Immunoaffinity extraction of a peptide modified by a small molecule

We investigated the affinity extraction conditions required to isolate peptide fragments modified with small molecules using an antibody that has a high affinity for the target small molecule. Investigation of antibody conformation and the retention behavior of the modified peptides on an immunosorbent matrix demonstrated the importance in efficient extraction of both the dissociation of hydrophobic interactions and the breakdown of the antibody conformation. Hydrophobic interactions, which anchor the small ligand to the paratope, were retained even when the three-dimensional structure of the antibody disintegrated in an acidic solution. For efficient extraction of a target peptide modified by a small molecule, it is therefore important to use an acidic solvent containing an organic modifier such as methanol at a concentration greater than 40% (v/v). We demonstrated the feasibility of this immunoaffinity extraction by application of this procedure to the analysis of modified peptide fragments obtained from a digestion of human serum albumin. The peptide fragments were affinity labeled with chenodeoxycholyl adenylate for analysis of the chenodeoxycholate binding site. This purification method could isolate the low levels of modified peptide contained in the reaction mixture, despite the presence of appreciable quantities of unlabeled peptide fragments.     

Nutrient-induced alpha-amylase and protease activity is regulated by crustacean cardioactive peptide (CCAP) in the cockroach midgut

The midgut plays a major role in digestion and absorption of nutrients in insects, and contains endocrine cells throughout the epithelial layer that express neuropeptides, including crustacean cardioactive peptide (CCAP). In the present study, we demonstrate regulation of digestive enzyme activities by CCAP in response to nutrient ingestion in the cockroach, Periplaneta americana. The midgut of the cockroach exhibits maximal alpha-amylase and protease activities 3 h after intake of either starch or casein, but not of non-nutrients. Similar time-dependent responses of CCAP expression in midgut endocrine cells were observed after feeding starch and casein, but not after non-nutrients. We also show that incubation of the dissected midgut with CCAP leads to an increase in alpha-amylase and protease activity in a time-dependent manner, with the maximal activity at 2 h. Taken together, our data indicate the existence of an inducible mechanism where endocrine cells in the midgut are stimulated to synthesize and secrete CCAP by nutrients, and CCAP then up-regulates the activity of digestive enzymes.     

Cellular uptake of S413-PV peptide occurs upon conformational changes induced by peptide-membrane interactions

In face of accumulated reports demonstrating that uptake of some cell-penetrating peptides occurs through previously described endocytic pathways, or is a consequence of cell fixation artifacts, we conducted a systematic analysis on the mechanism responsible for the cellular uptake of the S4₁₃-PV karyophilic cell-penetrating peptide. The results reviewed here show that the S4₁₃-PV peptide is able to very efficiently accumulate inside live cells in a rapid, non-toxic and dose-dependent manner, through a mechanism distinct from endocytosis. Comparative analysis of peptide uptake by mutant cells lacking heparan sulfate proteoglycans demonstrates that, although not mandatory, their presence at cell surface facilitates the cellular uptake of the S4₁₃-PV peptide. Furthermore, we demonstrate that upon interaction with lipid vesicles, the S4₁₃-PV peptide undergoes significant conformational changes that are consistent with the formation of helical structures. Such conformational changes occur concomitantly with a penetration of the peptide into the lipid bilayer, strongly suggesting that the resulting helical structures are crucial for the non-endocytic cellular uptake of the S4₁₃-PV peptide. Overall, our data support that, rather than endocytosis, the cellular uptake of the S4₁₃-PV cell-penetrating peptide is a consequence of its direct translocation through cell membranes following conformational changes induced by peptide-membrane interactions.     

Evidence for activation of Amh gene expression by steroidogenic factor 1

The anti-Müllerian hormone gene (Amh) is responsible for regression in males of the Müllerian ducts. The molecular mechanism of regulation of chicken Amh expression is poorly understood. To investigate the regulation of chicken Amh expression, we have cloned Amh cDNAs from quail and duck as well as the promoter regions of the gene from chicken, quail, and duck. The expression patterns of Amh during embryonic development in these three species were found to be similar, suggesting that the regulatory mechanisms of Amh expression are conserved. The sequence of the proximal promoter of Amh contains a putative binding site for steroidogenic factor 1 (SF1), the protein product of which can up-regulate Amh in mammals. We showed here that SF1 is able to activate the chicken Amh promoter and binds to its putative SF1 binding site. These results suggest that SF1 plays a role in regulation of Amh expression in avian species.     

The Involvement of Lipid Peroxide-Derived Aldehydes in Aluminum Toxicity of Tobacco Roots

Oxidative injury of the root elongation zone is a primary event in aluminum (Al) toxicity in plants, but the injuring species remain unidentified. We verified the hypothesis that lipid peroxide-derived aldehydes, especially highly electrophilic α,β-unsaturated aldehydes (2-alkenals), participate in Al toxicity. Transgenic tobacco (Nicotiana tabacum) overexpressing Arabidopsis (Arabidopsis thaliana) 2-alkenal reductase (AER-OE plants), wild-type SR1, and an empty vector-transformed control line (SR-Vec) were exposed to AlCl₃ on their roots. Compared with the two controls, AER-OE plants suffered less retardation of root elongation under AlCl₃ treatment and showed more rapid regrowth of roots upon Al removal. Under AlCl₃ treatment, the roots of AER-OE plants accumulated Al and H₂O₂ to the same levels as did the sensitive controls, while they accumulated lower levels of aldehydes and suffered less cell death than SR1 and SR-Vec roots. In SR1 roots, AlCl₃ treatment markedly increased the contents of the highly reactive 2-alkenals acrolein, 4-hydroxy-(E)-2-hexenal, and 4-hydroxy-(E)-2-nonenal and other aldehydes such as malondialdehyde and formaldehyde. In AER-OE roots, accumulation of these aldehydes was significantly less. Growth of the roots exposed to 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal were retarded more in SR1 than in AER-OE plants. Thus, the lipid peroxide-derived aldehydes, formed downstream of reactive oxygen species, injured root cells directly. Their suppression by AER provides a new defense mechanism against Al toxicity.Aluminum (Al) is the most abundant metal in the earth''s crust and is a major factor limiting plant growth and productivity in acid soils, which cover about 50% of the world''s potentially arable land surface (Kochian, 1995; Kochian et al., 2004). The primary site of Al accumulation and toxicity is the root meristem, and inhibition of root elongation is the most notable symptom of Al toxicity (Delhaize and Ryan, 1995; Yamamoto et al., 2003). Al causes various adverse effects, such as disruption of signal transduction pathways, inhibition of cell division and ion fluxes, disruption of cytoskeletal dynamics, induced generation of reactive oxygen species (ROS), and disturbance of plasma membrane stability and function (Jones and Kochian, 1995; Blancaflor et al., 1998; Yamamoto et al., 2001, 2002; Kochian et al., 2004; Ma et al., 2007). Of all these toxic effects, the generation of ROS is observed rapidly and sustainably in roots after Al exposure. Al-induced generation of ROS has been shown in maize (Zea mays) and Allium cepa roots (Jones et al., 2006; Achary et al., 2008). Tahara et al. (2008) showed that ROS generated to a greater degree in Al-sensitive species than in Al-tolerant species. Yamamoto et al. (2002, 2003) have shown a correlation between ROS level and inhibition of growth capacity in cultured tobacco (Nicotiana tabacum) cells. Furthermore, ROS generation increases with increasing Al concentration (Achary et al., 2008; Xue et al., 2008). Generation of ROS appears to be a cause, rather than a result, of Al-induced cell injury, because high ROS scavenging ability resulted in enhanced Al tolerance (Devi et al., 2003; Ezaki et al., 2008). In addition, overexpression of genes encoding antioxidant enzymes (peroxidase and superoxide dismutase) conferred Al tolerance to the transgenic plants (Ezaki et al., 2000; Basu et al., 2001). Thus, ROS appears to be the primary factors that cause growth inhibition in Al-stressed roots.Downstream of ROS generation, lipid peroxidation is a common symptom of Al toxicity (Yamamoto et al., 2001), and it increases with increasing Al concentration (Achary et al., 2008). From animal cell studies, it is now recognized that the toxicity of lipid peroxide (LOOH) is largely ascribable to LOOH-derived aldehydes. In particular, α,β-unsaturated aldehydes, such as 4-hydroxy-(E)-2-nonenal (HNE) and acrolein, are strong electrophiles and readily modify proteins and nucleic acids (Esterbauer et al., 1991; Taylor et al., 2002; O''Brien et al., 2005; Møller et al., 2007). HNE causes depletion of glutathione, a decrease in protein thiols, disturbance of calcium homeostasis, inhibition of DNA, RNA, and protein synthesis, lactate release, morphological changes of cells, and finally leading to cell death (Esterbauer et al., 1991; Burcham, 1998). Increase of HNE has been observed in a wide range of human diseases, including Alzheimer''s disease, Parkinson''s disease, and mitochondrial complex 1 deficiency (Poli and Schaur, 2000).In plants, too, a close correlation between the level of LOOH-derived aldehydes (determined as thiobarbituric acid-reactive substances [TBARS]) and cellular damage has been shown under environmental stresses caused by heat, chilling, UV-B radiation, salinity, heavy metals, and Al (Ma et al., 2007; Ezaki et al., 2008). Their involvement in cellular damage has been demonstrated by the protective effects of the aldehyde-scavenging enzymes aldehyde dehydrogenase (Sunkar et al., 2003; Kotchoni et al., 2006) and aldehyde reductase (Oberschall et al., 2000; Hideg et al., 2003; Hegedüs et al., 2004) to confer tolerance against various environmental stresses when they were overexpressed in plants. In barley (Hordeum vulgare) roots, the formation of HNE in association with Al treatment was observed (Sakihama and Yamasaki, 2002). Occurrence of HNE in Arabidopsis (Arabidopsis thaliana) leaves under oxidative stress has been also deduced by detection of modified proteins in the mitochondria (Winger et al., 2007). HNE rapidly inhibited respiration in isolated potato (Solanum tuberosum) mitochondria by inactivating pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, NAD-malic enzyme (Millar and Leaver, 2000), and alternative oxidase (Winger et al., 2005). HNE and other 2-alkenals also inactivated photosynthesis in isolated chloroplasts (Mano et al., 2009). Arabidopsis contains 2-alkenal reductase (AER; E.C. 1.3.1.74) that catalyzes the reduction of the α,β-unsaturated bond of 2-alkenals to produce n-alkanals (Mano et al., 2002). Overexpression of AER in tobacco (Mano et al., 2005) and in Arabidopsis (Papdi et al., 2008) improved the tolerance to photooxidative stress and NaCl stress, respectively. Thus, accumulated observation indicates that LOOH-derived aldehydes, especially 2-alkenals, are commonly involved in oxidative damage in plant cells. Considering the critical importance of ROS in Al toxicity to roots, it is expected that 2-alkenals are produced and mediate damage in the stressed root cells.To evaluate the roles of LOOH-derived aldehydes in root injury under Al stress, we employed transgenic tobacco plants that overexpress the AER gene (AER-OE plants; Mano et al., 2005). With Al treatment, the roots of AER-OE accumulated Al and H₂O₂ to the same levels as those of the wild type, but they showed resistance to inhibition of elongation. Aldehyde analysis revealed that the Al treatment increased the contents of several toxic aldehydes, including HNE and acrolein in wild-type plants, but these aldehydes were significantly suppressed in the AER-OE plants. On the basis of these results, we propose that the inhibition of root growth by Al ions is induced by toxic aldehydes generated with ROS.     

Extraction and physico-chemical characterization of a versatile biodegradable polysaccharide obtained from green algae

During the last years, considerable attention has been given to different marine organisms, like algae, as potential sources of valuable materials. The continuous demand for novel materials and technologies is high and research on the underexploited marine green algae, including its polysaccharidic part—ulvan, has increased accordingly. In this research work, a novel method for extraction of ulvan from green algae is proposed and demonstrated successfully. Different characterization techniques were employed to characterize the isolated algal polysaccharide, namely, on what concerns its thermal trace and crystallinity. Upon heating, ulvan behaves as a non-meltable polysaccharide that is thermally stable before degradation at 220 °C. Ulvan is semi-crystalline in nature and possesses high hygroscopic features, as revealed in this research work. Due to its properties, ulvan can be considered, pure or modified, as a versatile biodegradable polymer for different applications, including tissue engineering and regenerative medicine.