Biosynthesis of branched-chain fatty acid in bacilli: FabD (malonyl-CoA:ACP transacylase) is not essential for in vitro biosynthesis of branched-chain fatty acids

It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.     

neurotic,a novel maternal neurogenic gene,encodes an O-fucosyltransferase that is essential for Notch-Delta interactions

Notch signalling, which is highly conserved from nematodes to mammals, plays crucial roles in many developmental processes. In the Drosophila embryo, deficiency in Notch signalling results in neural hyperplasia, commonly referred to as the neurogenic phenotype. We identify a novel maternal neurogenic gene, neurotic, and show that it is essential for Notch signalling. neurotic encodes a Drosophila homolog of mammalian GDP-fucose protein O-fucosyltransferase, which adds fucose sugar to epidermal growth factor-like repeats and is known to play a crucial role in Notch signalling. neurotic functions in a cell-autonomous manner, and genetic epistasis tests reveal that Neurotic is required for the activity of the full-length but not an activated form of Notch. Further, we show that neurotic is required for Fringe activity, which encodes a fucose-specific beta1, 3 N-acetylglucosaminyltransferase, previously shown to modulate Notch receptor activity. Finally, Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. We present an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction. Our results suggest that O-fucosylation catalysed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions.     

Thyroid-hormone-dependent and fibroblast-specific expression of BMP-4 correlates with adult epithelial development during amphibian intestinal remodeling

We have identified one of the genes that are up-regulated by thyroid hormone (TH) in Xenopus laevis small intestine as the Xenopus homolog of bone morphogenetic protein-4 (BMP-4). To clarify possible roles of BMP-4 in intestinal remodeling during metamorphosis, we have examined its expression in X. laevis intestine by using in situ hybridization and organ culture techniques. At the beginning of metamorphic climax, BMP-4 mRNA first becomes detectable in the connective tissue, concurrently with the appearance of adult epithelial primordia. Subsequently, when the adult epithelial primordia are actively proliferating, BMP-4 mRNA becomes more abundant only in the connective tissue with a gradient toward the epithelium. Thereafter, as the adult primordia differentiate, the level of BMP-4 mRNA gradually decreases. Thus, BMP-4 expression correlates well with cell proliferation and/or initial differentiation of the adult epithelium, but not with apoptosis of the larval epithelium. Furthermore, the present culture study indicates that (1) TH-induced expression of BMP-4 mRNA is higher in the anterior part of the intestine than in the posterior part, which agrees with the better development of the adult epithelium in the more anterior part, and that (2) the expression of BMP-4 mRNA is up-regulated by TH in the presence of epithelium, but not in its absence. Therefore, BMP-4, which is indirectly induced by TH through some epithelial factor(s), probably plays important roles in adult epithelial development during amphibian intestinal remodeling.     

Failure to respond to interferon-α 2a therapy is associated with increased hepatic iron levels in patients with chronic hepatitis C

Recent reports suggest the hepatic iron concentration (HIC) may influence the activity of hepatitis and the response to interferon (IFN) therapy in patients with chronic hepatitis C (CH-C). We have evaluated iron status in 28 patients with CH-C and determined if pretreatment iron status can predict the response to IFN-α therapy in these patients. Increased serum iron, transferrin saturation, and ferritin levels were observed in 3 (11%), 11 (39%), and 5 (18%) patients, respectively. Hepatic iron deposits were histologically detected in 17 (61%) patients, and 14 of them had stainable hepatocytic iron. However, all HIC values were within the normal range (203–1279 μg/g). Seven of 17 patients treated with IFN-α for 6 mo had normalization of serum transaminases and disappearance of serum HCV-RNA (responders). Nonresponders had a significantly higher median HIC compared with responders (710 vs 343 μg/g, respectively;p < 0.05). There was no significant difference in other pretreatment iron parameters, serum HCV-RNA level, or HCV-genotype between responders and nonresponders. In conclusion, mild hepatic iron accumulation occurs in patients with CH-C. Increased hepatic iron stores are associated with poor response to IFN therapy. Pretreatment HIC may be an additional host-specific parameter with a predictive value for responsiveness to IFN therapy, in addition to well-known predictive viral factors.     

Induction of CD44 Variant 9-Expressing Cancer Stem Cells Might Attenuate the Efficacy of Chemoradioselection and Worsens the Prognosis of Patients with Advanced Head and Neck Cancer

Background

At our institute, a chemoradioselection strategy has been used to select patients for organ preservation on the basis of response to an initial 30–40 Gy concurrent chemoradiotherapy (CCRT). Patients with a favorable response (i.e., chemoradioselected; CRS) have demonstrated better outcomes than those with an unfavorable response (i.e., nonchemoradioselected; N-CRS). Successful targeting of molecules that attenuate the efficacy of chmoradioselection may improve results. Thus, the aim of this study was to evaluate the association of a novel cancer stem cell (CSC) marker, CD44 variant 9 (CD44v9), with cellular refractoriness to chemoradioselection in advanced head and neck squamous cell carcinoma (HNSCC).

Materials and Methods

Through a medical chart search, 102 patients with advanced HNSCC treated with chemoradioselection from 1997 to 2008 were enrolled. According to our algorithm, 30 patients were CRC following induction CCRT and 72 patients were N-CRS. Using the conventional immunohistochemical technique, biopsy specimens and surgically removed tumor specimens were immunostained with the anti-CD44v9 specific antibodies.

Results

The intrinsic expression levels of CD44v9 in the biopsy specimens did not correlate with the chemoradioselection and patient survival. However, in N-CRS patients, the CD44v9-positive group demonstrated significantly (P = 0.008) worse prognosis, than the CD44v9-negative group. Multivariate analyses demonstrated that among four candidate factors (T, N, response to CCRT, and CD44v9), CD44v9 positivity (HR: 3.145, 95% CI: 1.235–8.008, P = 0.0163) was significantly correlated with the poor prognosis, along with advanced N stage (HR: 3.525, 95% CI: 1.054–9.060, P = 0.0228). Furthermore, the survival rate of the CD44v9-induced group was significantly (P = 0.04) worse than the CD44v9-non-induced group.

Conclusions

CCRT-induced CD44v9-expressing CSCs appear to be a major hurdle to chemoradioselection. CD44v9-targeting seems to be a promising strategy to enhance the efficacy of chemoradioselection and consequent organ preservation and survival.     

Epstein-Barr Virus Infection in Chronically Inflamed Periapical Granulomas

Periapical granulomas are lesions around the apex of a tooth caused by a polymicrobial infection. Treatment with antibacterial agents is normally performed to eliminate bacteria from root canals; however, loss of the supporting alveolar bone is typically observed, and tooth extraction is often selected if root canal treatment does not work well. Therefore, bacteria and other microorganisms could be involved in this disease. To understand the pathogenesis of periapical granulomas more precisely, we focused on the association with Epstein-Barr virus (EBV) using surgically removed periapical granulomas (n = 32). EBV DNA was detected in 25 of 32 periapical granulomas (78.1%) by real-time PCR, and the median number of EBV DNA copies was approximately 8,688.01/μg total DNA. In contrast, EBV DNA was not detected in healthy gingival tissues (n = 10); the difference was statistically significant according to the Mann-Whitney U test (p = 0.0001). Paraffin sections were also analyzed by in situ hybridization to detect EBV-encoded small RNA (EBER)-expressing cells. EBER was detected in the cytoplasm and nuclei of B cells and plasma cells in six of nine periapical granulomas, but not in healthy gingival tissues. In addition, immunohistochemical analysis for latent membrane protein 1 (LMP-1) of EBV using serial tissue sections showed that LMP-1-expressing cells were localized to the same areas as EBER-expressing cells. These data suggest that B cells and plasma cells in inflamed granulomas are a major source of EBV infection, and that EBV could play a pivotal role in controlling immune cell responses in periapical granulomas.     

Schisandrin B Ameliorates ICV-Infused Amyloid β Induced Oxidative Stress and Neuronal Dysfunction through Inhibiting RAGE/NF-κB/MAPK and Up-Regulating HSP/Beclin Expression

Amyloid β (Aβ)-induced neurotoxicity is a major pathological mechanism of Alzheimer’s disease (AD). Our previous studies have demonstrated that schisandrin B (Sch B), an antioxidant lignan from Schisandra chinensis, could protect mouse brain against scopolamine- and cisplatin-induced neuronal dysfunction. In the present study, we examined the protective effect of Sch B against intracerebroventricular (ICV)-infused Aβ-induced neuronal dysfunction in rat cortex and explored the potential mechanism of its action. Our results showed that 26 days co-administration of Sch B significantly improved the behavioral performance of Aβ (1–40)-infused rats in step-through test. At the same time, Sch B attenuated Aβ-induced increases in oxidative and nitrosative stresses, inflammatory markers such as inducible nitric oxide syntheses, cyclooxygenase-2, interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α, and DNA damage. Several proteins such as receptor for advanced glycation end products (RAGE), nuclear factor-κB, mitogen-activated protein kinases, and apoptosis markers were over expressed in Aβ-infused rats but were significantly inhibited by Sch B treatment. Furthermore, Sch B negatively modulated the Aβ level with simultaneous up-regulation of HSP70 and beclin, autophagy markers in Aβ-infused rats. The aforementioned effects of Sch B suggest its protective role against Aβ-induced neurotoxicity through intervention in the negative cycle of RAGE-mediated Aβ accumulation during AD patho-physiology.     

Influence of Aging and Gender Differences on Feeding Behavior and Ghrelin-Related Factors during Social Isolation in Mice

Psychological stress due to social isolation is known to cause abnormal feeding behaviors, but the influences of gender and aging on subchronic stress-induced changes in feeding behaviors are unknown. Thus, we examined the changes in body weight, food intake, and orexigenic ghrelin-related factors during 2 weeks of isolation stress in young and aged mice. Food intake increased significantly in young mice in the isolation group compared with the group-housed control throughout the experimental period. This isolation-induced increase in food intake was not observed in aged mice. In young mice, there were no significant differences in body weight between the isolated group and group-housed control up to 2 weeks. However, aged male mice exhibited significant weight loss at 2 weeks and a similar tendency was observed in aged female mice. Young male mice, but not female mice, had significantly increased (2.2-fold) plasma acylated ghrelin levels after 1 week of isolation compared with the group-housed control. A significant but lower increase (1.3-fold) was also observed in aged male mice. Hypothalamic preproghrelin gene expression decreased significantly with isolation in young male mice, whereas it increased significantly in female mice. The expression levels of NPY and AGRP in the hypothalamus, which are transmitted by elevated peripheral ghrelin signals, increased significantly in isolated young male mice, whereas the AGRP expression levels decreased significantly in young female mice. Isolation caused no significant differences in the expression levels of these genes in aged mice. In isolation, young female mice exhibited markedly increased dark- and light-phase locomotor activities compared with male mice, whereas male and female aged mice exhibited no obvious increases in activity immediately after the dark phase started. We conclude that the gender-specific homeostatic regulatory mechanisms required to maintain body weight operated during subchronic psychological stress in young mice but not in aged mice.     

mTOR regulates the nucleoplasmic diffusion of Xrn2 under conditions of heat stress

Stress induces various responses, including translational suppression and tRNA degradation in mammals. Previously, we showed that heat stress induces degradation of initiator tRNA^Met (iMet) through 5′–3′ exoribonuclease Xrn1 and Xrn2, respectively. In addition, we found that rapamycin inhibits the degradation of iMet under heat stress conditions. Here, we report that the mammalian target of rapamycin (mTOR) regulates the diffusion of Xrn2 from the nucleolus to the nucleoplasm, facilitating the degradation of iMet under conditions of heat stress. Our results suggest a mechanism of translational suppression through mTOR-regulated iMet degradation in mammalian cells.     

N-type voltage-dependent Ca channel in non-excitable microglial cells in mice is involved in the pathophysiology of neuropathic pain

Peripheral nerve injury induces neuropathic pain which is characterized by tactile allodynia and thermal hyperalgesia. N-type voltage-dependent Ca²⁺ channel (VDCC) plays pivotal roles in the development of neuropathic pain, since mice lacking Ca_v2.2, the pore-forming subunit of N-type VDCC, show greatly reduced symptoms of both tactile allodynia and thermal hyperalgesia. Our study on gene expression profiles of the Ca_v2.2 knockout (KO) spinal cord after spinal nerve ligation (SNL)-injury revealed altered expression of genes known to be expressed in microglia, raising an odd idea that N-type VDCC may function in not only excitable (neurons) but also non-excitable (microglia) cells in neuropathic pain state. In the present study, we have tested this idea by using a transgenic mouse line, in which suppression of Ca_v2.2 expression can be achieved specifically in microglia/macrophage by the application of tamoxifen. We found SNL-operated transgenic mice exhibited greatly reduced signs of tactile allodynia, whereas the degree of thermal hyperalgesia was almost the same as that of control. Immunohistochemical analysis of the transgenic lumbar spinal cord revealed reduced accumulation of Iba1-positive cells (microglia/macrophage) around the injured neurons, indicating microglial N-type VDCC is important for accumulation of microglia at the lesion sites. Although the mechanism of its activation is not clear at present, activation of N-type VDCC expressed in non-excitable microglial cells contributes to the pathophysiology of neuropathic pain.