Extreme differences in charge changes during protein evolution

Summary The maintenance of a proper distribution of charged amino acid residues might be expected to be an important factor in protein evolution. We therefore compared the inferred changes in charge during the evolution of 43 protein families with the changes expected on the basis of random base substitutions. It was found that certain proteins, like the eye lens crystallins and most histones, display an extreme avoidance of changes in charge. Other proteins, like phospholipase A2 and ferredoxin, apparently have sustained more charged replacements than expected, suggesting a positive selection for changes in charge. Depending on function and structure of a protein, charged residues apparently can be important targets for selective forces in protein evolution. It appears that actual biased codon usage tends to decrease the proportion of charged amino acid replacements. The influence of nonrandomness of mutations is more equivocal. Genes that use the mitochondrial instead of the universal code lower the probability that charge changes will occur in the encoded proteins.     

Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase

A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies established that Nterm-E1p specifically competes with E1p for binding to the dihydrolipoyl transacetylase component (E2p) of PDHC. Moreover, the experiments show that the N-terminal region of E1p forms an independent folding domain that functions as a binding domain. CD measurements, two-dimensional (2D) (1)H NMR analysis, and secondary structure prediction all indicate that Nterm-E1p has a high alpha-helical content. Here a structural model of the N-terminal domain is proposed. The peptide is present in two conformations, the population of which depends on the sample conditions. The conformations are designated "unfolded" at pH > or =6 and "folded" at pH <5. The 2D (1)H TOCSY spectrum of a mixture of folded and unfolded Nterm-E1p shows exchange cross-peaks that "link" the folded and unfolded state of Nterm-E1p. The rate of exchange between the two species is in the range of 0.5-5 s(-1). Sharp resonances in the NMR spectra of wild-type E1p demonstrate that this 200 kDa enzyme contains highly flexible regions. The observed dynamic character of E1p and of Nterm-E1p is likely required for the binding of the E1p dimer to the two different binding sites on E2p. Moreover, the flexibility might be essential in sustaining the allosteric properties of the enzyme bound in the complex.     

The hydrochemical state of Lake Peipsi-Pihkva

The distribution and time dependence of total phosphorus (TP), dissolved inorganic phosphate (PO₄P), total nitrogen (TN), chlorophyll a (Chl), dichromate oxidizability (CODCr), permanganate oxidizability (CODMn), water colour (Col) and transparency (SD), pH, dissolved oxygen (O₂) and oxygen saturation (O₂%) in the surface water of Lake Peipsi-Pihkva are studied by using 65-parameter regression models with the help of the SAS system. The yearly means, polarity and seasonal dependence of each investigated parameter during 1985–1994 are estimated from fitted models. The bulk of data consists of 456 to 1149 measurements per parameter. L. Peipsi-Pihkva appears to be polar with respect to the majority of the studied parameters. The content of TP, PO₄P, TN, Chl, CODCr, CODMn, and Col decrease from south to north, while SD has an opposite trend. pH, O₂, and O₂% are quite uniform all over the lake. L. Peipsi is eutrophic, L. Pihkva is hypertrophic. The lake is influenced by significant yearly and seasonal changes. It is concluded that the Velikaja River is the main source of pollution for L. Peipsi-Pihkva.     

Solvent and electrolyte effects in enhancing the identification of intramolecular electronic communication in a multi redox-active diruthenium tetraferrocenoate complex, a triple-sandwiched dicadmium phthalocyanine and a ruthenocene-containing β-diketone

Enhanced electrochemical resolution of anodic processes is possible in the presence of [N(ⁿBu)₄][B(C₆F₅)₄], 1, as supporting electrolyte over that obtained in the presence of [N(ⁿBu)₄][PF₆]. By changing the anion of the supporting electrolyte to a salt having [B(C₆F₅)₄]⁻, anions, electrochemical processes of especially cationic analytes can benefit. Thus, the redox chemistry of 0.5 mmol dm⁻³ solutions of [Ru₂(μ-FcCOO)₄·(CH₃CH₂OH)₂][PF₆], 2, Fc = ferrocenyl, in CH₂Cl₂/[N(ⁿBu)₄][B(C₆F₅)₄] were found to involve four well-resolved ferrocenyl-based electrochemical reversible redox processes as well as reduction of Ru^III-Ru^II. At 1.0 mmol dm⁻³ concentrations of 2, or in the presence of [N(ⁿBu)₄][PF₆], the four ferrocenyl processes coalesced into only two waves as a result of (Fc⁺)?() ion paring. Seventeen of the possible 18 one-electron transfer processes of the biscadmium trisphthalocyaninato complex [Cd₂{Pc(C₆H₁₃)₈}₃], 3, could be observed in THF/[N(ⁿBu)₄][B(C₆F₅)₄], but the electrochemical window of CH₂Cl₂/[N(ⁿBu)₄][B(C₆F₅)₄] only allowed detection of 15 of these processes. Although reduction processes were unaffected, THF solvation leading to species such as (3ⁿ⁺)(THF)_x with 1 ? n ? 4 and x ? 1 as well as ion pair formation of the type (3ⁿ⁺)?() prevented good resolution of oxidation processes. The CH₂Cl₂/[N(ⁿBu)₄][B(C₆F₅)₄] system also allowed detection of reversible one-electron transfer ferrocenyl (Fc/Fc⁺) and ruthenocenyl-based (Rc/Rc⁺) processes for both enol and keto isomers of the β-diketone FcCOCH₂CORc, 4, Rc = ruthenocenyl. In CH₃CN/[N(ⁿBu)₄][PF₆], the ruthenocenyl moiety was oxidised to a Ru^IV species.     

Role of nitric oxide synthase/arginase balance in bronchial reactivity in patients with chronic obstructive pulmonary disease

Competition between nitric oxide synthases (NOSs) and arginases for their common substrate l-arginine could be involved in the regulation of cholinergic airway reactivity and subsequent airway remodeling. The aims of this study were to evaluate the relationships between the expression of this enzymatic balance and the effects of NOS and arginase inhibition on bronchoconstrictive response to acetylcholine of patients without and with early chronic obstructive pulmonary disease (COPD). Twenty-two human bronchi [15 COPD (9 GOLD-0, 6 GOLD-1, -2-A), 7 nonsmokers] were investigated for immunohistochemistry and modulation of acetylcholine-induced airway constriction. Significantly increased expression of NOS2 in immunoblots of bronchial tissue and staining in smooth muscle cells was evidenced in patients with COPD compared with control subjects, whereas no modification of arginase expression was evidenced. Forced expiratory volume in 1 s (FEV1) and NOS2 expression were negatively correlated (rho=-0.54, P=0.027). Pharmacological experiments demonstrated that resting tension was elevated in COPD compared with control subjects (2,243+/-154 vs. 1,574+/-218 mg, P=0.03) and was positively correlated with the expression of NOS2 (rho=0.61, P=0.044), whereas constrictor response to acetylcholine was similar [active tension, sensitivity (-logEC10), and reactivity (slope)]. The sole effect of the specific arginase inhibitor Nomega-hydroxy-nor-L-arginine (1 microM) was to decrease sensitivity in COPD patients, whereas 1 mM NG-nitro-L-arginine methyl ester unexpectedly decreased resting tension because of a non-cGMP-dependent effect. In conclusion, an upregulation of NOS2 expression in COPD patients is involved in airway tone regulation and functional airflow limitation, whereas increased arginase activity is involved in airway sensitivity.     

Correlation between Binding Affinity and Necrosis-Inducing Activity of Mutant AVR9 Peptide Elicitors

The race-specific peptide elicitor AVR9 of the fungus Cladosporium fulvum induces a hypersensitive response only in tomato (Lycopersicon esculentum) plants carrying the complementary resistance gene Cf-9 (MoneyMaker-Cf9). A binding site for AVR9 is present on the plasma membranes of both resistant and susceptible tomato genotypes. We used mutant AVR9 peptides to determine the relationship between elicitor activity of these peptides and their affinity to the binding site in the membranes of tomato. Mutant AVR9 peptides were purified from tobacco (Nicotiana clevelandii) inoculated with recombinant potato virus X expressing the corresponding avirulence gene Avr9. In addition, several AVR9 peptides were synthesized chemically. Physicochemical techniques revealed that the peptides were correctly folded. Most mutant AVR9 peptides purified from potato virus X::Avr9-infected tobacco contain a single N-acetylglucosamine. These glycosylated AVR9 peptides showed a lower affinity to the binding site than the nonglycosylated AVR9 peptides, whereas their necrosis-inducing activity was hardly changed. For both the nonglycosylated and the glycosylated mutant AVR9 peptides, a positive correlation between their affinity to the membrane-localized binding site and their necrosis-inducing activity in MoneyMaker-Cf9 tomato was found. The perception of AVR9 in resistant and susceptible plants is discussed.     

Rab11b and its effector Rip11 regulate the acidosis‐induced traffic of V‐ATPase in salivary ducts

Redistribution of acid‐base transporters is a crucial regulatory mechanism for many types of cells to cope with extracellular pH changes. In epithelial cells, however, translocation of acid‐base transporters ultimately leads to changes in vectorial transport of H⁺ and HCO. We have previously shown that the bicarbonate‐secreting epithelium of salivary ducts responds to changes of systemic acid‐base balance by adaptive redistribution of H⁺ and HCO transporters, thereby influencing the ionic composition and buffering capacity of saliva. However, the specific proteins involved in regulated vesicular traffic of acid‐base transporters are largely unknown. In the present study we have investigated the impact of Rab11 family members on the acidosis‐induced trafficking of the vacuolar‐type H⁺‐ATPase (V‐ATPase) in salivary duct cells in vitro using the human submandibular cell line of ductal origin HSG as an experimental model. The results show that Rab11b is expressed in salivary ducts and exhibits a significantly higher co‐localization with V‐ATPase than Rab11a and Rab25. We also show that Rab11 but not Rab25 interacts with the ε subunit of V‐ATPase. Extracellular acidosis up‐regulates Rab11b expression and protein abundance in HSG cells and causes translocation of the V‐ATPase from intracellular pools toward the plasma membrane. Loss‐of‐function experiments using specific siRNA either against Rab11b or against its effector Rip11 prevent acidosis‐induced V‐ATPase translocation. These data introduce Rab11b as a crucial regulator and Rip11 as mediator of acidosis‐induced V‐ATPase traffic in duct cells of submandibular gland. J. Cell. Physiol. 226: 638–651, 2011. © 2010 Wiley‐Liss, Inc.     

Recombination-Dependent Oligomerization of Human Papillomavirus Genomes upon Transient DNA Replication

We describe the extensive and progressive oligomerization of human papillomavirus (HPV) genomes after transfection into the U2OS cell line. The HPV genomic oligomers are extrachromosomal concatemeric molecules containing the viral genome in a head-to-tail orientation. The process of oligomerization does not depend on the topology of the input DNA, and it does not require any other viral factors besides replication proteins E1 and E2. We provide evidence that oligomerization of the HPV18 and HPV11 genomes involves homologous recombination. We also demonstrate oligomerization of the HPV18 and HPV11 genomes in SiHa, HeLa, and C-33 A cell lines and provide examples of oligomeric HPV genomes in clinical samples obtained from HPV-infected patients.     

Effect of the insecticidal Galanthus nivalis agglutinin on metabolism and the activities of brush border enzymes in the rat small intestine

With increasing use of lectin genes in crop plants to improve insect resistance, the dietary exposure of humans to lectins will rise and it is necessary to assess whether the presently most favored insecticidal lectin, Galanthus nivalis agglutinin, would be harmful for mammals. Effects of Galanthus nivalis agglutinin on gut and brush border enzymes were studied in rats over a 10-day dietary exposure and compared with those of a known antinutrient, phytohaemagglutinin. At a level that provides insecticidal protection for plants but did not reduce the growth of young rats, Galanthus nivalis agglutinin had negligible effects on the weight and length of the small intestine even though there was a slight, but significant hypertrophy of this tissue. However, the activities of brush border enzymes were affected; sucrase-isomaltase activity was nearly halved and those of alkaline phosphatase and aminopeptidase were significantly increased. Although most of the changes in gut metabolism caused by the incorporation of Galanthus nivalis agglutinin in the diet were less extensive than those found with toxic phytohaemagglutinin, some of them may be potentially deleterious. Thus, further and longer animal studies are needed to establish whether it is safe to use Galanthus nivalis agglutinin in transgenic plants destined for human consumption.