Circulating Levels of Human salusin-β,a Potent Hemodynamic and Atherogenesis Regulator

Using bioinformatics analysis, we previously identified salusin-β, an endogenous bioactive peptide with diverse physiological activities. Salusin-β is abundantly expressed in the neuroendocrine system and in systemic endocrine cells/macrophages. Salusin-β acutely regulates hemodynamics and chronically induces atherosclerosis, but its unique physicochemical characteristics to tightly adhere to all types of plastic and glassware have prevented elucidation of its precise pathophysiological role. To quantitate plasma total salusin-β concentrations, we produced rabbit and chicken polyclonal antibodies against the C- and N-terminal end sequences, circumvented its sticky nature, and successfully established a sandwich enzyme-linked immunosorbent assay (ELISA). Salusin-β was abundantly present in the plasma of healthy volunteers, ranging from 1.9 to 6.6 nmol/L. Reverse phase-high performance liquid chromatography analysis showed that a single immunoreactive salusin-β peak coincided with synthetic authentic salusin-β. Plasma salusin-β concentrations were unaffected by postural changes and by potent vasopressin release stimuli, such as hypertonic saline infusion or smoking. However, salusin-β concentrations showed significant circadian variation; concentrations were high during the daytime and reached the lowest concentrations in the early morning. Plasma salusin-β levels in subjects with diabetes mellitus, coronary artery disease, and cerebrovascular disease showed distinctly higher levels than healthy controls. Patients with panhypopituitarism combined with complete central diabetes insipidus also showed significantly higher plasma salusin-β levels. Therefore, the ELISA system developed in this study will be useful for evaluating circulating total salusin-β levels and for confirming the presence of authentic salusin-β in human plasma. The obtained results suggest a limited contribution of the neuroendocrine system to peripheral total salusin-β concentrations and a role for plasma total salusin-β concentrations as an indicator of systemic vascular diseases.     

Production of Hybrid-IgG/IgA Plantibodies with Neutralizing Activity against Shiga Toxin 1

Shiga toxin 1 (Stx1) is a virulence factor of enterohemorrhagic Escherichia coli, such as the O157:H7 strain. In the intestines, secretory IgA (SIgA) is a major component of the immune defense against pathogens and toxins. To form SIgA, the production of dimeric IgA that retains biological activity is an important step. We previously established hybrid-IgG/IgA having variable regions of the IgG specific for the binding subunit of Stx1 (Stx1B) and the heavy chain constant region of IgA. If hybrid-IgG/IgA cDNAs can be expressed in plants, therapeutic or preventive effects may be expected in people eating those plants containing a “plantibody”. Here, we established transgenic Arabidopsis thaliana expressing dimeric hybrid-IgG/IgA. The heavy and light chain genes were placed under the control of a bidirectional promoter and terminator of the chlorophyll a/b-binding protein of Arabidopsis thaliana (expression cassette). This expression cassette and the J chain gene were subcloned into a single binary vector, which was then introduced into A. thaliana by means of the Agrobacterium method. Expression and assembly of the dimeric hybrid-IgG/IgA in plants were revealed by ELISA and immunoblotting. The hybrid-IgG/IgA bound to Stx1B and inhibited Stx1B binding to Gb₃, as demonstrated by ELISA. When Stx1 holotoxin was pre-treated with the resulting plantibody, the cytotoxicity of Stx1 was inhibited. The toxin neutralization was also demonstrated by means of several assays including Stx1-induced phosphatidylserine translocation on the plasma membrane, caspase-3 activation and 180 base-pair DNA ladder formation due to inter-nucleosomal cleavage. These results indicate that edible plants containing hybrid-IgG/IgA against Stx1B have the potential to be used for immunotherapy against Stx1-caused food poisoning.     

Cysteine Proteinase from Streptococcus pyogenes Enables Evasion of Innate Immunity via Degradation of Complement Factors

Streptococcus pyogenes is an important human pathogen that causes invasive diseases such as necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome. We investigated the function of a major cysteine protease from S. pyogenes that affects the amount of C1-esterase inhibitor (C1-INH) and other complement factors and aimed to elucidate the mechanism involved in occurrence of streptococcal toxic shock syndrome from the aspect of the complement system. First, we revealed that culture supernatant of a given S. pyogenes strain and recombinant SpeB degraded the C1-INH. Then, we determined the N-terminal sequence of the C1-INH fragment degraded by recombinant SpeB. Interestingly, the region containing one of the identified cleavage sites is not present in patients with C1-INH deficiency. Scanning electron microscopy of the speB mutant incubated in human serum showed the abnormal superficial architecture and irregular oval structure. Furthermore, unlike the wild-type strain, that mutant strain showed lower survival capacity than normal as compared with heat-inactivated serum, whereas it had a significantly higher survival rate in serum without the C1-INH than in normal serum. Also, SpeB degraded multiple complement factors and the membrane attack complex. Flow cytometric analyses revealed deposition of C9, one of the components of membrane the attack complex, in greater amounts on the surface of the speB mutant, whereas lower amounts of C9 were bound to the wild-type strain surface. These results suggest that SpeB can interrupt the human complement system via degrading the C1-INH, thus enabling S. pyogenes to evade eradication in a hostile environment.     

The Distribution of TRPV1 and TRPV2 in the Rat Pharynx

Immunohistochemistry for two nociceptive transducers, the transient receptor potential cation channel subfamily V members 1 (TRPV1) and 2 (TRPV2), was performed on the pharynx and its adjacent regions. TRPV1-immunoreactivity (IR) was detected in nerve fibers beneath and within the epithelium and/or taste bud-like structure. In the pharynx, these nerve fibers were abundant in the naso-oral part and at the border region of naso-oral and laryngeal parts. They were also numerous on the laryngeal side of the epiglottis and in the soft palate. TRPV2-IR was expressed by dendritic cells in the pharynx and epiglottis, as well as in the root of the tongue and soft palate. These cells were located in the epithelium and lamina propria. TRPV2-immunoreactive (IR) dendritic cells were numerous in the naso-oral part of the pharynx, epiglottis, and tongue. Abundance of TRPV2-IR dendritic processes usually obscured the presence of TRPV2-IR nerve fibers in these portions. However, some TRPV2-IR nerve fibers could be observed in the epithelium of the soft palate. Retrograde tracing method also revealed that sensory neurons which innervate the pharynx or soft palate were abundant in the jugular–petrosal ganglion complex and relatively rare in the nodose ganglion. In the jugular–petrosal ganglion complex, TRPV1- and TRPV2-IR were expressed by one-third of pharyngeal and soft palate neurons. TRPV2-IR was also detected in 11.5 % pharyngeal and 30.9 % soft palate neurons in the complex. Coexpression of TRPV1 and CGRP was frequent among pharyngeal and soft palate neurons. The present study suggests that TRPV1- and TRPV2-IR jugular–petrosal neurons may be associated with the regulation of the swallowing reflex.     

Alterations in growth and fatty acid profiles under stress conditions of Hansenula polymorpha defective in polyunsaturated fatty acid synthesis

Using chemical mutagenesis, mutants of Hansenula polymorpha that were defective in fatty acid synthesis were selected based on their growth requirements on saturated fatty acid mixtures. One mutant (S7) was incapable of synthesizing polyunsaturated fatty acids (PUFA), linoleic and α-linolenic acids. A genetic analysis demonstrated that the S7 strain had a double lesion affecting fatty acid synthesis and Δ¹²-desaturation. A segregant with a defect in PUFA synthesis (H69-2C) displayed normal growth characteristics in the temperature range of 20–42 °C through a modulation of the cellular fatty acid composition. Compared with the parental strain, this yeast mutant had increased sensitivity at low and high temperatures (15 and 48 °C, respectively) with an increased tolerance to oxidative stress. The responses to ethanol stress were similar for the parental and PUFA-defective strains. Myristic acid was also determined to play an essential role in the cell growth of H. polymorpha. These findings suggest that both the type of cellular fatty acids and the composition of fatty acids might be involved in the stress responsive mechanisms in this industrially important yeast.     

Decreased Succinate Dehydrogenase Activity of Gamma and Alpha Motoneurons in Mouse Spinal Cords Following 13 Weeks of Exposure to Microgravity

Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm²) gamma and medium-sized (300–700 μm²) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm²) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity.     

Advantages of AlaGln as an additive to cell culture medium: use with anti-CD20 chimeric antibody-producing POTELLIGENT™ CHO cell lines

l-alanyl-l-glutamine (AlaGln) is dipeptide that has better solubility and stability than Glutamine (Gln). In this study, we evaluated the utility of this dipeptide during culture of POTELLIGENT™ Chinese hamster ovary (CHO) cells expressing anti-CD20 chimeric antibody. Although AlaGln in the culture medium lowered the specific growth rate, the MAb titer was maximized when Gln was completely replaced by AlaGln in both the basal and feed media. Moreover, AlaGln augmented production of antibody not only at flask scale but also at spinner scale, although the extent of this effect was dependent on the cell clone. To explore the mechanism responsible for the effect of AlaGln on cell growth, we measured apoptosis in the early phase of cell culture on days 8, 9, and 10. The apoptotic ratio was reduced in medium containing AlaGln. Ammonia was generated in medium containing Gln when it was maintained at 37 °C, which impeded the growth and productivity of the cells. In contrast, AlaGln produced less ammonia under these conditions, which may have been one of the properties associated with its beneficial effects. We conclude that certain dipeptides can serve as superior alternative sources of amino acids in cell culture and antibody production.     

Combination of BCAAs and glutamine enhances dermal collagen protein synthesis in protein-malnourished rats

Skin collagen decreases in protein-malnourished states. Amino acids regulate protein metabolism, glutamine stimulates collagen synthesis through the conversion process to proline and provides 75 % of the intracellular free proline in fibroblasts. However, the impact of these amino acids on collagen synthesis under malnutrition has not been examined. We investigated the effect of amino acids on dermal tropocollagen synthesis in protein-malnourished rats. The fractional synthesis rate (FSR, %/h) of dermal tropocollagen was evaluated by the incorporation of l-[ring-²H₅]-phenylalanine after 4 h infusion of each amino acid and the stable isotope. None of the infused 12 single amino acids (glutamine, proline, alanine, arginine, glutamate, glycine, aspartate, serine, histidine, lysine, phenylalanine and threonine) significantly increased the FSR (P = 0.343, one-way ANOVA). In contrast, amino acid mixtures of essential amino acids + glutamine + arginine (EAARQ) and branched-chain amino acids + glutamine (BCAAQ) significantly increased the FSR compared to saline, but the branched-chain amino acids (BCAAs) and amino acid mixture of collagen protein (AAC) did not alter the FSR (saline, 0.96 ± 0.24 %/h; EAARQ, 1.76 ± 0.89 %/h; BCAAQ 1.71 ± 0.36 %/h; BCAAs, 1.08 ± 0.20 %/h and AAC 1.39 ± 0.35 %/h, P < 0.05, Tukey’s test). Proline conversion from glutamine represented only 3.9 % of the free proline in skin, as evaluated by the primed-constant infusion of l-d7-proline and l-α-15N-glutamine in rats. These results suggested that the combination of BCAAQ is a key factor for the enhancement of skin collagen synthesis in protein-malnourished rats. The contribution of extracellular free glutamine on de novo proline synthesis and collagen synthesis is very low in vivo compared to the contribution in vitro.     

Practical application of flavonoid-poor menu meals to the study of the bioavailability of bilberry anthocyanins in human subjects

Practical application of flavonoid-poor menus was evaluated on the bioavailability of anthocyanins as model flavonoids. Detectable amounts of flavonoids were not found in plasma and urine collected from 13 participants, who took the menus. After ingesting bilberry anthocyanins (919 μmol), average plasma AUC_0-6h, C_max, T_max values and urinary recovery were 386.0 nmol h/mL, 139.1 nM, 1.31 h and 0.21%, respectively.