In situ studies on AMP deaminase as a control system of the adenylate energy charge in yeasts

The role of AMP deaminase reaction in the stabilization of the adenylate energy charge was investigated using permeabilized yeast cells. The addition of P_i or Zn²⁺, which inhibits AMP deaminase, remarkably retarded the depletion of total adenylate pool and the recovery of the adenylate energy charge. Polyamine, an activator of the enzyme, decreased total adenylates, resulting in the enhanced recovery of the energy charge in situ. AMP deaminase can act as a regulatory enzyme in the system that stabilizes the adenylate energy charge in yeast cells under the conditions of severe metabolic stress.     

Expression of gastric gland mucous cell-type mucin in normal and neoplastic human tissues.

Gastric gland mucous cells produce class III mucin, which is also found in Brunner's glands and mucous glands along the pancreaticobiliary tract, and in metaplasia and adenocarcinomas differentiating towards gastric mucosa. Recently, we showed that class III mucin possesses GlcNAcalpha1-->4Galbeta-->R, formed by alpha1,4-N-acetylglucosaminyltransferase (alpha4GnT). Examining the tissue-specific expression of mucin epitopes is useful to clarify cell-lineage differentiation and to identify the site of origin of metastatic carcinomas in histological specimens. Formalin-fixed, paraffin-embedded tissue sections from esophagus, stomach, colon, liver, pancreas, lung, kidney, prostate, breast, and salivary gland resected for carcinoma, as well as salivary gland adenoma, colon adenoma, and metastatic adenocarcinoma of lymph nodes from stomach, pancreas, colon, and breast, were immunostained for MUC6, alpha4GnT, and GlcNAcalpha1-->4Galbeta-->R. These were all expressed in normal, metaplastic, and adenocarcinoma tissues of stomach, pancreas, and bile duct, and in pulmonary mucinous bronchioloalveolar carcinomas. Cells expressing alpha4GnT uniformly expressed GlcNAcalpha1-->4Galbeta-->R. Only MUC6 was expressed in normal salivary glands, pancreas, seminal vesicles, renal tubules, and colon adenomas, and in normal tissue and adenocarcinomas of prostate and breast. No tissues showed immunoreactivity for alpha4GnT alone. Immunohistochemistry (IHC) profiles were similar for metastatic carcinomas and primary carcinoma tissues. The IHC profiles for MUC6, alpha4GnT, and GlcNAcalpha1-->4Galbeta-->R may be diagnostically relevant.     

Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model

Baloxavir is approved in several countries for the treatment of uncomplicated influenza in otherwise-healthy and high-risk patients. Treatment-emergent viruses with reduced susceptibility to baloxavir have been detected in clinical trials, but the likelihood of widespread occurrence depends on replication capacity and onward transmission. We evaluated the fitness of A/H3N2 and A/H1N1pdm09 viruses with the polymerase acidic (PA) I38T-variant conferring reduced susceptibility to baloxavir relative to wild-type (WT) viruses, using a competitive mixture ferret model, recombinant viruses and patient-derived virus isolates. The A/H3N2 PA/I38T virus showed a reduction in within-host fitness but comparable between-host fitness to the WT virus, while the A/H1N1pdm09 PA/I38T virus had broadly similar within-host fitness but substantially lower between-host fitness. Although PA/I38T viruses replicate and transmit between ferrets, our data suggest that viruses with this amino acid substitution have lower fitness relative to WT and this relative fitness cost was greater in A/H1N1pdm09 viruses than in A/H3N2 viruses.     

Metabolism of a liver-selective nitric oxide-releasing agent, V-PYRRO/NO, by human microsomal cytochromes P450

Endogenously generated nitric oxide (NO) mediates a host of important physiological functions, playing roles in the vascular, immunological, and neurological systems. As a result, exogenous agents that release NO have become important therapeutic interventions and research tools. O(2)-Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a prodrug designed with the hypothesis that it might release nitric oxide via epoxidation of the vinyl group by cytochrome P450, followed by enzymatic and/or spontaneous epoxide hydration to release the ultimate NO-donating moiety, 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO) ion. In this study, we investigated this hypothetical activation mechanism quantitatively for V-PYRRO/NO using cDNA-expressed human cytochrome P450 (CYP)2E1. Incubation with CYP2E1 and an NADPH-regenerating system resulted in a time-dependent decomposition of V-PYRRO/NO, with a turnover rate of 2.0 nmol/min/pmol CYP2E1. Nitrate and nitrite were detected in high yield as metabolites of NO. The predicted organic metabolites pyrrolidine and glycolaldehyde were also detected in near-quantitative yields. The enzymatic decomposition of V-PYRRO/NO was also catalyzed, albeit at lower rates, by CYP2A6 and CYP2B6. We conclude that the initial step in the metabolism of V-PYRRO/NO to NO in the liver is catalyzed efficiently but not exclusively by the alcohol-inducible form of cytochrome P450 (CYP2E1). The results confirm the proposed activation mechanism involving enzymatic oxidation of the vinyl group in V-PYRRO/NO followed by epoxide hydration and hydrolytic decomposition of the resulting PYRRO/NO ion to generate nitric oxide.     

Network modeling of the transcriptional effects of copy number aberrations in glioblastoma

DNA copy number aberrations (CNAs) are a hallmark of cancer genomes. However, little is known about how such changes affect global gene expression. We develop a modeling framework, EPoC (Endogenous Perturbation analysis of Cancer), to (1) detect disease‐driving CNAs and their effect on target mRNA expression, and to (2) stratify cancer patients into long‐ and short‐term survivors. Our method constructs causal network models of gene expression by combining genome‐wide DNA‐ and RNA‐level data. Prognostic scores are obtained from a singular value decomposition of the networks. By applying EPoC to glioblastoma data from The Cancer Genome Atlas consortium, we demonstrate that the resulting network models contain known disease‐relevant hub genes, reveal interesting candidate hubs, and uncover predictors of patient survival. Targeted validations in four glioblastoma cell lines support selected predictions, and implicate the p53‐interacting protein Necdin in suppressing glioblastoma cell growth. We conclude that large‐scale network modeling of the effects of CNAs on gene expression may provide insights into the biology of human cancer. Free software in MATLAB and R is provided.     

Isolation and partial characterization of a novel disulfoglycosphingolipid from rat kidney

A novel sulfoglycosphingolipid containing two sulfate ester groups was isolated from the lipid extract of rat kidney by a procedure involving mild alkaline methanolysis and column chromatographies on DEAE-Sephacel and silicic acid. The component carbohydrates were galactose, glucose and $\text{N}$-acetylgalactosamine in equimolar amounts. Infrared spectroscopy, permethylation study, periodate oxidation and solvolysis suggested that the sulfoglycolipid was GalNAc1-4Gal1-4GlcCer sulfated at the C3 hydroxyls of both galactose and $\text{N}$-acetylgalactosamine. The yield of this sulfoglycolipid was 11.2 nmol/g tissue.     

Effects of p35 Mutations Associated with Mental Retardation on the Cellular Function of p35-CDK5

p35 is an activation subunit of the cyclin-dependent kinase 5 (CDK5), which is a Ser/Thr kinase that is expressed predominantly in neurons. Disruption of the CDK5 or p35 (CDK5R1) genes induces abnormal neuronal layering in various regions of the mouse brain via impaired neuronal migration, which may be relevant for mental retardation in humans. Accordingly, mutations in the p35 gene were reported in patients with nonsyndromic mental retardation; however, their effect on the biochemical function of p35 has not been examined. Here, we studied the biochemical effect of mutant p35 on its known properties, i.e., stability, CDK5 activation, and cellular localization, using heterologous expression in cultured cells. We also examined the effect of the mutations on axon elongation in cultured primary neurons and migration of newborn neurons in embryonic brains. However, we did not detect any significant differences in the effects of the mutant forms of p35 compared with wild-type p35. Therefore, we conclude that these p35 mutations are unlikely to cause mental retardation.     

Intermolecular crosslinking of abnormal prion protein is efficiently induced by a primuline-sensitized photoreaction

In prion diseases, infectious pathogenic particles that are composed of abnormal prion proteins (PrP^Sc) accumulate in the brain. PrP^Sc is biochemically characterized by its protease-resistance core (PrP^res), but its structural features have not been fully elucidated. Here, we report that primuline, a fluorescent dye with photosensitization activity, dramatically enhances UV-irradiation-induced SDS-resistant PrP^Sc/res oligomer formation that can be detected by immunoblot analysis of prion-infected materials. This oligomer formation occurs specifically with PrP^Sc/res but not with normal prion protein, and it was demonstrated using purified PrP^Sc/res as well as unpurified materials. The oligomer formation proceeded in both primuline-dose- and UV irradiation time-dependent manners. Treatment with urea or formic acid did not break oligomers into monomers. Neither did the presence of aromatic amino acids modify oligomer formation. Analysis with a panel of anti-prion protein antibodies showed that the antibodies against the N-terminal region of PrP^res were less reactive in the dimer than the monomer. These findings suggest that the primuline-sensitized photoreaction enhances intermolecular crosslinking of PrP^Sc/res molecules at a hydrophobic area of the N-terminal region of PrP^res. In the screening of other compounds, photoreactive compounds such as luciferin exhibited a similar but lower activity with respect to oligomer formation than primuline. The enhanced photoreaction with these compounds will be useful for evaluating the structural features of PrP^Sc/res, especially the interactions between PrP^Sc/res molecules.     

Rhodopsin in the Dark Hot Sea: Molecular Analysis of Rhodopsin in a Snailfish,Careproctus rhodomelas,Living near the Deep-Sea Hydrothermal Vent

Visual systems in deep-sea fishes have been previously studied from a photobiological aspect; however, those of deep-sea fish inhabiting the hydrothermal vents are far less understood due to sampling difficulties. In this study, we analyzed the visual pigment of a deep-sea snailfish, Careproctus rhodomelas, discovered and collected only near the hydrothermal vents of oceans around Japan. Proteins were solubilized from the C. rhodomelas eyeball and subjected to spectroscopic analysis, which revealed the presence of a pigment characterized by an absorption maximum (λ_max) at 480 nm. Immunoblot analysis of the ocular protein showed a rhodopsin-like immunoreactivity. We also isolated a retinal cDNA encoding the entire coding sequence of putative C. rhodomelas rhodopsin (CrRh). HEK293EBNA cells were transfected with the CrRh cDNA and the proteins extracted from the cells were subjected to spectroscopic analysis. The recombinant CrRh showed the absorption maximum at 480 nm in the presence of 11-cis retinal. Comparison of the results from the eyeball extract and the recombinant CrRh strongly suggests that CrRh has an A₁-based 11-cis-retinal chromophore and works as a photoreceptor in the C. rhodomelas retina, and hence that C. rhodomelas responds to dim blue light much the same as other deep-sea fishes. Because hydrothermal vent is a huge supply of viable food, C. rhodomelas likely do not need to participate diel vertical migration and may recognize the bioluminescence produced by aquatic animals living near the hydrothermal vents.