Partial purification and characterization of taurodeoxycholate 7α-monooxygenase

Taurodeoxycholate 7α-monooxygenase was partially purified from rat liver microsomes. The enzyme was solubilized with cholate, fractionated with polyethylene glycol and chromatographed on a Sepharose 4B column with cholate as ligand. The enzyme activity was eluted from the column into the fraction eluted with 50 mM phosphate buffer containing cholate and KCl, whereas the benzphetamine demethylase activity was eluted in the non-bound fraction. Thus it was established that both enzymes are different entities. The taurodeoxycholate 7α-monooxygenase activity was reconstituted from the partially purified cytochrome P-450, highly purified NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine and NADPH.     

Loss of functional MYO1C/myosin 1c,a motor protein involved in lipid raft trafficking,disrupts autophagosome-lysosome fusion

MYO1C, a single-headed class I myosin, associates with cholesterol-enriched lipid rafts and facilitates their recycling from intracellular compartments to the cell surface. Absence of functional MYO1C disturbs the cellular distribution of lipid rafts, causes the accumulation of cholesterol-enriched membranes in the perinuclear recycling compartment, and leads to enlargement of endolysosomal membranes. Several feeder pathways, including classical endocytosis but also the autophagy pathway, maintain the health of the cell by selective degradation of cargo through fusion with the lysosome. Here we show that loss of functional MYO1C leads to an increase in total cellular cholesterol and its disrupted subcellular distribution. We observe an accumulation of autophagic structures caused by a block in fusion with the lysosome and a defect in autophagic cargo degradation. Interestingly, the loss of MYO1C has no effect on degradation of endocytic cargo such as EGFR, illustrating that although the endolysosomal compartment is enlarged in size, it is functional, contains active hydrolases, and the correct pH. Our results highlight the importance of correct lipid composition in autophagosomes and lysosomes to enable them to fuse. Ablating MYO1C function causes abnormal cholesterol distribution, which has a major selective impact on the autophagy pathway.     

Induced pluripotent stem cell‐derived melanocyte precursor cells undergoing differentiation into melanocytes

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self‐renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte‐specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high‐level expression of WNT5A, ROR2, which are non‐canonical WNT pathway markers, and its related receptor TGFβR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3β inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non‐canonical WNT signaling pathway.     

Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.     

Cynomolgus monkeys (Macaca fascicularis) may not become infected with equine herpesvirus 9

Background It was suggested that Equine herpesvirus 9 (EHV‐9) could be transmitted to higher non‐human primates. Methods Four cynomolgus monkeys (Macaca fascicularis) were inoculated with EHV‐9 by the nasal route. Results No abnormalities were observed pathologically, immunohistochemically, and genetically. Conclusions These findings indicate that cynomolgus monkeys are not susceptible to EHV‐9.     

The aglomerular kidney of the deep-sea gulper eel <Emphasis Type="Italic">Saccopharynx ampullaceus</Emphasis> (Saccopharyngiformes: Saccopharyngidae)

We report the presence of an aglomerular kidney in the pelagic deep-sea fish Saccopharynx ampullaceus (Saccopharyngiformes: Saccopharyngidae). The thin kidney is unpaired and ribbon-like rostrally, while it is thicker caudally with a rod-like shape. Light microscopic observation of serial sections revealed no glomeruli at all. The kidney is composed of renal tubules, sinusoids and capillaries of the renal portal system and extensive interstitial lymphoid tissues. Each renal tubule is surrounded by well-developed renal portal sinusoids, and the tubules are well separated from each other. There is a large space dorsal to the vertebrae, similar to the situation in the closely related Eurypharynx pelecanoides. We consider that S. ampullaceus possesses an aglomerular kidney to gain neutral buoyancy. The urinary bladder of S. ampullaceus is a distinct vesicular structure, unlike that of E. pelecanoides.     

Arabidopsis dynamin-related proteins DRP3A and DRP3B are functionally redundant in mitochondrial fission, but have distinct roles in peroxisomal fission

Two similar Arabidopsis dynamin-related proteins, DRP3A and DRP3B, are thought to be key factors in both mitochondrial and peroxisomal fission. However, the functional and genetic relationships between DRP3A and DRP3B have not been fully investigated. In a yeast two-hybrid assay, DRP3A and DRP3B interacted with themselves and with each other. DRP3A and DRP3B localized to mitochondria and peroxisomes, and co-localized with each other in leaf epidermal cells. In two T-DNA insertion mutants, drp3a and drp3b , the mitochondria are a little longer and fewer in number than those in the wild-type cells. In the double mutant, drp3a/drp3b , mitochondria are connected to each other, resulting in massive elongation. Overexpression of either DRP3A or DRP3B in drp3a/drp3b restored the particle shape of mitochondria, suggesting that DRP3A and DRP3B are functionally redundant in mitochondrial fission. In the case of peroxisomal fission, DRP3A and DRP3B appear to have different functions: peroxisomes in drp3a were larger and fewer in number than those in the wild type, whereas peroxisomes in drp3b were as large and as numerous as those in the wild type, and peroxisomes in drp3a/drp3b were as large and as numerous as those in drp3a . Although overexpression of DRP3A in drp3a/drp3b restored the shape and number of peroxisomes, overexpression of DRP3B did not restore the phenotypes, and often caused elongation instead. These results suggest that DRP3B and DRP3A have redundant molecular functions in mitochondrial fission, whereas DRP3B has a minor role in peroxisomal fission that is distinct from that of DRP3A.     

Hepatitis C Virus Impairs p53 via Persistent Overexpression of 3��-Hydroxysterol ��24-Reductase

Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes. To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3β-hydroxysterol Δ24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus.     

Time course of vocal modulation during isolation in common marmosets (Callithrix jacchus)

Common marmosets vocalize phee calls as isolation calls, which seem to facilitate their reunion with family groups. To identify multiple acoustic properties with different time courses, we examined acoustic modulations of phees during different social contexts of isolation. Subject marmosets were totally isolated in one condition, were visually isolated and could exchange vocalizations in another condition, and were visually isolated and subsequently totally isolated in a third condition. We recorded 6,035 phees of 10 male–female marmoset pairs and conducted acoustic analysis. The marmosets frequently vocalized phees that were temporally elongated and louder during isolation, with varying time courses of these changes in acoustic parameters. The vocal rates and sound levels of the phees increased as soon as the marmosets saw their pair mates being taken away, and then gradually calmed down. The phee duration was longer in conditions during which there were no vocal responses from their pair mates. Louder vocalizations are conspicuous and seem to be effective for long‐distance transmission, whereas shorter call duration during vocal exchanges might avoid possible vocal overlap between mates. Am. J. Primatol. 72:681–688, 2010. © 2010 Wiley‐Liss, Inc.