Immunocytochemical demonstration of serine: pyruvate amino-transferase in peroxisomes and mitochondria of rat kidney

The light- and electron-microscopic localization of serine: pyruvate aminotransferase (SPT) in rat kidney was studied using immunoenzyme and protein A-gold techniques. Rat kidneys were fixed by perfusion through the abdominal aorta and small tissue slices were embedded in Epon, Lowicryl K4M, or LR Gold. The Epon was removed from the semithin sections, which were then stained using the immunoenzyme technique. Ultrathin sections of Lowicryl K4M- or LR gold-embedded materials were labeled using the protein A-gold technique. At light microscopy, discrete granular reaction deposits were exclusively present in the proximal tubule, all of whose segments were positive for SPT. A weakly positive reaction was observed in the distal tubules. At electron microscopy, gold particles indicating the antigenic sites for SPT were confined to the peroxisomes and mitochondria. The labeling intensity of both organelles was dependent on the embedding resins used. The labeling of Lowicryl K4M-embedded material was weaker than that of LR gold-embedded material; Quantitative analysis confirmed this result. Our results indicate that, in rat kidney, the main intracellular sites for SPT are peroxisomes and mitochondria of the proximal tubule.     

Cell Growth and Antimicrobial Activity of Mouse Peritoneal Macrophages in Response to Glucocorticoids,Choleragen and Lipopolysaccharide

Normal, thioglycollate-stimulated and BCG-activated mouse peritoneal macrophages were cultivated in vitro with the conditioned medium of mouse L-929 cells. The thioglycollate- and BCG-macrophages rapidly proliferated, whereas normal macrophages grew more slowly. A clear morphological difference between the three types of macrophages in the culture was observed. Glucocorticoids inhibited the growth of the macrophages at pharmacological concentrations. Other steroids, progesterone, diethylstilbesterol and testosterone in that order, had a far lower growth-inhibiting effect. Macrophages cultured with 10^-6 M dexamethasone had a reduced antimicrobial effect on Candida parapsilosis compared with that of the untreated cells. Choleragen had the same effect on the macrophages as glucocorticoids. The toxin inhibited growth at a concentration as low as 10 pg/ml and cells treated with 1 ng of choleragen per ml had decreased antifungal activity. Similarly, Escherichia coli lipopolysaccharide at 10 ng/ml inhibited the growth of thioglycollate-macrophages. However, macrophages incubated with the lipopolysaccharide had enhanced anticandida activity. Thus, the immunosuppressors glucocorticoid and choleragen inhibited both the increase in the number of macrophages and the microbicidal activity of the phagocytes. Lipopolysaccharide, an immunostimulant, stimulated macrophage activity, but was toxic for cell growth.     

Microbicidal Activity of Bone Marrow Cells. Augmentation of Candida Killing Activity of the Cells by Culturing with Fibroblast Conditioned Medium

Mouse bone marrow cells were seeded in 96-well trays and infected with twofold serially diluted Candida parapsilosis. Outgrowth of the fungi in each well was determined after a 48-hr incubation period. Freshly collected cells demonstrated a candidacidal activity which increased with increase in the number of cells seeded. When fresh cells were cultivated with conditioned medium of mouse embryo fibroblasts, the candidacidal activity continuously increased for a few days and reached a plateau. The activity was augmented more than 1,000-fold compared with that of fresh marrow cells. It is suggested that augmentation is due not to the increase in number of effector cells but to the maturation of effector cells. Carrageenan, which is specifically cytotoxic to macrophages, inhibited augmentation of the activity. Therefore, it appears that most effector cells that matured by cultivation with the conditioned medium belong to the monocyte-macrophage lineage.     

Zinc is released by cultured astrocytes as a gliotransmitter under hypoosmotic stress-loaded conditions and regulates microglial activity

Aim

Astrocytes contribute to the maintenance of brain homeostasis via the release of gliotransmitters such as ATP and glutamate. Here we examined whether zinc was released from astrocytes under stress-loaded conditions, and was involved in the regulation of microglial activity as a gliotransmitter.

Main methods

Hypoosmotic stress was loaded to astrocytes using balanced salt solution prepared to 214–314 mOsmol/L, and then intra- and extra-cellular zinc levels were assessed using Newport Green DCF diacetate (NG) and ICP-MS, respectively. Microglial activation by the astrocytic supernatant was assessed by their morphological changes and poly(ADP-ribose) (PAR) polymer accumulation.

Key findings

Exposure of astrocytes to hypoosmotic buffer, increased the extracellular ATP level in osmolarity-dependent manners, indicating a load of hypoosmotic stress. In hypoosmotic stress-loaded astrocytes, there were apparent increases in the intra- and extra-cellular zinc levels. Incubation of microglia in the astrocytic conditioned medium transformed them into the activated “amoeboid” form and induced PAR formation. Administration of an extracellular zinc chelator, CaEDTA, to the astrocytic conditioned medium almost completely prevented the microglial activation. Treatment of astrocytes with an intracellular zinc chelator, TPEN, suppressed the hypoosmotic stress-increased intracellular, but not the extracellular, zinc level, and the increase in the intracellular zinc level was blocked partially by a nitric oxide synthase inhibitor, but not by CaEDTA, indicating that the mechanisms underlying the increases in the intra- and extra-cellular zinc levels might be different.

Significance

These findings suggest that under hypoosmotic stress-loaded conditions, zinc is released from astrocytes and then plays a primary role in microglial activation as a gliotransmitter.     

Intracranial arachnoid cysts in a chimpanzee (Pan troglodytes)

An intracranial arachnoid cyst was detected in a 32-year-old, 44.6-kg, female chimpanzee at the Primate Research Institute, Kyoto University. Magnetic resonance imaging (MRI) and computed tomography (CT) were performed and the cognitive studies in which she participated were reviewed. MRI revealed that the cyst was present in the chimpanzee’s right occipital convexity, and was located in close proximity to the posterior horn of the right lateral ventricle without ventriculomegaly. CT confirmed the presence of the cyst and no apparent signs indicating previous skull fractures were found. The thickness of the mandible was asymmetrical, whereas the temporomandibular joints and dentition were symmetrical. She showed no abnormalities in various cognitive studies since she was 3 years old, except a different behavioural pattern during a recent study, indicating a possible visual field defect. Detailed cognitive studies, long-term observation of her physical condition and follow-up MRI will be continued.     

Evidence for participation of GCS1 in fertilization of the starlet sea anemone Nematostella vectensis: Implication of a common mechanism of sperm–egg fusion in plants and animals

It has been reported that GCS1 (Generative Cell Specific 1) is a transmembrane protein that is exclusively expressed in sperm cells and is essential for gamete fusion in flowering plants. The GCS1 gene is present not only in angiosperms but also in unicellular organisms and animals, implying the occurrence of a common or ancestral mechanism of GCS1-mediated gamete fusion. In order to elucidate the common mechanism, we investigated the role of GCS1 in animal fertilization using a sea anemone (Cnidaria), Nematostella vectensis. Although the existence of the GCS1 gene in N. vectensis has been reported, the expression of GCS1 in sperm and the role of GCS1 in fertilization are not known. In this study, we showed that the GCS1 gene is expressed in the testis and that GCS1 protein exists in sperm by in situ hybridization and proteomic analysis, respectively. Then we made four peptide antibodies against the N-terminal extracellular region of NvGCS1. These antibodies specifically reacted to NvGCS1 among sperm proteins on the basis of Western analysis and potently inhibited fertilization in a concentration-dependent manner. These results indicate that sperm GCS1 plays a pivotal role in fertilization, most probably in sperm–egg fusion, in a starlet sea anemone, suggesting a common gamete-fusion mechanism shared by eukaryotic organisms.     

Aberrant histone acetylation contributes to elevated interleukin-6 production in rheumatoid arthritis synovial fibroblasts

Accumulating evidence indicates that epigenetic aberrations have a role in the pathogenesis of rheumatoid arthritis (RA). However, reports on histone modifications are as yet quite limited in RA. Interleukin (IL)-6 is an inflammatory cytokine which is known to be involved in the pathogenesis of RA. Here we report the role of histone modifications in elevated IL-6 production in RA synovial fibroblasts (SFs). The level of histone H3 acetylation (H3ac) in the IL-6 promoter was significantly higher in RASFs than osteoarthritis (OA) SFs. This suggests that chromatin structure is in an open or loose state in the IL-6 promoter in RASFs. Furthermore, curcumin, a histone acetyltransferase (HAT) inhibitor, significantly reduced the level of H3ac in the IL-6 promoter, as well as IL-6 mRNA expression and IL-6 protein secretion by RASFs. Taken together, it is suggested that hyperacetylation of histone H3 in the IL-6 promoter induces the increase in IL-6 production by RASFs and thereby participates in the pathogenesis of RA.     

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.