High MET copy number and MET overexpression: poor outcome in non-small cell lung cancer patients

The aim of this study was to evaluate the prevalence and prognostic role of increased gene copy number and protein expression of MET and EGFR in non-small cell lung cancer (NSCLC) patients. Samples were collected from 380 patients with surgically resected NSCLC, and fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) were performed. EGFR amplification and high polysomy (EGFR FISH-positive) were observed in 9.7% and 17.4% of the patients, respectively. EGFR was overexpressed (EGFR IHC-positive) in 19.2% of the patients. Neither EGFR FISH-positive nor EGFR IHC-positive status affected survival after resection. Increased MET copy number (MET FISH-positive by University of Colorado Cancer Center criteria) was observed in 11.1% of the patients (high polysomy, 8.7%; gene amplification, 2.4%). According to the Cappuzzo system, 7.1% of the patients were MET FISH-positive. MET FISH positivity was a negative prognostic factor, especially in patients with adenocarcinoma histology (p=0.040), female gender (p=0.010), old age (p=0.084), and EGFR FISH negativity (p=0.020) at the univariate level but not at the multivariate level. MET was overexpressed (MET IHC-positive) in 13.7% of the patients and associated with shorter overall and disease-free survival (p=0.010 and p=0.056, respectively). Multivariate analysis revealed that MET IHC-positive patients had a significantly increased risk of death (hazard ratio, 1.618; 95% confidence interval, 1.066-2.456; p=0.024). Increased MET copy number and MET overexpression are negative prognostic factors for surgically resected NSCLCs.     

Prehension synergy: use of mechanical advantage during multifinger torque production on mechanically fixed and free objects

The aim of this study was to test the mechanical advantage (MA) hypothesis in multifinger torque production tasks in humans: fingers with longer moment arms produce greater force magnitudes during torque production tasks. There were eight experimental conditions: two prehension types determined by different mechanical constraints (i.e., fixed- and free-object prehension) with two torque directions (supination and pronation) and two torque magnitudes (0.24 and 0.48 N·m). The subjects were asked to produce prescribed torques during the fixed-object prehension or to maintain constant position of the free hand-held object against external torques. The index of MA was calculated for agonist and antagonist fingers, which produce torques in the same and opposite directions to the target torques, respectively. Within agonist fingers, the fingers with longer moment arms produced greater grasping forces while within antagonist fingers, the fingers with shorter moment arms produced greater forces. The MA index was greater in the fixed-object condition as compared with the free-object condition. The MA index was greater in the pronation condition than in the supination condition. This study supports the idea that the CNS utilizes the MA of agonist fingers, but not of antagonist fingers, during torque production in both fixed- and free-object conditions.     

Expression of peroxiredoxin 1, 2, and 6 in the rat brain during perinatal development and in response to dexamethasone

Peroxiredoxins (Prdx), a family of antioxidant proteins, have important defensive roles in the degenerative brain diseases and neuronal cell death in adult subjects. However, little is known in the neonatal brain. Here, we studied the developmental expression of Prdxs and their response to dexamethasone in the perinatal rat brain. Prdx 1 expression increased during late gestations and peaked at postnatal-day 1, when its expression gradually decreased. Prdx 2 expression remained largely unchanged. Prdx 6 expression continually increased as growing. Using immunohistochemistry, each Prdx showed a strong expression in the cerebral cortex and hippocampus. Prdx 1 was strongly expressed in the corpus callosum. The dexamethasone injection increased the expression of Prdx 6. In conclusion, we reveal for the first time that Prdx 1, 2 and 6 are found in abundance in the perinatal rat brain and are differentially expressed during development. The expression of Prdx 6 was affected by dexamethasone treatment.     

PURIFICATION OF A SEX‐SPECIFIC LECTIN INVOLVED IN GAMETE BINDING OF AGLAOTHAMNION CALLOPHYLLIDICOLA (RHODOPHYTA)1

Egg and sperm binding and correct recognition is the first stage for successful fertilization. In red algae, spermatial attachment to female trichogynes is mediated by a specific binding between the lectin(s) distributed on the surface of trichogyne and the complementary carbohydrates on the spermatial surface. A female‐specific lectin was isolated from Aglaothamnion callophyllidicola by agarose‐bound fetuin affinity chromatography. Two proteins, 50 and 14 kDa, eluted from the fetuin column were separated using a native‐polyacrylamide gel electrophoresis method and subjected to a gamete binding assay. The 50 kDa protein, which blocked spermatial binding to female trichogynes, was used for further analysis. Internal amino acid sequence of the 50 kDa protein was analyzed using matrix‐assisted laser desorption/ionization‐mass spectrometry and degenerated primers were designed based on the information. A full‐length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends polymerase chain reaction (PCR). The cDNA was 1552 bp in length and coded for a protein of 450 amino acids with a deduced molecular mass of 50.7 kDa, which agreed well with the protein data. Real‐time PCR analysis showed that this protein was up‐regulated about 10‐fold in female thalli. As the protein was novel and showed no significant homology to any known proteins, it was designated Rhodobindin.     

Over-expression of human clusterin increases stress resistance and extends lifespan in Drosophila melanogaster

Clusterin is a disulfide-linked heterodimeric glycoprotein that has been implicated in a variety of biological processes. Its expression has been shown to be elevated during cellular senescence and normal aging, but it is uncertain whether clusterin protects against aging or whether its expression is a consequence of aging. To investigate the functions of clusterin during organismal aging, we established transgenic Drosophila alleles to induce the expression of the secretory form of human clusterin (hClu(S)) using the Gal4/UAS system. hClu(S) protein (~60 kDa) was detected in both adult homogenates and larval hemolymphs of flies ubiquitously overexpressing hClu(S) (da-Gal4>UAS-hClu(S)) and in motoneurons (D42-Gal4>UAS-hClu(S)). Interestingly, the mean lifespans of these hClu(S)-overexpressing flies were significantly greater than those of control flies that exhibited no hClu(S) induction. hClu(S)-overexpressing flies also showed significantly greater tolerance to heat shock, wet starvation, and oxidative stress. Furthermore, amounts of reactive oxygen species (ROS) in whole bodies were significantly lower in hClu(S)-overexpressing flies. In addition, clusterin was found to prevent the inactivation of glutamine synthetase (GS) by metal-catalyzed oxidation (MCO) in vitro, and this protection was only supported by thiol-reducing equivalents, such as, DTT or GSH, and not by ascorbate (a non-thiol MCO system). Furthermore, this protection against GS inactivation by clusterin was abolished by reacting clusterin with N-ethylmaleimide, a sulfhydryl group-modifying agent. Taken together, these results suggest that a disulfide-linked form of clusterin functions as an antioxidant protein via its cysteine sulfhydryl groups to reduce ROS levels and delay the organismal aging in fruit flies.     

Regulatory mechanism for expression of IL1B receptors in the uterine endometrium and effects of IL1B on prostaglandin synthetic enzymes during the implantation period in pigs

During the implantation period, the porcine conceptus secretes interleukin-1beta (IL1B) that may be involved in the establishment of pregnancy in pigs. However, the regulatory mechanism for IL1B receptor expression and the function of IL1B in the uterine endometrium are not well elucidated. In this study, we determined IL1B receptor expression in the uterine endometrium of pigs during pregnancy. IL1B receptor subtypes, IL1 receptor type I (IL1R1) and IL1 receptor accessory protein (IL1RAP) were expressed in the uterine endometrium with the expression being most abundant on Day 12 of pregnancy primarily in the luminal and glandular epithelial cells. Expression of IL1R1 mRNA increased in response to IL1B in a dose-dependent manner, and expression of IL1RAP mRNA increased in response to both IL1B and estradiol, indicating that expression of endometrial IL1B receptors was regulated cooperatively by IL1B and estrogen of conceptus origin. During the peri-implantation period, the porcine uterine endometrium actively synthesizes and secretes prostaglandins (PGs). IL1B increased expression of PTGS1 and PTGS2 genes that are rate-limiting for PG synthesis in the uterine endometrium. Collectively, the results indicated that IL1B regulates expression of IL1R1 and IL1RAP and stimulates expression of PTGS1 and PTGS2 that are considered to be the most rate-limiting enzymes for endometrial synthesis of PGs during the peri-implantation period of pregnancy in pigs.     

Heliorhodopsin binds and regulates glutamine synthetase activity

Photoreceptors are light-sensitive proteins found in various organisms that respond to light and relay signals into the cells. Heliorhodopsin, a retinal-binding membrane protein, has been recently discovered, however its function remains unknown. Herein, we investigated the relationship between Actinobacteria bacterium IMCC26103 heliorhodopsin (AbHeR) and an adjacent glutamine synthetase (AbGS) in the same operon. We demonstrate that AbHeR binds to AbGS and regulates AbGS activity. More specifically, the dissociation constant (K_d) value of the binding between AbHeR and AbGS is 6.06 μM. Moreover, the absence of positively charged residues within the intracellular loop of AbHeR impacted K_d value as they serve as critical binding sites for AbGS. We also confirm that AbHeR up-regulates the biosynthetic enzyme activity of AbGS both in vitro and in vivo in the presence of light. GS is a key enzyme involved in nitrogen assimilation that catalyzes the conversion of glutamate and ammonia to glutamine. Hence, the interaction between AbHeR and AbGS may be critical for nitrogen assimilation in Actinobacteria bacterium IMCC26103 as it survives in low-nutrient environments. Overall, the findings of our study describe, for the first time, to the best of our knowledge, a novel function of heliorhodopsin as a regulatory rhodopsin with the capacity to bind and regulate enzyme activity required for nitrogen assimilation.

A study of heliorhodopsin, an actinobacterial photoreceptor of unknown function, reveals that it interacts with glutamine synthetase, an enzyme involved in nitrogen assimilation, and regulates its activity in the presence of light, highlighting the diverse functions of rhodopsins in different organisms.     

Selection of 3'-template bases and initiating nucleotides by hepatitis C virus NS5B RNA-dependent RNA polymerase

De novo RNA synthesis by hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase has been investigated using short RNA templates. Various templates including those derived from the HCV genome were evaluated by examining the early steps of de novo RNA synthesis. NS5B was shown to be able to produce an initiation dinucleotide product from templates as short as 4-mer and from the 3'-terminal sequences of both plus and minus strands of the HCV RNA genome. GMP, GDP, and guanosine were able to act as an initiating nucleotide in de novo RNA synthesis, indicating that the triphosphate moiety is not absolutely required by an initiating nucleotide. Significant amounts of the initiation product accumulated in de novo synthesis, and elongation from the dinucleotide was observed when large amounts of dinucleotide were available. This result suggests that NS5B, a template, and incoming nucleotides are able to form an initiation complex that aborts frequently by releasing the dinucleotide product before transition to an elongation complex. The transition is rate limiting. Furthermore, we discovered that the secondary structure of a template was not essential for de novo initiation and that 3'-terminal bases of a template conferred specificity in selection of an initiation site. Initiation can occur at the +1, +2, or +3 position numbered from the 3' end of a template depending on base composition. Pyrimidine bases at any of the three positions are able to serve as an initiation site, while purine bases at the +2 and +3 positions do not support initiation. This result implies that HCV possesses an intrinsic ability to ensure that de novo synthesis is initiated from the +1 position and to maintain the integrity of the 3' end of its genome. This assay system should be an important tool for investigating the detailed mechanism of de novo initiation by HCV NS5B as well as other viral RNA polymerases.     

Heavy and light chain variable single domains of an anti-DNA binding antibody hydrolyze both double- and single-stranded DNAs without sequence specificity

Anti-DNA antibodies (Abs) are of biomedical interest because they are associated with autoimmune diseases in human and mice. Previously we isolated an anti-DNA monoclonal Ab 3D8 from an autoimmune-prone MRL-lpr/lpr mouse. Here we have characterized DNA binding kinetics and hydrolyzing activities of the recombinant single chain variable fragment (scFv) and the single variable domains of heavy chain (VH) and light chain (VL) using various single-stranded (ss) and double-stranded (ds) DNA substrates. All the Abs bound to both ds- and ssDNAs without significant preferential sequence specificity showing scFv higher affinities (KD = approximately 17-74 nm) than VH (KD = approximately 2.4-8.4 microm) and VL (KD = approximately 3.2-72 microm), and efficiently hydrolyzed both ds- and ssDNAs without sequence specificity in a Mg2+-dependent manner, except for the poor activity of 3D8 scFv for ss-(dT)40. Elucidated crystal structure-based His to Ala mutations on the complementarity determining regions of VH (His-H35 --> Ala) and/or VL (His-L94 --> Ala) of 3D8 scFv significantly inhibited the catalytic activities, indicating that the His residues are involved in the catalytic mechanism of 3D8 scFv. However, the DNA hydrolyzing activities of single domain VH and VL were not affected by the mutations, indicative of their different catalytic mechanisms from that of 3D8 scFv. Our results demonstrate single domain Abs with DNase activities for the first time, which might provide new insights into substrate recognition and catalytic mechanisms of anti-DNA Abs.