper yield.     

The Physics of Gas Exchange Across the Avian Eggshell

The principles which govern gas exchange by diffusion acrossthe pores of the avian eggshell are reviewed and compared withconvective gas exchange. The concept of conductance is definedfor both diffusive and convective gas exchange through pores,and methods of calculating pore size are described. Estimatesof conductances of the elements in the gas transfer path fromatmosphere to chorioallantoic capillary blood are discussed,and recent studies on the role of ternary diffusion and a convectivecomponent to gas fluxes are presented.