Cholesterol controls lipid endocytosis through Rab11

Cellular cholesterol increases when cells reach confluency in Chinese hamster ovary (CHO) cells. We examined the endocytosis of several lipid probes in subconfluent and confluent CHO cells. In subconfluent cells, fluorescent lipid probes including poly(ethylene glycol)derivatized cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol, and fluorescent sphingomyelin analogs were internalized to pericentriolar recycling endosomes. This accumulation was not observed in confluent cells. Internalization of fluorescent lactosylceramide was not affected by cell confluency, suggesting that the endocytosis of specific membrane components is affected by cell confluency. The crucial role of cellular cholesterol in cell confluency-dependent endocytosis was suggested by the observation that the fluorescent sphingomyelin was transported to recycling endosomes when cellular cholesterol was depleted in confluent cells. To understand the molecular mechanism(s) of cell confluency- and cholesterol-dependent endocytosis, we examined intracellular distribution of rab small GTPases. Our results indicate that rab11 but not rab4, altered intracellular localization in a cell confluency-associated manner, and this alteration was dependent on cell cholesterol. In addition, the expression of a constitutive active mutant of rab11 changed the endocytic route of lipid probes from early to recycling endosomes. These results thus suggest that cholesterol controls endocytic routes of a subset of membrane lipids through rab11.     

Molecular cloning and histological localization of LH-like substances in a bottlenose dolphin (Tursiops truncatus) placenta

All mammals exhibit pituitary-specific expression of LH and FSH, whereas placental expression of gonadotropins has been reported only in primates and equids. Some cetaceans, such as dolphins, have a long gestational period and a sexual cycle of about 27 days almost comparable with that of humans. Histologically, dolphins have an epitheliochorial placentae that resembles placentas of Perissodactyla including horses. In the present study, we cloned cDNAs encoding gonadotropins and observed their immunohistochemical localization in the placenta of bottlenose dolphin. The cDNAs obtained encoded 120 amino acids for the alpha-subunit (including 96 amino acids of mature proteins), and 141 amino acids for the beta-subunit (including 121 amino acids of mature proteins). The sequence of the alpha-subunit was similar to that in the pig (Artiodactyla) pituitary glycoprotein hormone [96.7% homology at amino acids (aa) level], and the sequence of the beta-subunit was similar to that of luteinizing hormone (LH) in the pig [94.3% homology at aa level] and white rhinoceros (Perissodactyla) [93.3% homology at aa level]. Of interest, dolphin LHbeta lacks carboxyl-terminal-peptides (CTP). This fact suggests that CTP are not essential for placental expression of gonadotropin in dolphins. Immunohistochemical observations employing anti-ovine LHbeta antibody revealed positive staining in the villositycal tissue. Our observations suggest placental expression of gonadotropin homologues in cetaceans and possible evolutionary conservation of placentae-derived hormonal control of ovarian functions during pregnancy.     

In vitro antioxidant,anti-glycation and immunomodulation activities of fermented blue-green algae Aphanizomenon flos-aquae

Molecular Biology Reports - To clarify the antioxidant, anti-glycation and immunomodulatory capacities of fermented blue-green algae Aphanizomenon flos-aquae (AFA), hot aqueous extract suspensions...     

Probing the Catalytic Mechanism of Copper Amine Oxidase from Arthrobacter globiformis with Halide Ions

The catalytic reaction of copper amine oxidase proceeds through a ping-pong mechanism comprising two half-reactions. In the initial half-reaction, the substrate amine reduces the Tyr-derived cofactor, topa quinone (TPQ), to an aminoresorcinol form (TPQ_amr) that is in equilibrium with a semiquinone radical (TPQ_sq) via an intramolecular electron transfer to the active-site copper. We have analyzed this reductive half-reaction in crystals of the copper amine oxidase from Arthrobacter globiformis. Anerobic soaking of the crystals with an amine substrate shifted the equilibrium toward TPQ_sq in an “on-copper” conformation, in which the 4-OH group ligated axially to the copper center, which was probably reduced to Cu(I). When the crystals were soaked with substrate in the presence of halide ions, which act as uncompetitive and noncompetitive inhibitors with respect to the amine substrate and dioxygen, respectively, the equilibrium in the crystals shifted toward the “off-copper” conformation of TPQ_amr. The halide ion was bound to the axial position of the copper center, thereby preventing TPQ_amr from adopting the on-copper conformation. Furthermore, transient kinetic analyses in the presence of viscogen (glycerol) revealed that only the rate constant in the step of TPQ_amr/TPQ_sq interconversion is markedly affected by the viscogen, which probably perturbs the conformational change. These findings unequivocally demonstrate that TPQ undergoes large conformational changes during the reductive half-reaction.     

Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum

Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process.     

Effects of phylogeny,leaf traits,and the altitudinal distribution of host plants on herbivore assemblages on congeneric Acer species

Historical, niche-based, and stochastic processes have been proposed as the mechanisms that drive community assembly. In plant–herbivore systems, these processes can correspond to phylogeny, leaf traits, and the distribution of host plants, respectively. Although patterns of herbivore assemblages among plant species have been repeatedly examined, the effects of these factors among co-occurring congeneric host plant species have rarely been studied. Our aim was to reveal the process of community assembly for herbivores by investigating the effects of phylogeny, leaf traits, and the altitudinal distribution of closely related host plants of the genus Acer. We sampled leaf functional traits for 30 Acer species in Japan. Using a newly constructed phylogeny, we determined that three of the six measured leaf traits (leaf thickness, C/N ratio, and condensed tannin content) showed a phylogenetic signal. In a field study, we sampled herbivore communities on 14 Acer species within an elevation gradient and examined relationships between herbivore assemblages and host plants. We found that herbivore assemblages were significantly correlated with phylogeny, leaf traits, phylogenetic signals, and the altitudinal distribution of host plants. Our results indicate that the interaction between historical and current ecological processes shapes herbivore community assemblages.     

HLA Class I Expression and Its Alteration by Preoperative Hyperthermo-Chemoradiotherapy in Patients with Rectal Cancer

Objective

Enhancing immunologic responses, including human leukocyte antigen (HLA) class I expression on tumor cells and recognition and elimination of tumor cells by tumor-specific cytotoxic T lymphocyte (CTL), is considered a novel concept of radiotherapy. The present study examined patients who underwent preoperative hyperthermo-chemoradiotherapy (HCRT) for locally advanced rectal cancer to assess the correlation between HLA class I expression and clinical outcome.

Materials and Methods

Seventy-eight patients with locally advanced rectal adenocarcinoma who received preoperative HCRT were enrolled. The median age of the patients was 64 years (range, 33–85 years) and 4, 18, and 56 patients had clinical stage I, II and III disease, respectively. Formalin-fixed and paraffin-embedded tissues excised before and after HCRT were subjected to immunohistochemical analysis with an anti-HLA class I-A, B, C antibody. HLA class I expression was graded according to tumor cell positivity.

Results

In pre-HCRT, the number of specimens categorized as Grade 0 and 1 were 19 (24%) and 58 (74%), respectively. Only 1 patient (1%) showed Grade 2 expression. However, 6 (8%), 27 (35%), 7 (9%), and 12 (15%) post-HCRT specimens were graded as Grade 0, 1, 2, and 3, respectively. There was a significant increase in HLA class I expression in post-HCRT specimens (p<0.01). However, neither pre- nor post-HCRT HLA class I expression affected overall survival and distant metastasis-free survival in clinical stage III patients. Univariate analysis revealed that Post-HCRT HLA class I expression showed a significant negative relationship with LC (p<0.05). Nevertheless, multivariate analysis showed that there was no correlation between HLA class I expression and clinical outcome.

Conclusion

HCRT increased HLA class I expression in rectal cancer patients. However, multivariate analysis failed to show any correlation between the level of HLA class I expression and prognosis.     

The Effects of Harvesting Media on Biological Characteristics and Repair Potential of Neural Stem Cells after Traumatic Brain Injury

Various solutions are utilized widely for the isolation, harvesting, sorting, testing and transplantation of neural stem cells (NSCs), whereas the effects of harvesting media on the biological characteristics and repair potential of NSCs remain unclear. To examine some of these effects, NSCs were isolated from cortex of E14.5 mice and exposed to the conventional harvesting media [0.9% saline (Saline), phosphate-buffered saline (PBS) or artificial cerebrospinal fluid (ACSF)] or the proliferation culture medium (PCM) for different durations at 4°C. Treated NSCs were grafted by in situ injection into the lesion sites of traumatic brain injury (TBI) mice. In vitro, harvesting media-exposed NSCs displayed time-dependent reduction of viability and proliferation. S phase entry decreased in harvesting media-exposed cells, which was associated with upregulation of p53 protein and downregulation of cyclin E1 protein. Moreover, harvesting media exposure induced the necrosis and apoptosis of NSCs. The levels of Fas-L, cleaved caspase 3 and 8 were increased, which suggests that the death receptor signaling pathway is involved in the apoptosis of NSCs. In addition, exposure to Saline did not facilitate the neuronal differentiation of NSCs, suggesting that Saline exposure may be disadvantageous for neurogenesis. In vivo, NSC-mediated functional recovery in harvesting media-exposed NSC groups was notably attenuated in comparison with the PCM-exposed NSC group. In conclusion, harvesting media exposure modulates the biological characteristics and repair potential of NSCs after TBI. Our results suggest that insight of the effects of harvesting media exposure on NSCs is critical for developing strategies to assure the successful long-term engraftment of NSCs.     

Hsp40 Gene Therapy Exerts Therapeutic Effects on Polyglutamine Disease Mice via a Non-Cell Autonomous Mechanism

The polyglutamine (polyQ) diseases such as Huntington’s disease (HD), are neurodegenerative diseases caused by proteins with an expanded polyQ stretch, which misfold and aggregate, and eventually accumulate as inclusion bodies within neurons. Molecules that inhibit polyQ protein misfolding/aggregation, such as Polyglutamine Binding Peptide 1 (QBP1) and molecular chaperones, have been shown to exert therapeutic effects in vivo by crossing of transgenic animals. Towards developing a therapy using these aggregation inhibitors, we here investigated the effect of viral vector-mediated gene therapy using QBP1 and molecular chaperones on polyQ disease model mice. We found that injection of adeno-associated virus type 5 (AAV5) expressing QBP1 or Hsp40 into the striatum both dramatically suppresses inclusion body formation in the HD mouse R6/2. AAV5-Hsp40 injection also ameliorated the motor impairment and extended the lifespan of R6/2 mice. Unexpectedly, we found even in virus non-infected cells that AAV5-Hsp40 appreciably suppresses inclusion body formation, suggesting a non-cell autonomous therapeutic effect. We further show that Hsp40 inhibits secretion of the polyQ protein from cultured cells, implying that it inhibits the recently suggested cell-cell transmission of the polyQ protein. Our results demonstrate for the first time the therapeutic effect of Hsp40 gene therapy on the neurological phenotypes of polyQ disease mice.