Characterization of Low pH-induced Catecholamine Secretion in the Rat Adrenal Medulla

Abstract: Catecholamine (CA) secretion was evoked when the isolated rat adrenal gland was perfused with HEPES-buffered Krebs solution acidified by the addition of HCI or by gassing with 95% O₂/5% CO₂. The secretion was detectable at pH 7.0 and increased with decreasing pH until at ～6.4. The low pH-induced CA secretion consisted of two phases, an initial transient response followed by a sustained phase. An intracellular Ca²⁺ antagonist, 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octyl ester, selectively inhibited the initial phase of secretion. Both of the responses were resistant to nifedipine, a blocker of voltage-gated Ca²⁺ channel, but were completely inhibited in Ca²⁺-free (1 mM EGTA containing) solution. Adrenaline was an exclusive component in CAs released by low pH. The time course and extent of intracellular acidification caused either by low pH in the external medium or by the offset of a transitory NH₄CI application had no correlation with those of the secretory responses in the corresponding period. These results suggest that extracellular acidification preferentially activates adrenaline secretive cells to evoke CA secretion and that this low pH-induced CA secretion may be mediated by dihydropyridine-insensitive Ca²⁺ influx. Furthermore, the initial transient phase of the low pH-induced CA secretion might be caused by a Ca²⁺ release from intracellular stores, which is also induced by the Ca²⁺ influx.     

Collagen adhesin–nanoparticle interaction impairs adhesin's ligand binding mechanism

Background

Pathogenic bacteria specifically recognize extracellular matrix (ECM) molecules of the host (e.g. collagen, fibrinogen and fibronectin) through their surface proteins known as MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) and initiate colonization. On implantation, biomaterials easily get coated with these ECM molecules and the MSCRAMMs mediate bacterial adherence to biomaterials. With the rapid rise in antibiotic resistance, designing alternative strategies to reduce/eliminate bacterial colonization is absolutely essential.

Methods

The Rhusiopathiae surface protein B (RspB) is a collagen‐binding MSCRAMM of Erysipelothrix rhusiopathiae. It also binds to abiotic surfaces. The crystal structure of the collagen‐binding region of RspB (rRspB_31–348) reported here revealed that RspB also binds collagen by a unique ligand binding mechanism called “Collagen Hug” which is a common theme for collagen‐binding MSCRAMMs of many Gram-positive bacteria. Here, we report the interaction studies between rRspB_31–348 and silver nanoparticles using methods like gel shift assay, gel permeation chromatography and circular dichroism spectroscopy.

Results

The “Collagen Hug” mechanism was inhibited in the presence of silver nanoparticles as rRspB_31–348 was unable to bind to collagen. The total loss of binding was likely because of rRspB_31–348 and silver nanoparticle protein corona formation and not due to the loss of the structural integrity of rRspB_31–348 on binding with nanoparticles as observed from circular dichroism experiments.

General significance

Interaction of rRspB_31–348 with silver nanoparticle impaired its ligand binding mechanism. Details of this inhibition mechanism may be useful for the development of antimicrobial materials and antiadhesion drugs.     

Enzymatic nitration of phytophenolics: evidence for peroxynitrite-independent nitration of plant secondary metabolites

Peroxynitrite (ONOO(-)), a reactive nitrogen species, is capable of nitrating tyrosine residue of proteins. Here we show in vitro evidence that plant phenolic compounds can also be nitrated by an ONOO(-)-independent mechanism. In the presence of NaNO(2), H(2)O(2), and horseradish peroxidase (HRP), monophenolic p-coumaric acid (p-CA, 4-hydroxycinnamic acid) was nitrated to form 4-hydroxy-3-nitrocinnamic acid. The reaction was completely inhibited by KCN, an inhibitor for HRP. The antioxidant ascorbate suppressed p-CA nitration and its suppression time depended strongly on ascorbate concentration. We conclude that nitrogen dioxide radical (NO(2)(radical)), but not ONOO(-), produced by a guaiacol peroxidase is the intermediate for phytophenolic nitration.     

NADPH dependent activation of microsomal glutathione transferase 1

Microsomal glutathione transferase 1 (MGST1) can become activated up to 30-fold by several mechanisms in vitro (e.g. covalent modification by reactive electrophiles such as N-ethylmaleimide (NEM)). Activation has also been observed in vivo during oxidative stress. It has been noted that an NADPH generating system (g.s.) can activate MGST1 (up to 2-fold) in microsomal incubations, but the mechanism was unclear. We show here that NADPH g.s treatment impaired N-ethylmaleimide activation, indicating a shared target (identified as cysteine-49 in the latter case). Furthermore, NADPH activation was prevented by sulfhydryl compounds (glutathione and dithiothreitol). A well established candidate for activation would be oxidative stress, however we could exclude that oxidation mediated by cytochrome P450 2E1 (or flavine monooxygenase) was responsible for activation under a defined set of experimental conditions since superoxide or hydrogen peroxide alone did not activate the enzyme (in microsomes prepared by our routine procedure). Actually, the ability of MGST1 to become activated by hydrogen peroxide is critically dependent on the microsome preparation method (which influences hydrogen peroxide decomposition rate as shown here), explaining variable results in the literature. NADPH g.s. dependent activation of MGST1 could instead be explained, at least partly, by a direct effect observed also with purified enzyme (up to 1.4-fold activation). This activation was inhibited by sulfhydryl compounds and thus displays the same characteristics as that of the microsomal system. Whereas NADPH, and also ATP, activated purified MGST1, several nucleotide analogues did not, demonstrating specificity. It is thus an intriguing possibility that MGST1 function could be modulated by ligands (as well as reactive oxygen species) during oxidative stress when sulfhydryls are depleted.     

Adhesive surface proteins of Erysipelothrix rhusiopathiae bind to polystyrene,fibronectin, and type I and IV collagens

Erysipelothrix rhusiopathiae is a gram-positive bacterium that causes erysipelas in animals and erysipeloid in humans. We found two adhesive surface proteins of E. rhusiopathiae and determined the nucleotide sequences of the genes, which were colocalized and designated rspA and rspB. The two genes were present in all of the serovars of E. rhusiopathiae strains examined. The deduced RspA and RspB proteins contain the C-terminal anchoring motif, LPXTG, which is preceded by repeats of consensus amino acid sequences. The consensus sequences are composed of 78 to 92 amino acids and repeat 16 and 3 times in RspA and RspB, respectively. Adhesive surface proteins of other gram-positive bacteria, including Listeria monocytogenes adhesin-like protein, Streptococcus pyogenes protein F2 and F2-like protein, Streptococcus dysgalactiae FnBB, and Staphylococcus aureus Cna, share the same consensus repeats. Furthermore, the N-terminal regions of RspA and RspB showed characteristics of the collagen-binding domain that was described for Cna. RspA and RspB were expressed in Escherichia coli as histidine-tagged fusion proteins and purified. The recombinant proteins showed a high degree of capacity to bind to polystyrene and inhibited the binding of E. rhusiopathiae onto the abiotic surface in a dose dependent manner. In a solid-phase binding assay, both of the recombinant proteins bound to fibronectin, type I and IV collagens, indicating broad spectrum of their binding ability. It was suggested that both RspA and RspB were exposed on the cell surface of E. rhusiopathiae, as were the bacterial cells agglutinated by the anti-RspA immunoglobulin G (IgG) and anti-RspB IgG. RspA and RspB were present both in surface-antigen extracts and the culture supernatants of E. rhusiopathiae Fujisawa-SmR (serovar 1a) and SE-9 (serovar 2). The recombinant RspA, but not RspB, elicited protection in mice against experimental challenge. These results suggest that RspA and RspB participate in initiation of biofilm formation through their binding abilities to abiotic and biotic surfaces.     

Four alkaloids, lucidine B, oxolucidine A, lucidine A, and lucidulinone from Lycopodium lucidulum

The structures of four alkaloids extracted from Lycopodium lucidulum (Lycopodiaceae) were established by X-ray and 2D NMR spectroscopic analyses. The dihydro-derivative of oxolucidine A, which was obtained by NaBH4 reduction of oxolucidine A, was treated with p-bromobenzoyl chloride to afford crystals, whose X-ray crystallographic analysis established the stereostructure, including the absolute configuration. The 2D NMR spectra of tetrahydrodeoxylucidine B were fully analyzed to establish the full structure of lucidine B, and the hitherto unknown stereochemistry at the C-14 position was established as beta-H. The structure of a new alkaloid, lucidulinone, was determined by spectroscopic analysis to be luciduline lactam.     

Local and global cerebral blood flow and glucose utilization in the α-galactosidase A knockout mouse model of Fabry disease

Fabry disease is an X-linked lysosomal disorder characterized by deficient alpha-galactosidase A activity and intracellular accumulations of glycosphingolipids, mainly globotriaosylceramide (Gb3). Clinically, patients occasionally present CNS dysfunction. To examine the pathophysiology underlying brain dysfunction, we examined glucose utilization (CMR(glc)) and cerebral blood flow (CBF) globally and locally in 18 brain structures in the alpha-galactosidase A gene knockout mouse. Global CMR(glc) was statistically significantly reduced by 22% in Fabry mice (p < 0.01). All 18 structures showed decreases in local CMR(glc) ranging from 14% to 33%. The decreases in all structures of the diencephalon, caudate-putamen, brain stem, and cerebellar cortex were statistically significant (p < 0.05). Global cerebral blood flow (CBF) and local CBF measured in the same 18 structures were lower in Fabry mice than in control mice, but none statistically significantly. Histological examination of brain revealed no cerebral infarcts but abundant Gb3 deposits in the walls of the cerebral vessels with neuronal deposits localized to the medulla oblongata. These results indicate an impairment in cerebral energy metabolism in the Fabry mice, but one not necessarily due to circulatory insufficiency.     

Thy-1-Mediated phosphatidylinositol turnover in cultured rat glomerular mesangial cell

Thy-1 glycoprotein is expressed in rat glomerular mesangial cells, and anti-Thy-1 nephritis induced by anti-Thy-1 antibodies is a model of human renal diseases. In this study, we examined Thy-1-mediated biological reactions in cultured rat glomerular mesangial cells utilizing two anti-Thy-1 monoclonal antibodies (mAbs), 1-22-3 and OX-7. Incubation of the cells with these mAbs resulted in increased inositol trisphosphate (IP₃) levels. The rise in IP₃ produced by mAb 1-22-3 was greater than that produced by mAb OX-7 at the same dose. Incubation of mesangial cells with these mAbs resulted in an increase in the intracellular free calcium concentration ([Ca²⁺]i). mAb 1-22-3 induced a sustained increase in [Ca²⁺]i, while that induced by mAb OX-7 lasted 1-2 min, then decreased to the basal level. An transient increase in [Ca²⁺]i was also observed in Ca²⁺-free medium, indicating that these [Ca²⁺]i increases are due to release of Ca²⁺ from internal stores by IP₃ without calcium flux across cell membrane. When cells were pretreated with protein tyrosine kinase (PTK) inhibitors (herbimycin A or genistein), Thy-1-mediated increases in [Ca²⁺]i were inhibited. These data suggest that Thy-1 induces the production of IP₃ (including inositol 1,4,5-triphosphate, an intracellular Ca²⁺-releasing factor) and that PTKs may contribute to the Thy-1-mediated elevation of [Ca²⁺]i which presumably results from phospholipase C activation following Thy-1-mediated signaling in rat mesangial cells. © 1996 Wiley-Liss, Inc.     

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.