Optimizing the MALDI-TOF-MS Observation of Peptides Containing Disulfide Bonds

The observation of peaks corresponding to both disulfide-bonded and reduced peptides in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of disulfides could suggest that the samples are either mixtures prior to analysis or that the measurement process has converted single compounds into mixtures. This is an important distinction when characterizing potentially disulfide-bonded peptides obtained from proteolyzed proteins or from oxidized synthetic peptides. It is well documented that disulfides can undergo in-source decay (ISD) when using a 337-nm laser. However, the mixed matrix 2-(4-hydroxyphenylazo)benzoic acid:α-cyano-4-hydroxycinnamic acid (1:10) not only suppresses the ISD reduction of disulfides to thiols but allows the same low threshold laser power generally used with α-cyano-4-hydroxycinnamic acid to be applied.     

Reduced genetic diversity associated with the northern expansion of an amphibian species with high habitat specialization,Ascaphus truei,resolved using two types of genetic markers

Reconstruction of historical relationships between geographic regions within a species’ range can indicate dispersal patterns and help predict future responses to shifts in climate. Ascaphus truei (coastal tailed frog) is an indicator species of the health of forests and perennial streams in the Coastal and Cascade Mountains of the Pacific Northwest of North America. We used two genetic techniques—microsatellite and genotype‐by‐sequencing (GBS)—to compare the within‐region genetic diversity of populations near the northern extent of the species’ range (British Columbia, Canada) to two geographic regions in British Columbia and two in Washington, USA, moving toward the core of the range. Allelic richness and heterozygosity declined substantially as latitude increased. The northernmost region had the lowest mean expected heterozygosities for both techniques (microsatellite, M = 0.20, SE = 0.080; GBS, M = 0.025, SE = 0.0010) and the southernmost region had the highest (microsatellite, M = 0.88, SE = 0.054; GBS, M = 0.20, SE = 0.0029). The northernmost regions (NC and MC) clustered together in population structure models for both genetic techniques. Our discovery of reduced diversity may have important conservation and management implications for population connectivity and the response of A. truei to climate change.     

Muscle acidosis during static exercise is associated with calf vasoconstriction

In this study we measured (n = 6) the phosphocreatine-to-inorganic phosphate ratio (PCr/Pi), Pi, and pH with 31P-nuclear magnetic resonance (31P-NMR) in the human forearm during static work at 30% of maximal voluntary contraction (MVC) for 2 min followed immediately by 3 min of circulatory arrest (forearm arterial occlusion). Static exercise, with its central volitional and skeletal muscle metabolic and mechanical afferent components, caused a rise in heart rate (HR, 32%), blood pressure (BP, 29%), and calf vascular resistance (calf R, 30%). During forearm occlusion after static exercise, HR returned to base line, the increase in BP was attenuated by 30%, and calf R remained elevated and unchanged. The percent change in calf R was correlated with forearm cellular pH (R = 0.56, P less than 0.001) but only weakly associated with PCr/Pi (R = 0.33, P less than 0.042). 30% MVC for 1 min followed by arterial occlusion (3 min) reduced PCr/Pi by 65% and pH by 0.16 U (P less than 0.05). Calf R was unchanged. Circulatory arrest alone (20 min) caused no change in either pH or calf R but large changes in PCr/Pi (50% reduction). We conclude that 1) there is an association between forearm cellular acidosis and calf vasconstriction during static forearm exercise and 2) large changes in PCr/Pi without concomitant changes in pH are not associated with changes in calf R.     

On-Off Kinetics of Engagement of FNI Modules of Soluble Fibronectin by β-Strand Addition

Intrinsically disordered sequences within bacterial adhesins bind to E-strands in the β-sheets of multiple FNI modules of fibronectin (FN) by anti-parallel β-strand addition, also called tandem β-zipper formation. The FUD segment of SfbI of Streptococcus pyogenes and Bbk32 segment of BBK32 of Borrelia burgdorferi, despite being imbedded in different adhesins from different bacteria, target the same FNI modules, ^2–5,8–9FNI, in the N-terminal 70-kDa region (FN70K) of FN. To facilitate further comparisons, FUD, Bbk32, two other polypeptides based on SfbI that target ^1–5FNI (HADD) and ^2–5FNI (FRD), and mutant Bbk32 (ΔBbk32) were produced with fluorochromes placed just outside of the binding sequences. Unlabeled FUD competed ~1000-fold better for binding of labeled Bbk32 to FN than unlabeled Bbk32 competed for binding of labeled FUD to FN. Binding kinetics were determined by fluorescence polarization in a stopped-flow apparatus. On-rates for FUD, Bbk32, HADD, and FRD were similar, and all bound more rapidly to FN70K fragment than to full length FN. In stopped-flow displacement and size exclusion chromatographic assays, however, k_off for FUD or HADD to FN70K or FN was considerably lower compared to k_off of FRD or Bbk32. FUD and Bbk32 differ in the spacing between sequences that interact with ³FNI and ⁴FNI or with ⁵FNI and ⁸FNI. ΔBbk32, in which 2 residues were removed from Bbk32 to make the spacing more like FUD, had a k_off intermediate between that of Bbk32 and FUD. These results indicate a “folding-after-binding” process after initial association of certain polypeptide sequences to FN that results in formation of a stable complex and is a function of number of FNI modules engaged by the polypeptide, spacing of engagement sites, and perhaps flexibility within the polypeptide-FN complex. We suggest that contributions of SfbI and BBK32 adhesins to bacterial pathogenicity may be determined in part by stability of adhesin-FN complexes.     

The Lesion-Mimic Mutant cpr22 Shows Alterations in Abscisic Acid Signaling and Abscisic Acid Insensitivity in a Salicylic Acid-Dependent Manner

A number of Arabidopsis (Arabidopsis thaliana) lesion-mimic mutants exhibit alterations in both abiotic stress responses and pathogen resistance. One of these mutants, constitutive expresser of PR genes22 (cpr22), which has a mutation in two cyclic nucleotide-gated ion channels, is a typical lesion-mimic mutant exhibiting elevated levels of salicylic acid (SA), spontaneous cell death, constitutive expression of defense-related genes, and enhanced resistance to various pathogens; the majority of its phenotypes are SA dependent. These defense responses in cpr22 are suppressed under high-humidity conditions and enhanced by low humidity. After shifting plants from high to low humidity, the cpr22 mutant, but not the wild type, showed a rapid increase in SA levels followed by an increase in abscisic acid (ABA) levels. Concomitantly, genes for ABA metabolism were up-regulated in the mutant. The expression of a subset of ABA-inducible genes, such as RD29A and KIN1/2, was down-regulated, but that of other genes, like ABI1 and HAB1, was up-regulated in cpr22 after the humidity shift. cpr22 showed reduced responsiveness to ABA not only in abiotic stress responses but also in germination and stomatal closure. Double mutant analysis with nahG plants that degrade SA indicated that these alterations in ABA signaling were attributable to elevated SA levels. Furthermore, cpr22 displayed suppressed drought responses by long-term drought stress. Taken together, these results suggest an effect of SA on ABA signaling/abiotic stress responses during the activation of defense responses in cpr22.Plants have evolved a large number of defense systems to protect themselves against pathogen invasion. Whether these defenses are successful depends on the speed and intensity of their activation. The first line of defense is the basal immune system that is activated by molecules that are conserved among many pathogens (microbe-associated molecular patterns). Pathogens in turn have evolved a number of effector molecules that can block the basal resistance response (Jones and Dangl, 2006; Bent and Mackey, 2007). A second, stronger response to pathogen infection is mediated by resistance (R) genes that can interact with particular effectors (previously termed avirulence factors) from the pathogen or that can recognize effector-induced modifications of plant proteins (Flor, 1971; Bent and Mackey, 2007). One defense mechanism activated by R gene-mediated pathogen recognition is the hypersensitive response (HR), which is characterized by apoptosis-like cell death at and around the site of pathogen entry (Hammond-Kosack and Jones, 1996; Heath, 2000). HR development is usually accompanied by an increase in salicylic acid (SA) and the accumulation of defense-related proteins such as the pathogenesis-related (PR) proteins (Vlot et al., 2008). At later times after infection, elevated SA levels and PR gene expression are also detected in the uninoculated leaves, concurrent with the development of systemic acquired resistance (SAR), a long-lasting, broad-based resistance to subsequent infection (Durrant and Dong, 2004; Grant and Lamb, 2006; Vlot et al., 2008).Many studies have demonstrated that SA is an important signaling molecule in the pathways conferring local and systemic resistance (Dempsey et al., 1999; Vlot et al., 2008). To identify other components in the pathogen resistance signal transduction pathway, many Arabidopsis (Arabidopsis thaliana) mutants with altered resistance to pathogens have been isolated. One class exhibits constitutively increased SA levels and PR gene expression as well as heightened resistance to pathogen infection. This group includes dnd1, dnd2/hlm1, copine1 (cpn1), constitutive expresser of PR genes22 (cpr22), and ssi4 (Yu et al., 1998; Jambunathan et al., 2001; Yoshioka et al., 2001; Shirano et al., 2002; Balague et al., 2003; Jurkowski et al., 2004). The majority of these mutants share similar phenotypes such as spontaneous HR-like lesions and thus are categorized as lesion-mimic mutants (Moeder and Yoshioka, 2008). Interestingly, it has been reported that some lesion-mimic mutants are environmentally sensitive (i.e. their resistance phenotypes are conditional; Moeder and Yoshioka, 2009). For instance, under high-humidity conditions such as on agar plates or when grown at high temperature, both the spontaneous HR and the enhanced pathogen resistance are suppressed (Jambunathan et al., 2001; Yoshioka et al., 2001; Jambunathan and McNellis, 2003; Xiao et al., 2003; Zhou et al., 2004; Noutoshi et al., 2005). On the other hand, relatively low humidity or cold temperature enhances their SA-related phenotypes, including HR-like cell death (Jambunathan et al., 2001; Zhou et al., 2004).Some of these lesion-mimic phenotypes are caused by mutations in R genes, such as SSI4 and SLH1 (Shirano et al., 2002; Noutoshi et al., 2005), or by the overexpression of an R gene, such as RPW8 (Xiao et al., 2003), indicating the involvement of environmental factors on R gene-mediated signaling pathway(s). Indeed, similar environmental effects were also reported for the response of wild-type R genes. It is well known that the HR induced by the recognition of Tobacco mosaic virus by the N protein can be completely suppressed when plants are kept above 28°C. When plants are shifted back to 22°C, the HR starts to develop, indicating that there is a temperature-sensitive step in the signaling pathway (Samuel, 1931). Both basal and R gene-mediated resistance against the bacterial pathogen, Pseudomonas syringae, is attenuated by a moderate increase in temperature (Wang et al., 2009). It has also been reported that high humidity (greater than 95% relative humidity [RH]) delayed or reduced the HR and other resistance responses induced by the interaction of the Cladosporium fulvum avirulence factors Avr2, Avr4, and Avr9 and their cognate tomato R proteins Cf-2, Cf-4, and Cf-9, respectively (Hammond-Kosack et al., 1996; May et al., 1996; Wang et al., 2005). These findings suggest that there is a universal factor(s) in defense signaling that is environmentally sensitive.Abscisic acid (ABA) controls various environmental (abiotic) stress responses, including drought, salinity, and temperature stress, and many components involved in these responses have been identified (Shinozaki et al., 2003). Additionally, it is becoming clear that ABA is also involved in biotic stress responses in a complex manner. For instance, treatment with exogenous ABA prior to pathogen infection induces enhanced susceptibility in various plant species (Mauch-Mani and Mauch, 2005). Mohr and Cahill (2003, 2006) suggested that the mechanisms behind this phenomenon are likely related to the antagonistic effect of ABA on SA signaling. Similarly, several groups have reported that virulent P. syringae DC3000 enhances the production of ABA during pathogenesis (Schmelz et al., 2003; de Torres-Zabala et al., 2007). Furthermore, Yasuda et al. (2008) suggested the antagonism between SA and ABA signaling in SAR. These studies suggest that ABA plays a negative role in pathogen resistance. In contrast, Melotto and colleagues (2006) reported that ABA-dependent stomata closure is part of plant innate immunity against bacterial invasion and that SA is required for this response. They also reported that aba3-1, an ABA-deficient mutant, was more susceptible to P. syringae DC3000, suggesting a positive role of ABA in innate immunity (Melotto et al., 2006).Here, we attempt to characterize the effects of humidity on pathogen resistance responses using the lesion-mimic mutant cpr22. Previously, we reported that most phenotypes of cpr22, such as spontaneous lesion formation, SA accumulation, and constitutive PR gene expression, were suppressed under high RH (Yoshioka et al., 2001). cpr22 contains a deletion that fuses two cyclic nucleotide-gated ion channel (CNGC)-encoding genes, AtCNGC11 and AtCNGC12, generating the novel chimeric AtCNGC11/12 (Yoshioka et al., 2006). We proposed that the expression of AtCNGC11/12 activates pathogen resistance responses through the same signal transduction pathway used by R genes and that cell death induced by the expression of AtCNGC11/12 is HR-like programmed cell death (Yoshioka et al., 2006; Urquhart et al., 2007). Here, we report intriguing alterations in ABA-related phenotypes in cpr22. Our data demonstrate that elevated SA accumulation is the cause of these alterations, suggesting complex SA-ABA cross talk during lesion formation.     

Binding and degradation of α2-macroglobulin by cultured fibroblasts

We studied the interactions of α₂-macroglobulin, a major protease inhibitor of plasma and of serum-containing culture medium, with cultured fibroblasts. Iodinated human α₂-macroglobulin bound specifically to washed cell layers of cultured human fibroblasts. At 0–4°C, binding was saturated at a concentration of 10–20 μg/ml. At 37°C, radiolabel appered in the medium in a form soluble in 10% trichloroacetic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that ingested iodinated α₂-macroglobulin transiently forms a complex with a trypsin-like protease. Indirect immunofluorescence demonstrated α₂-macroglobulin in vacuoles of fibroblasts grown in 10% human serum or incubated with purified α₂-macroglobulin. Fibroblasts transformed by SV-40 (VA-13 cells) bound and degraded less ¹²⁵I-labeled α₂-macroglobulin than non-transformed fibroblasts and had fewer vacuoles containing α₂-macroglobulin. These observations indicate that cultured fibroblasts bind, take up by endocytosis, and degrade α₂-macroglobulin. Binding and endocytosis of α₂-macroglobulin by a cell may be a means of modulating proteases in the micro-environment of the cell and during endocytosis.