Cellular localization of thiol-proteinase inhibitor in the epidermis of the newborn rat

Summary In cichlid, poecilid and centrarchid fishes luteinizing hormone releasing hormone (LHRH)-immunoreactive neurons are found in a cell group (nucleus olfactoretinalis) located at the transition between the ventral telencephalon and olfactory bulb. Processes of these neurons project to the contralateral retina, traveling along the border between the internal plexiform and internal nuclear layer, and probably terminating on amacrine or bipolar cells. Horseradish peroxidase (HRP) injected into the eye or optic nerve is transported retrogradely in the optic nerve to the contralateral nucleus olfactoretinalis where neuronal perikarya are labeled. Labeled processes leave this nucleus in a rostral direction and terminate in the olfactory bulb. The nucleus olfactoretinalis is present only in fishes, such as cichlids, poecilids and centrarchids, in which the olfactory bulbs border directly the telencephalic hemispheres. In cyprinid, silurid and notopterid fishes, in which the olfactory bulbs lie beneath the olfactory epithelium and are connected to the telencephalon via olfactory stalks, the nucleus olfactoretinalis or a comparable arrangement of LHRH-immunoreactive neurons is lacking. After retrograde transport of HRP in the optic nerve of these fishes no labeling of neurons in the telencephalon occurred. It is proposed that the nucleus olfactoretinalis anatomically and functionally interconnects and integrates parts of the olfactory and optic systems.     

Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants

Key message

The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements.

Abstract

Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.

     

Proton receptor GPR68 expression in dendritic-cell-like S100β-positive cells of rat anterior pituitary gland: GPR68 induces interleukin-6 gene expression in extracellular acidification

S100β-positive cells, which do not express the classical pituitary hormones, appear to possess multifunctional properties and are assumed to be heterogeneous in the anterior pituitary gland. The presence of several protein markers has shown that S100β-positive cells are composed of populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. Recently, we succeeded in separating S100β-positive cells into round-cell (dendritic-cell-like) and process-cell types. We also found the characteristic expression of anti-inflammatory factors (interleukin-6, Il-6) and membrane receptors (integrin β-6) in the round type. Here, we further investigate the function of the subpopulation of S100β-positive cells. Since IL-6 is also a paracrine factor that regulates hormone producing-cells, we examine whether a correlation exists among extracellular acid stress, IL-6 and hormone production by using primary cultures of anterior pituitary cells. Dendritic-cell-like S100β-positive cells notably expressed Gpr68 (proton receptor) and Il-6. Furthermore, the expression of Il-6 and proopiomelanocortin (Pomc) was up-regulated by extracellular acidification. The functional role of IL-6 and GPR68 in the gene expression of Pomc during extracellular acidification was also examined. Small interfering RNA for Il-6 up-regulated Pomc expression and that for Gpr68 reversed the down-regulation of Il-6 and up-regulated Pomc expression by extracellular acidification. Thus, S100β-positive dendritic-like cells can sense an increase in extracellular protons via GPR68 and respond by the production of IL-6 in order to suppress the up-regulation of Pomc expression.     

Purification and Properties of α-Glucosidase and Glucoamylase from Lentinus edodes (Berk.) Sing.

An α-glucosidase and a glucoamylase have been isolated from fruit bodies of Lentinus edodes (Berk.) Sing., by a procedure including fractionation with ammonium sulfate, DEAE-cellulose column chromatography, and preparative gel electrofocusing. Both of them were homogeneous on gel electrofocusing and ultracentrifugation. The molecular weight of α-glucosidase and glucoamylase was 51,000 and 55,000, respectively. The α-glucosidase hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose, and soluble starch, but did not act on sucrose. The glucoamylase hydrolyzed maltose, maltotriose, phenyl α-maltoside, soluble starch, amylose, amylopectin, and glycogen, glucose being the sole product formed in the digests of these substrates. Both enzymes hydrolyzed phenyl a-maltoside into glucose and phenyl α-glucoside. The glucoamylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen, converting them almost completely into glucose. It was found that β-glucose was liberated from amylose by the action of glucoamylase, while α-glucose was produced by the α-glucosidase.

Maltotriose was the main α-glucosyltransfer product formed from maltose by the α-glucosidase.     

β-Hydroxy-L-valine and 4-Amino-3,6-dihydroxy-2-methylhexanoic Acid,Constitutional Amino Acids of the Antibiotic YA–56

From the acid hydrolyzate of the antibiotic YA–56 X (Zorbamycin) and Y belonging to the phleomycin-bleomycin group, 3 unique amino acids were isolated and their structures were determined to be β-hydroxy-L-valine and 2-(3′-amino-tetrahydrofuryl-2′-yl)-propionic acid and an isomer of the latter compound.

Considerations of NMR of the antibiotic YA-56 and of the reaction conditions led us to a view that the latter two new amino acids were not an integral part of the YA–56 molecule but were artifacts derived from 4-amino-3,6-dihydroxy-2-methyhexanoic acid constituting of YA–56 X and Y.     

Structural Analysis of Sydowic Acid by X-ray Diffraction

Streptomyces sp. No. 280 produced several kinds of amylase inhibitors (amylase inhibitor A, B, B' and C). Two amylase inhibitors (designated as AI-A₁ and AI-A₂) were obtained from an amylase inhibitor A fraction by paper chromatography. AI-A₁ inhibited muscle phosphorylase a much more than AI-A₂ and was hydrolyzed by sweet potato β-amylase whereas AI-A₂ was not. Both amylase inhibitors had a carbohydrate and were hydrolyzed by some kinds of amylases or acids. They lost their inhibitory activity against phosphorylase a after treatment with acids or hog pancreatic α-amylase, but they showed increased inhibitory activity toward porcine small intestinal sucrase.

Both AI-A₁ and AI-A₂ were composed of glucose and a basic moiety which gave a positive ninhydrin reaction. The molecular weights of AI-A₁ and AI-A₂ were estimated to be approximately 1300 ? 1500 by gel filtration on a Sephadex G-15 column. The nitrogen content of the amylase inhibitors was found to be about 1.3% by elementary analysis     

Telmisartan inhibits methylglyoxal-mediated cell death in human vascular endothelium

Methylglyoxal (MGO) is a metabolite of glucose. Since serum MGO level is increased in diabetic patients, MGO is implicated in diabetic complications related to vascular injury. We have recently demonstrated that glucose metabolite is a more powerful stimulant for endothelial cells (ECs) injury rather than glucose or advanced glycation-end products. Recent clinical trials suggest that angiotensin receptor blockers are effective to prevent diabetes-associated cardiovascular disorders beyond blood pressure lowering effect. To explore the mechanisms, we examined effects of telmisartan on MGO-induced ECs injury. Treatment of human umbilical vein ECs with MGO (560 μM) induced time-dependent (0-24 h) cell death. MGO-induced cell death was apoptosis since MGO increased cleaved caspase-3 expression. Telmisartan (0.1-10 μM) inhibited MGO-induced cell death and caspase-3 activation. These results indicate that telmisartan prevents MGO-induced apoptosis by inhibiting caspase-3 activation, which might explain at least in part the beneficial effects of telimisartan against diabetes-related cardiovascular diseases.     

Effect of tea catechins on mitochondrial DNA 4977-bp deletions in human leucocytes

The catechins in green tea have antioxidative and antimutagenic effects. We examined the effect of green tea enriched with catechins on the presence of mitochondrial DNA (mtDNA) with a common 4977-bp deletion mutation (mtDNA4977) in human leucocytes. Ten healthy females [aged 20.80 +/- 1.03 years] drank 350 ml of catechin-rich tea daily after supper for 5 weeks. Blood samples were collected twice before, and twice after 5 weeks of consuming the tea. Deletions in mtDNA were analyzed using the nested polymerase chain reaction (PCR). We identified a common mtDNA4977 deletion in nine participants before drinking the tea. However, this mtDNA4977 deletion was not evident in leucocytes from most of the participants 5 weeks after drinking the tea. Catechins found in tea might contribute to the maintenance of health status by reducing damage to mtDNA and by maintaining the capacity of mtDNA for oxidative phosphorylation.