Isomer-specific anti-obese and hypolipidemic properties of conjugated linoleic acid in obese OLETF rats

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid, has attracted considerable attention because of its potentially beneficial biologic effects both in vitro and in vivo. Our results clearly show the specific action of the 10trans,12cis-CLA isomer against hyperlipidemia and obesity in obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. After 2 weeks of feeding with 10t,12c-CLA, but not 9cis,11trans-CLA, abdominal adipose tissue weight and serum and hepatic lipid levels in OLETF rats were lower than those in linoleic acid-fed rats. These effects were attributable to suppressed fatty acid synthesis and enhanced fatty acid beta oxidation in the liver on a 10t,12c-CLA diet. Additionally, we showed that mRNA expression of fatty acid synthase, carnitine palmitoyltransferase, leptin, and sterol regulatory element binding protein-1 was also regulated by 10t,12c-CLA. We suppose that 10t,12c-CLA reveals hypolipidemic and anti-obese activity through the alteration of mRNA expressions in the liver and white adipose tissue.     

Target molecules of molecular chaperone (HSP70 family) in injured gastric mucosa in vivo

AimsSeveral recent studies, including ours, have indicated the importance of heat shock proteins (HSPs) in cytoprotection against cytotoxic agents and environmental stresses mediated by the chaperone function of HSPs (molecular chaperones). However, the target molecule that is recognized by HSPs in damaged cells currently remains unknown. As HSPs rapidly recognize and bind to degenerated protein in cells, target molecules of HSPs might be key molecules for the initiation and pathogenesis of cellular damage. In the present study, gastric mucosal proteins that specifically bind to the HSP70 family (HSC70) were analyzed using HSC70-affinity chromatography.Main methodsThe gastric mucosa was removed from Sprague–Dawley rats after exposure to water immersion-stress for 0, 1, 3 or 5 h. Soluble fractions of each gastric mucosa were applied to the HSC70-affinity column separately. After washing off non-specific binding proteins, specific binding proteins were eluted by ATP-containing buffer. Binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In addition, the amino acid sequence of purified proteins was also analyzed.Key findingsSpecific HSC70-binding proteins with a molecular weight of 200-kDa and 45-kDa were eluted from an affinity column when gastric mucosal homogenate of 1-h stress exposure was applied. The amino acid sequencing showed that these binding proteins were cytoskeletal myosin (heavy chain) and actin, respectively.SignificanceDuring the pathogenesis of stress-induced gastric mucosal damage, structurally degenerated cytoskeletal myosin (heavy chain) and actin may be key or initiation molecules which structural changes were firstly recognized by molecular chaperone.     

Synthesis, characterization and redox reactivity of l-tartrato bridged dinuclear manganese complex with 2,2′-bipyridine

A new l-tartrato manganese(III) complex was synthesized and characterized as the dinuclear dimanganese(III) structure with a stereospefically formed [Λ-Λ] absolute configuration around Mn(III) ions. The thermal and photo decomposition gave the first example of dihydrogen gas evolution besides CO and CO₂ gas associated with cis-[Mn^II(ox)(bpy)(H₂O)₂]. A proposed redox reaction proceeds from Mn(III) to Mn(II) via intermediate Mn(IV) and Mn(II) with CO anion radical species followed by oxidation of tartrate ligands.     

HCHL expression in hairy roots of Beta vulgaris yields a high accumulation of p-hydroxybenzoic acid (pHBA) glucose ester,and linkage of pHBA into cell walls

As part of a study to explore the potential for new or modified bio-product formation, Beta vulgaris (sugar beet) has been genetically modified to express in root-organ culture a bacterial gene of phenylpropanoid catabolism. The HCHL gene, encoding p-hydroxycinnamoyl-CoA hydratase/lyase, was introduced into B. vulgaris under the control of a CaMV 35S promoter, using Agrobacterium rhizogenes LBA 9402. Hairy root clones expressing the HCHL gene, together with non-expressing clones, were analysed and revealed that one expression-positive clone accumulated the glucose ester of p-hydroxybenzoic acid (pHBA) at about 14% on a dry weight basis. This is the best yield achieved in plant systems so far. Determination of cell-wall components liberated by alkaline hydrolysis confirmed that the ratio of pHBA to ferulic acid was considerably higher in the HCHL-expressing clones, whereas only ferulic acid was detected in a non-expressing clone. The change in cell-wall components also resulted in a decrease in tensile strength in the HCHL-expressing clones.     

Involvement of Ca in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca²⁺ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca²⁺ ([Ca²⁺]_i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca²⁺]_i occurred 30 min after gAd treatment, after which [Ca²⁺]_i increased continually and exceeded the initial level. The mitochondrial Ca²⁺ ([Ca²⁺]_m) detected by Rhod-2 fluorescence started to increase at 6 h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca²⁺]_i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca²⁺]_m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.     

Transferrin secretory pathways in rat parotid acinar cells

Transferrin is the major iron transporter in blood plasma, and is also found, at lower concentrations, in saliva. We studied the synthesis and secretion of transferrin in rat parotid acinar cells in order to elucidate its secretory pathways. Two sources were identified for transferrin in parotid acinar cells: synthesis by the cells (endogenous), and absorption from blood plasma (exogenous). Transferrin from both sources is secreted from the apical side of parotid acinar cells. Endogenous transferrin is transported to secretory granules. It is secreted from mature secretory granules upon stimulation with a β-adrenergic reagent and from smaller vesicles in the absence of stimulation. Exogenous transferrin is internalized from the basolateral side of parotid acinar cells, transported to the apical side by transcytosis, and secreted from the apical side. Secretory processes for exogenous transferrin include transport systems involving microfilaments and microtubules.     

Cytoplasmic expression of the JM403 antigen GlcA-GlcNH 3 + on heparan sulfate glycosaminoglycan in mammary carcinomas—a novel proliferative biomarker for breast cancers with high malignancy

The expressions of heparan sulfate glycosaminoglycans (HSGAGs) in breast carcinoma specimens from 60 patients were immunohistochemically investigated using monoclonal antibodies (mAbs) that recognized different epitopes of the glycan structure. Cytoplasmic expression of GlcA-GlcNH ₃ ⁺ on HSGAG was detected in carcinomas at high frequency (58.3%) using mAb JM403, whereas it was almost undetectable in normal breast ducts. This cytoplasmic expression was confirmed using confocal laser scanning microscopy. The expression of JM403 antigen in invasive carcinomas significantly correlated with nuclear atypia score (p?=?0.0004), mitotic counts score (p?=?0.0018), nuclear grade (p?=?0.0061) and the incidence of metastasis to axillary lymph nodes (p?=?0.0061). Furthermore, its expression was significantly correlated with the Ki67-labeling index in 55 invasive carcinomas (p?p? ₃ ⁺ was also expressed in the cytoplasm of normal crypt epithelial cells where Ki67 protein was expressed in the cell nuclei in the proliferative compartment of the human small intestines. To date, HSGAGs have generally been found to exist on cell surface membranes and in extracellular matrices as components of HS proteoglycans, and the negatively-charged sulfated domains on HSGAGs are considered to be important for their functions. However, our present findings indicate that the cytoplasmic expression of the JM403 antigen GlcA-GlcNH ₃ ⁺ on positively charged, non-sulfated HSGAG may be involved in cell proliferation and associated with increased degrees of malignancy. The unordinary carbohydrate antigen of GlcA-GlcNH ₃ ⁺ on HSGAGs recognized by mAb JM403 may represent a novel proliferative biomarker for highly malignant mammary carcinomas.     

Kinesin‐1/Hsc70‐dependent mechanism of slow axonal transport and its relation to fast axonal transport

Cytoplasmic protein transport in axons (‘slow axonal transport’) is essential for neuronal homeostasis, and involves Kinesin‐1, the same motor for membranous organelle transport (‘fast axonal transport’). However, both molecular mechanisms of slow axonal transport and difference in usage of Kinesin‐1 between slow and fast axonal transport have been elusive. Here, we show that slow axonal transport depends on the interaction between the DnaJ‐like domain of the kinesin light chain in the Kinesin‐1 motor complex and Hsc70, scaffolding between cytoplasmic proteins and Kinesin‐1. The domain is within the tetratricopeptide repeat, which can bind to membranous organelles, and competitive perturbation of the domain in squid giant axons disrupted cytoplasmic protein transport and reinforced membranous organelle transport, indicating that this domain might have a function as a switchover system between slow and fast transport by Hsc70. Transgenic mice overexpressing a dominant‐negative form of the domain showed delayed slow transport, accelerated fast transport and optic axonopathy. These findings provide a basis for the regulatory mechanism of intracellular transport and its intriguing implication in neuronal dysfunction.