The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

Influence of calcium on proliferation and phenotype alteration of cardiomyocyte in vitro

An accelerated weight gain is noted in the heart of Ca-deficient, hypertensive chick embryos maintained in a shell-less culture in vitro. We previously observed that the Ca handling property of cardiomyocytes isolated from the shell-less embryo is altered, i.e., faster Ca uptake, suggesting a requirement for adequate Ca supply and/or proper Ca handling in embryonic cardiac development. In this study, we have examined the function of Ca on cardiomyocytes by analyzing the effects of (1) various Ca concentration in the culture medium (NCa, 1.8 mmol/L; HCa, 2.8 mmol/L; LCa, 0.9 mmol/L), and (2) various modulators of Ca handling on cell proliferation and phenotype regulation in chick embryonic cardiomyocytes. The analytical parameters included cell number, DNA content, expression of cell cycle–specific and cardiomyocyte-specific proteins, and creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) enzyme activities. Cell number and total DNA were significantly larger (P < 0.01) in LCa cultures compared with those in NCa. The level of LDH was elevated (P < 0.01), but that of CPK was lowered in LCa. Expression of the G1-S–specific protein PCNA was raised, but that of the contractile proteins myosin and tropomyosin was substantially suppressed in LCa; in HCa, the cells did not proliferate as well, whereas the level of contractile proteins was higher. Thapsigargin, a sarcoplasmic reticulum (SR)-specific, Ca-ATPase inhibitor, simulated the effects of LCa by enhancing cell proliferation and lowering the expression of tropomyosin. These results suggest that culturing in low Ca concentration and inhibition of SR Ca pumping enhance myocardial cell proliferation and suppress sarcomeric protein expression, perhaps by inducing cellular de-differentiation. The in vitro effects of medium Ca concentration and Ca handling modulators on cardiomyocytes also suggest that the in vivo cardiomegaly of the SL embryos is a direct result of Ca-deficiency, and that Ca is important in the phenotype regulation of cardiomyocytes. J. Cell. Physiol. 177:289–298, 1998. © 1998 Wiley-Liss, Inc.     

Bacterial symbionts of the giant jewel stinkbug Eucorysses grandis (Hemiptera: Scutelleridae)

Microbiological characterization of gut symbiotic bacteria in a limited number of stinkbugs of the families Acanthosomatidae, Plataspidae, Pentatomidae, Scutelleridae, Parastrachiidae, Alydidae and Pyrrhocoridae has shown symbiotic association with midgut bacteria to be common in phytophagous taxa of these heteropteran insects. Here we investigated the midgut bacterial symbiont of Eucorysses grandis, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified in insects from five different geographic origins. The bacterium was detected in 64 of 64 insects sampled from three host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the Gammaproteobacteria, neither closely related to the gut symbiont of another scutellerid stinkbug, Cantao ocellatus, nor to gut symbionts of other stinkbugs. Diagnostic PCR, in situ hybridization and electron microscopy demonstrated that the bacterium is located extracelluarly, in the midgut fourth section, which possesses crypts. These results indicate that the primary gut symbionts have multiple evolutionary origins in the Scutelleridae. A Sodalis-allied facultative symbiont was also identified in some insects from natural populations. Biological aspects of the primary gut symbiont and the secondary Sodalis-allied symbiont are discussed.     

Primary Gut Symbiont and Secondary,Sodalis-Allied Symbiont of the Scutellerid Stinkbug Cantao ocellatus

Symbiotic associations with midgut bacteria have been commonly found in diverse phytophagous heteropteran groups, where microbiological characterization of the symbiotic bacteria has been restricted to the stinkbug families Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae. Here we investigated the midgut bacterial symbiont of Cantao ocellatus, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified from the insects of different geographic origins. The bacterium was detected in all 116 insects collected from 9 natural host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the Gammaproteobacteria, not closely related to gut symbionts of other stinkbugs. Diagnostic PCR and in situ hybridization demonstrated that the bacterium is extracellularly located in the midgut 4th section with crypts. Electron microscopy of the crypts revealed a peculiar histological configuration at the host-symbiont interface. Egg sterilization experiments confirmed that the bacterium is vertically transmitted to stinkbug nymphs via egg surface contamination. In addition to the gut symbiont, some individuals of C. ocellatus harbored another bacterial symbiont in their gonads, which was closely related to Sodalis glossinidius, the secondary endosymbiont of tsetse flies. Biological aspects of the primary gut symbiont and the secondary Sodalis-allied symbiont are discussed.Insects are among the largest animal groups on the earth, embracing 750,000 to several millions of species (37, 52). Diverse insects are symbiotically associated with microorganisms, especially bacteria (5-7). In some insects, symbiotic bacteria are harbored in specialized host cells called bacteriocytes (or mycetocytes), constituting obligate mutualistic associations. For example, Buchnera aphidicola is harbored within bacteriocytes in the abdominal body cavity of almost all aphids and provides essential amino acids that are lacking in the phloem sap diet of the insects (9, 47). Wigglesworthia glossinidia is localized in a midgut-associated bacteriome of tsetse flies and plays pivotal roles in biosynthesis of B vitamins that are deficient in the vertebrate blood diet of the insects (2, 34). These obligate endocellular symbionts are often collectively referred to as “primary symbionts.”In contrast, there are facultative endosymbiotic microorganisms not essential for their host insects, often collectively called “secondary symbionts.” For example, many aphids are known to harbor various facultative symbionts, which belong to distinct lineages in the Gamma- and Alphaproteobacteria (33, 43) and the Mollicutes (10). While the majority of those facultative bacteria are either parasitic or commensalistic for their hosts, some of them affect the host fitness beneficially in particular ecological contexts (29, 32, 36, 44, 51). In addition to the obligate primary symbiont Wigglesworthia, tsetse flies harbor the facultative secondary symbiont Sodalis glossinidius, whose biological function for the hosts is currently elusive (3, 8).Members of the suborder Heteroptera, known as true bugs and consisting of over 38,000 described species, are characterized by their sucking mouthparts, half-membranous forewings, and incomplete metamorphosis (46). In the Heteroptera, symbiotic associations with bacteria are mainly found in phytophagous groups, especially in stinkbugs of the infraorder Pentatomomorpha. These stinkbugs generally possess many sacs or tubular outgrowths, called crypts or ceca, in a posterior region of the midgut, whose lumen is densely populated by a specific bacterial symbiont (7, 16). In some cases, experimental elimination of the symbiotic bacteria resulted in retarded growth and high mortality of the host insects (1, 13, 21, 26, 27, 39), indicating that these gut symbionts play important biological roles. Most of the gut symbionts are vertically transmitted through host generations by such mechanisms as egg surface contamination in the families Pentatomidae and Acanthosomatidae (1, 27, 39, 40, 42), coprophagy in the Cydnidae and Coreidae (22, 45), and capsule transmission in the Plataspidae (20), whereas a case of environmental acquisition has been reported from the Alydidae (26). Thus far, gut symbiotic bacteria of some members of the Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae have been characterized using molecular techniques (21, 23, 25, 27, 38), while phylogenetic and biological aspects of gut symbiotic bacteria have been untouched in many other stinkbug groups.These gut symbiotic bacteria are, despite their extracellular localization, regarded as “primary symbionts” of the stinkbugs. On the other hand, some stinkbugs may, in addition to the gut symbiotic bacteria, also be associated with facultative “secondary symbionts.” For example, Wolbachia infections have been detected from diverse stinkbugs, most of which are probably of parasitic or commensalistic nature (24). Besides Wolbachia, there has been no report on facultative, secondary symbionts from stinkbugs.Members of the family Scutelleridae, often referred to as jewel bugs or shield-backed bugs, are stinkbugs characterized by their greatly enlarged convex scutellum that usually covers the entire abdomen. Some tropical species are also known for their vivid and beautiful body coloration (46). The family contains approximately 80 genera and 450 species, and in Japan, at least 7 genera and 9 species have been recorded (50). In the early 20th century, the presence of symbiotic bacteria was histologically described in midgut crypts of several scutellerid species (16, 31, 42). Since these pioneer works, however, no studies have been conducted on the symbiotic bacteria of scutellerid stinkbugs.Here we investigated the midgut symbiont of Cantao ocellatus, a scutellerid stinkbug widely distributed in Asian countries, including Japan, and known to guard their eggs and newborn nymphs (Fig. ​(Fig.1A)1A) (50). In addition to the gut symbiont, we also identified a Sodalis-allied facultative secondary symbiont from gonads of the insect.Open in a separate windowFIG. 1.(A) Adult female of Cantao ocellatus, guarding hatchlings under her body. (B) Dissected midgut from an adult female of C. ocellatus. 1st, midgut 1st section; 2nd, midgut 2nd section; 3rd, midgut 3rd section; 4th, midgut 4th section with crypts; hg, hindgut. (C) Enlarged image of the midgut 4th section with crypts. Arrowheads indicate three rows of crypts, while a fourth row is hidden behind. Glandular crypts (gc) are developed in adult females specifically, which may be involved in egg surface contamination with the symbiont. (D) An in situ hybridization image of the midgut 4th section, in which red and green signals indicate the gut symbiont and the host nucleus, respectively. Each arrow shows a crypt. (E) An enlarged image of the symbiotic bacteria in the crypts.     

Is parainfluenza virus a threatening virus for human cancer cell lines?

Immortalized cell lines, such as human cancer cell lines, are an indispensable experimental resource for many types of biological and medical research. However, unless the cell line has been authenticated prior to use, interpretation of experimental results may be problematic. The potential problems this may cause are illustrated by studies in which authentication of cell lines has not been carried out. For example, immortalized cell lines may unknowingly be infected with viruses that alter their characteristics. In fact, parainfluenza virus type 5 (PIV5) poses a threat to the use of immortalized cell lines in biological and medical research; PIV5 infection significantly alters cellular physiology associated with the response to interferon. If PIV5 infection is widespread in immortalized cell lines, then a very large number of published studies might have to be re-evaluated. Fortunately, analyses of a large number of immortalized cell lines indicate that PIV5 infection is not widespread.     

Esophageal stimulation by hydrochloric acid causes neurogenic inflammation in the airways in guinea pigs

Hamamoto, Junji, Hirotsugu Kohrogi, Osamu Kawano,Hajime Iwagoe, Kazuhiko Fujii, Nahomi Hirata, and Masayuki Ando.Esophageal stimulation by hydrochloric acid causes neurogenicinflammation in the airways in guinea pigs. J. Appl.Physiol. 82(3): 738-745, 1997.Toinvestigate whether tachykinins are released in the airways in responseto stimulation of the esophagus, we studied the airway plasmaextravasation induced by intraesophageal HCl in the presence or absenceof neutral endopeptidase inhibitor phosphoramidon and NK₁-receptor antagonist FK-888 inanesthetized guinea pigs. The airway plasma leakage wasevaluated by measuring extravasated Evans blue dye in the animalspretreated with propranolol and atropine. Infusion of 1 N HCl into theesophagus significantly increased plasma extravasation in the trachea.Phosphoramidon significantly potentiated plasma extravasation in thetrachea and main bronchi, whereas FK-888 significantly inhibited that extravasation in a dose-related manner. In the capsaicin-treated animals, airway plasma extravasation was completely inhibited even inthe presence of phosphoramidon. Tracheal plasma extravasation potentiated by phosphoramidon was significantly inhibited in the bilateral vagotomized animals. These results suggest that1) tachykinin-like substances arereleased to cause plasma extravasation in the airways as a result ofintraesophageal HCl stimulation and2) there are neural pathwayscommunicating between the esophagus and airways, including the vagusnerve.

     

Acetylation of αA-crystallin in the human lens: effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(ε)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

Aggrelyte-2 promotes protein solubility and decreases lens stiffness through lysine acetylation and disulfide reduction: Implications for treating presbyopia

Aging proteins in the lens become increasingly aggregated and insoluble, contributing to presbyopia. In this study, we investigated the ability of aggrelyte-2 (N,S-diacetyl-L-cysteine methyl ester) to reverse the water insolubility of aged human lens proteins and to decrease stiffness in cultured human and mouse lenses. Water-insoluble proteins (WI) of aged human lenses (65–75 years) were incubated with aggrelyte-2 (500 μM) for 24 or 48 h. A control compound that lacked the S-acetyl group (aggrelyte-2C) was also tested. We observed 19%–30% solubility of WI upon treatment with aggrelyte-2. Aggrelyte-2C also increased protein solubility, but its effect was approximately 1.4-fold lower than that of aggrelyte-2. The protein thiol contents were 1.9- to 4.9-fold higher in the aggrelyte-2- and aggrelyte-2C-treated samples than in the untreated samples. The LC–MS/MS results showed N^ε-acetyllysine (AcK) levels of 1.5 to 2.1 nmol/mg protein and 0.6 to 0.9 nmol/mg protein in the aggrelyte-2- and aggrelyte-2C-treated samples. Mouse (C57BL/6J) lenses (incubated for 24 h) and human lenses (incubated for 72 h) with 1.0 mM aggrelyte-2 showed significant decreases in stiffness with simultaneous increases in soluble proteins (human lenses) and protein-AcK levels, and such changes were not observed in aggrelyte-2C-treated lenses. Mass spectrometry of the solubilized protein revealed AcK in all crystallins, but more was observed in α-crystallins. These results suggest that aggrelyte-2 increases protein solubility and decreases lens stiffness through acetylation and disulfide reduction. Aggrelyte-2 might be useful in treating presbyopia in humans.     

Comparative proteomic analysis of proliferating and functionally differentiated mammary epithelial cells

Proliferation and differentiation of mammary epithelial cells are governed by hormonal stimuli, cell-cell, and cell-matrix interactions. Terminal differentiation of mammary epithelial cells depends upon the action of the lactogenic hormones, insulin, glucocorticoids, and prolactin that enable them to synthesize and secrete milk proteins. These differentiated cells are polarized and carry out vectorial transport of milk constituents across the apical plasma membrane. To gain additional insights into the mechanisms governing differentiation of mammary epithelial cells, we identified proteins whose expression distinguishes proliferating from differentiated mammary epithelial cells. For this purpose we made use of the HC11 mammary epithelial line, which is capable of differentiation in response to lactogenic hormones. Using two-dimensional gel electrophoresis and mass spectrometry, we found about 60 proteins whose expression levels changed in between these two differentiation states. Bioinformatic analysis revealed differential expression of cytoskeletal components, molecular chaperones and regulators of protein folding and stability, calcium-binding proteins, and components of RNA-processing pathways. The actin cytoskeleton is asymmetrically distributed in differentiated epithelial cells, and the identification of proteins involved in mRNA binding and localization suggests that asymmetry might in part be achieved by controlling cellular localization of mRNAs. The proteins identified provide insights into the differentiation of mammary epithelial cells and the regulation of this process.