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Productive infection of Trichoplusia ni cells by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) leads to expression of ~156 viral genes and results in dramatic cell remodeling. How the cell transcriptome responds to viral infection was unknown due to the lack of a reference genome and transcriptome for T. ni. We used an~60-Gb RNA sequencing (RNA-seq) data set from infected and uninfected T. ni cells to generate and annotate a de novo transcriptome assembly of approximately 70,322 T. ni unigenes (assembled transcripts), representing the 48-h infection cycle. Using differential gene expression analysis, we found that the majority of host transcripts were downregulated after 6 h postinfection (p.i.) and throughout the remainder of the infection. In contrast, 5.7% (4,028) of the T. ni unigenes were upregulated during the early period (0 to 6 h p.i.), followed by a decrease through the remainder of the infection cycle. Also, a small subset of genes related to metabolism and stress response showed a significant elevation of transcript levels at 18 and 24 h p.i. but a decrease thereafter. We also examined the responses of genes belonging to a number of specific pathways of interest, including stress responses, apoptosis, immunity, and protein trafficking. We identified specific pathway members that were upregulated during the early phase of the infection. Combined with the parallel analysis of AcMNPV expression, these results provide both a broad and a detailed view of how baculovirus infection impacts the host cell transcriptome to evade cellular defensive responses, to modify cellular biosynthetic pathways, and to remodel cell structure. [ABSTRACT FROM AUTHOR]

Autophagy is the principal catabolic prosurvival pathway during nutritional starvation. However, excessive autophagy could be cytotoxic, contributing to cell death, but its mechanism remains elusive. Arginine starvation has emerged as a potential therapy for several types of cancers, owing to their tumor-selective deficiency of the arginine metabolism. We demonstrated here that arginine depletion by arginine deiminase induces a cytotoxic autophagy in argininosuccinate synthetase (ASS1)-deficient prostate cancer cells. Advanced microscopic analyses of arginine-deprived dying cells revealed a novel phenotype with giant autophagosome formation, nucleus membrane rupture, and histone-associated DNA leakage encaptured by autophagosomes, which we shall refer to as chromatin autophagy, or chromatophagy. In addition, nuclear inner membrane (lamin A/C) underwent localized rearrangement and outer membrane (NUP98) partially fused with autophagosome membrane. Further analysis showed that prolonged arginine depletion impaired mitochondrial oxidative phosphorylation function and depolarized mitochondrial membrane potential. Thus, reactive oxygen species (ROS) production significantly increased in both cytosolic and mitochondrial fractions, presumably leading to DNA damage accumulation. Addition of ROS scavenger N-acetyl cysteine or knockdown of ATG5 or BECLIN1 attenuated the chromatophagy phenotype. Our data uncover an atypical autophagy-related death pathway and suggest that mitochondrial damage is central to linking arginine starvation and chromatophagy in two distinct cellular compartments. [ABSTRACT FROM AUTHOR]

Background: Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3' end adapter containing a 5′,5′-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3′ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available. Results: We demonstrate that DNA oligo with 5′ phosphate and 3′ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum) using the T4 RNA ligase 1 adenylated adapter. Conclusion: We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples. [ABSTRACT FROM AUTHOR]

Background: Hepatitis C virus (HCV) core protein is a multi-functional viral protein that interacts with several target proteins of both viral and cellular origin. Aim and Methods: To gain insight into the mechanism of action of HCV core protein, we used a yeast two-hybrid system to identify the core protein-interacting cellular targets. Results: A cDNA clone encoding an aspartoacylase was obtained, termed aspartoacylase 3 (ACY3). Interaction between ACY3 and HCV core protein was verified using a co-immunoprecipitation assay in vitro, and a mammalian two-hybrid system in vivo. Fluorescence microscopy showed green fluorescence protein-fused ACY3 localized in the cytoplasm. Conclusion: Our data suggest that ACY3 is an HCV core binding protein, which may play a role in the development of HCV-associated diseases. [ABSTRACT FROM AUTHOR]

Significant variations in the frequency of single nucleotide polymorphisms (SNPs) of human leukocyte antigen among Taiwan's aboriginal tribes and high homogeneity within each tribe have been reported. To identify variations in genetic polymorphisms of drug-metabolizing enzyme in seven of Taiwan's ethnic groups, the SNPs of N-acetyl transferase 2, a conjugation enzyme in phase II metabolism, were investigated. Methods: Three SNPs of NAT2, NAT2*5, NAT2*6, and NAT2*7, were determined by the TaqMan® method and subsequently confirmed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Our results showed statistically significant variations in the frequency of NAT2 genotype categories according to the 3 SNPs of NAT2 within 7 Taiwanese ethnic groups (Chi-square 32.08, p<0.05). The distribution of the frequency of the slow acetylator genotype ranged from 32.0% in the Hakka population to 75.5% in the Paiwan population. A lack of NAT2*5 SNP alleles was found in the Atayal population, while a NAT2*5 allele frequency of 31.6% was observed in the Paiwan group. The frequencies of allele NAT2*6 and NAT2*7 in the investigated ethnic groups ranged from 11.2-37.0% and 17.0-53%, respectively. Our results also showed the considerable variations in drug-metabolizing enzyme NAT2 genotypes among Taiwan's aboriginal and general population. The variation in the genetic polymorphisms of drug-metabolizing enzyme in Taiwan's groups is worthy of further study to understand different therapeutic and adverse drug responses in ethnic groups. [ABSTRACT FROM AUTHOR]

The amyloid β peptide (Aβ), composed of 40 or 42 amino acids, is a critical component in the etiology of the neurodegenerative Alzheimer disease. Aβ is prone to aggregate and forms amyloid fibrils progressively both in vitro and in vivo. To understand the process of amyloidogenesis, it is pivotal to examine the initial stages of the folding process. We examined the equilibrium folding properties, assembly states, and stabilities of the early folding stages of A1340 and A1342 prior to fibril formation. We found that Aβ40 and Aβ42 have different conformations and assembly states upon refolding from their unfolded ensembles. Aβ40 is predominantly an unstable and collapsed monomeric species, whereas Aβ42 populates a stable structured trimeric or tetrameric species at concentrations above ~12.5 µM. Thermodynamic analysis showed that the free energies of Aβ40 monomer and Aβ42 trimer/tetramer are ~1.1 and ~15/~22 kcal/mol, respectively. The early aggregation stages of Aβ40 and Aβ42 contain different solvent-exposed hydrophobic surfaces that are located at the sequences flanking its protease-resistant segment. The amyloidogenic folded structure of Aβ is important for the formation of spherical β oligomenic species. However, β oligomers are not an obligatory intermediate in the process of fibril formation because oligomerization is inhibited at concentrations of urea that have no effect on fibril formation. The distinct initial folding properties of Aβ40 and Aβ42 may play an important role in the higher aggregation potential and pathological significance of Aβ42. [ABSTRACT FROM AUTHOR]

We have characterized the equilibrium and kinetic folding of a unique protein domain, caspase recruitment domain (CARD), of the RIP-like interacting CLARP kinase (RICK) (RICK-CARD), which adopts a α-helical Greek key fold. At equilibrium, the folding of RICK-CARD is well described by a two-state mechanism representing the native and unfolded ensembles. The protein is marginally stable, with a ΔG[sup H[sub 2]O] of 3.0 ± 0.15 kcal/mol and an m-value of 1.27 ± 0.06 kcal mol[sup -1] M[sup -1] (30 mM Tris-HCl, pH 8, 1 mM DTT, 25 °C). While the m-value is constant, the protein stability decreases in the presence of moderate salt concentrations (below 200 mM) and then increases at higher salt concentrations. The results suggest that electrostatic interactions are stabilizing in the native protein, and the favorable Coulombic interactions are reduced at low ionic strength. Above 200 mM salt, the results are consistent with Hofmeister effects. The unfolding pathway of RICK-CARD is complex and contains at least three non-native conformations. The refolding pathway of RICK-CARD also is complex, and the data suggest that the unfolded protein folds via two intermediate conformations prior to reaching the native state. Overall, the data suggest the presence of kinetically trapped, or misfolded, species that are on-pathway both in refolding and in unfolding. [ABSTRACT FROM AUTHOR]

Examines the oligomeric properties of procaspase-3 and pro-lee variant. Interactions between amino acid pro-peptide in trans and pro-less variant; Estimation of the procaspase limitation for the equilibrium dissociation constant; Expression levels of caspase-3 in Jurkat cells.

Proteins can spontaneously tie a variety of intricate topological knots through twisting and threading of the polypeptide chains. Recently developed artificial intelligence algorithms have predicted several new classes of topological knotted proteins, but the predictions remain to be authenticated experimentally. Here, we showed by X-ray crystallography and solution-state NMR spectroscopy that Q9PR55, an 89-residue protein from Ureaplasma urealyticum, possesses a novel 71 knotted topology that is accurately predicted by AlphaFold 2, except for the flexible N terminus. Q9PR55 is monomeric in solution, making it the smallest and most complex knotted protein known to date. In addition to its exceptional chemical stability against urea-induced unfolding, Q9PR55 is remarkably robust to resist the mechanical unfolding-coupled proteolysis by a bacterial proteasome, ClpXP. Our results suggest that the mechanical resistance against pulling-induced unfolding is determined by the complexity of the knotted topology rather than the size of the molecule. [ABSTRACT FROM AUTHOR]

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus that encodes approximately 156 genes and is highly pathogenic to a variety of larval lepidopteran insects in nature. Oral infection of larval midgut cells is initiated by the occlusion-derived virus (ODV), while secondary infection of other tissues is mediated by the budded virus (BV). Global viral gene expression has been studied in detail in BV-infected cell cultures, but studies of ODV infection in the larval midgut are limited. In this study, we examined expression of the ~156 AcMNPV genes in Trichoplusia ni midgut tissue using a transcriptomic approach. We analyzed expression profiles of viral genes in the midgut and compared them with profiles from a T. ni cell line (Tnms42). Several viral genes (p6.9, orf76, orf75, pp31, Ac-bro, odv-e25, and odv-ec27) had high expression levels in the midgut throughout the infection. Also, the expression of genes associated with occlusion bodies (polh and p10) appeared to be delayed in the midgut in comparison with the cell line. Comparisons of viral gene expression profiles revealed remarkable similarities between the midgut and cell line for most genes, although substantial differences were observed for some viral genes. These included genes associated with high level BV production (fp-25k), acceleration of systemic infection (vfgf), and enhancement of viral movement (arif-1/orf20). These differential expression patterns appear to represent specific adaptations for virus infection and transmission through the polarized cells of the lepidopteran midgut. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins comprise the minimal machinery that triggers fusion of transport vesicles with their target membranes. Comparative studies revealed that genes encoding the components of the SNARE system are highly conserved in yeast, insect, and human genomes. Upon infection of insect cells by the virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the transcript levels of most SNARE genes initially were upregulated. We found that overexpression of dominant-negative (DN) forms of NSF or knockdown of the expression of NSF, the key regulator of the SNARE system, significantly affected infectious AcMNPV production. In cells expressing DN NSF, entering virions were trapped in the cytoplasm or transported to the nucleus with low efficiency. The presence of DN NSF also moderately reduced trafficking of the viral envelope glycoprotein GP64 to the plasma membrane but dramatically inhibited production of infectious budded virions (BV). Transmission electron microscopy analysis of infections in cells expressing DN NSF revealed that progeny nucleocapsids were retained in a perinuclear space surrounded by inner and outer nuclear membranes. Several baculovirus conserved (core) proteins (Ac76, Ac78, GP41, Ac93, and Ac103) that are important for infectious budded virion production were found to associate with NSF, and NSF was detected within the assembled BV. Together, these data indicate that the cellular SNARE system is involved in AcMNPV infection and that NSF is required for efficient entry and nuclear egress of budded virions of AcMNPV. IMPORTANCE Little is known regarding the complex interplay between cellular factors and baculoviruses during viral entry and egress. Here, we examined the cellular SNARE system, which mediates the fusion of vesicles in healthy cells, and its relation to baculovirus infection. Using a DN approach and RNA interference knockdown, we demonstrated that a general disruption of the SNARE machinery significantly inhibited the production of infectious BV of AcMNPV. The presence of a DN NSF protein resulted in low-efficiency entry of BV and the retention of progeny nucleocapsids in the perinuclear space during egress. Combined with these effects, we also found that several conserved (core) baculovirus proteins closely associate with NSF, and these results suggest their involvement in the egress of BV. Our findings are the first to demonstrate that the SNARE system is required for efficient entry of BV and nuclear egress of progeny nucleocapsids of baculoviruses. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the most common dementia affecting tens of million people worldwide. The primary neuropathological hallmark in AD is amyloid plaques composed of amyloid-β peptide (Aβ). Several familial mutations found in Aβ sequence result in early onset of AD. Previous studies showed that the mutations located at N-terminus of Aβ, such as the English (H6R) and Tottori (D7N) mutations, promote fibril formation and increase cytotoxicity. However, A2T mutant located at the very N-terminus of Aβ shows low-prevalence incidence of AD, whereas, another mutant A2V causes early onset of AD. To understand the molecular mechanism of the distinct effect and develop new potential therapeutic strategy, here, we examined the effect of full-length and N-terminal A2V/T variants to wild type (WT) Aβ40 by fibrillization assays and NMR studies. We found that full-length and Nterminal A2V accelerated WT fibrillization and induced large chemical shifts on the N-terminus of WT Aβ, whereas, full-length and N-terminal A2T retarded the fibrillization. We further examined the inhibition effect of various N-terminal fragments (NTFs) of A2T to WT Aβ. The A2T NTFs ranging from residue 1 to residue 7 to 10, but not 1 to 6 or shorter, are capable to retard WT Aβ fibrillization and rescue cytotoxicity. The results suggest that in the presence of full-length or specific N-terminal A2T can retard Aβ aggregation and the A2T NTFs can mitigate its toxicity. Our results provide a novel targeting site for future therapeutic development of AD. [ABSTRACT FROM AUTHOR]

Hexanucleotide expansions, GGGGCC, in the non-coding regions of the C9orf72 gene were found in major frontotemporal lobar dementia and amyotrophic lateral sclerosis patients (C9FTD/ALS). In addition to possible RNA toxicity, several dipeptide repeats (DPRs) are translated through repeat-associated non-ATG-initiated translation. The DPRs, including poly(GA), poly(GR), poly(GP), poly(PR), and poly(PA), were found in the brains and spinal cords of C9FTD/ALS patients. Among the DPRs, poly(GA) is highly susceptible to form cytoplasmic inclusions, which is a characteristic of C9FTD/ALS. To elucidate DPR aggregation, we used synthetic (GA)15 DPR as a model system to examine the aggregation and structural properties in vitro. We found that (GA)15 with 15 repeats fibrillates rapidly and ultimately forms flat, ribbon-type fibrils evidenced by transmission electron microscopy and atomic force microscopy. The fibrils are capable of amyloid dye binding and contain a characteristic cross-β sheet structure, as revealed by x-ray scattering. Furthermore, using neuroblastoma cells, we demonstrated the neurotoxicity and cell-to-cell transmission property of (GA)15 DPR. Overall, our results show the structural and toxicity properties ofGADPR to facilitate future DPR-related therapeutic development. [ABSTRACT FROM AUTHOR]

Background: Cell membranes exhibit abundant types of responses to external stimulations. Intuitively, membrane topography should be sensitive to changes of physical or chemical factors in the microenvironment. We employed the non-interferometric wide-field optical profilometry (NIWOP) technique to quantify the membrane roughness of living neuroblastoma cells under various treatments that could change the mechanical properties of the cells. Results: The membrane roughness was reduced as the neuroblastoma cell was treated with paclitaxel, which increases cellular stiffness by translocating microtubules toward the cell membranes. The treatment of positively charged gold nanoparticles (AuNPs) showed a similar effect. In contrast, the negatively charged AuNPs did not cause significant changes of the membrane roughness. We also checked the membrane roughness of fixed cells by using scanning electron microscopy (SEM) and confirmed that the membrane roughness could be regarded as a parameter reflecting cellular mechanical properties. Finally, we monitored the temporal variations of the membrane roughness under the treatment with a hypertonic solution (75 mM sucrose in the culture medium). The membrane roughness was increased within 1 h but returned to the original level after 2 h. Conclusions: The results in the present study suggest that the optical measurement on membrane roughness can be regarded as a label-free method to monitor the changes in cell mechanical properties or binding properties of nanoparticles on cell surface. Because the cells were left untouched during the measurement, further tests about cell viability or drug efficacy can be done on the same specimen. Membrane roughness could thus provide a quick screening for new chemical or physical treatments on neuronal cells. [ABSTRACT FROM AUTHOR]

Cytosine methylation at CG sites (mCG) plays critical roles in development, epigenetic inheritance, and genome stability in mammals and plants. In the dicot model plant Arabidopsis thaliana, methyltransferase 1 (MET1), a principal CG methylase, functions to maintain mCG during DNA replication, with its null mutation resulting in global hypomethylation and pleiotropic developmental defects. Null mutation of a critical CG methylase has not been characterized at a whole-genome level in other higher eukaryotes, leaving the generality of the Arabidopsis findings largely speculative. Rice is a model plant of monocots, to which many of our important crops belong. Here we have characterized a null mutant of OsMet1-2, the major CG methylase in rice. We found that seeds homozygous for OsMet1-2 gene mutation (OsMET1-2-/-), which directly segregated from normal heterozygote plants (OsMET1-2+/-), were seriously maldeveloped, and all germinated seedlings underwent swift necrotic death. Compared with wild type, genome-wide loss of mCG occurred in the mutant methylome, which was accompanied by a plethora of quantitative molecular phenotypes including dysregulated expression of diverse protein-coding genes, activation and repression of transposable elements, and altered small RNA profiles. Our results have revealed conservation but also distinct functional differences in CG methylases between rice and Arabidopsis. [ABSTRACT FROM AUTHOR]

Background Triple negative breast cancer (TNBC) has higher rates of recurrence and distant metastasis, and poorer outcome as compared to non-TNBC. Aberrant activation of WNT signaling has been detected in TNBC, which might be important for triggering oncogenic conversion of breast epithelial cell. Therefore, we directed our focus on identifying the WNT ligand and its underlying mechanism in TNBC cells. Methods We performed large-scale analysis of public microarray data to screen the WNT ligands and the clinical significance of the responsible ligand in TNBC. WNT5B was identified and its overexpression in TNBC was confirmed by immunohistochemistry staining, Western blot and ELISA. ShRNA was used to knockdown WNT5B expression (shWNT5B). Cellular functional alteration with shWNT5B treatment was determined by using wound healing assay, mammosphere assay; while cell cycle and apoptosis were examined by flowcytometry. Mitochondrial morphology was photographed by electron microscope. Biological change of mitochondria was detected by RT-PCR and oxygen consumption assay. Activation of WNT pathway and its downstream targets were evaluated by liciferase assay, immunohistochemistry staining and immunoblot analysis. Statistical methods used in the experiments besides microarray analysis was two-tailed t-test. Results WNT5B was elevated both in the tumor and the patients' serum. Suppression of WNT5B remarkably impaired cell growth, migration and mammosphere formation. Additionally, G0/G1 cell cycle arrest and caspase-independent apoptosis was observed. Study of the possible mechanism indicated that these effects occurred through suppression of mitochondrial biogenesis, as evidenced by reduced mitochondrial DNA (MtDNA) and compromised oxidative phosphorylation (OXPHOS). In Vivo and in vitro data uncovered that WNT5B modulated mitochondrial physiology was mediated by MCL1, which was regulated by WNT/ß-catenin responsive gene, Myc. Clinic data analysis revealed that both WNT5B and MCL1 are associated with enhanced metastasis and decreased disease-free survival. Conclusions All our findings suggested that WNT5B/MCL1 cascade is critical for TNBC and understanding its regulatory apparatus provided valuable insight into the pathogenesis of the tumor development and the guidance for targeting therapeutics. [ABSTRACT FROM AUTHOR]

The membrane roughness of Neuro-2a neroblastoma cells is measured by using noninterferometric widefield optical profilometry. The cells are treated with the fibril and oligomer conformers of amyloid-beta (Aβ) 42, which is a peptide of 42 amino acids related to the development of Alzheimer's disease. We find that both the A β42 fibrils and Aβ42 oligomers reduced the cell membrane roughness, but the effect of Aβ42 oligomers was faster and stronger than that of the fibrils. We also apply direct-current electric field (dcEF) stimulations on the cells. A dcEF of 300 mV/mm can increase the membrane roughness under the treatment of Aβ42. These results suggest that Aβ42 can decrease the membrane compliance of live neuroblastoma cells, and dcEFs may counteract this effect. [ABSTRACT FROM AUTHOR]

TDP-43 is the major pathological protein identified in the cellular inclusions in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The pathogenic forms of TDP-43 are processed C-terminal fragments containing a truncated RNA-recognition motif (RRM2) and a glycine-rich region. Although extensive studies have focused on this protein, it remains unclear how the dimeric full-length TDP-43 is folded and assembled and how the processed C-terminal fragments are misfolded and aggregated. Here, using size-exclusion chromatography, pulldown assays, and small angle x-ray scattering, we show that the C-terminal-deleted TDP-43 without the glycine-rich tail is sufficient to form a head-to-head homodimer primarily via its N-terminal domain. The truncated RRM2, as well as two β-strands within the RRM2, form fibrils in vitro with a similar amyloid-negative staining property to those of TDP-43 pathogenic fibrils in diseases. In addition to the glycine-rich region, the truncated RRM2, but not the intact RRM2, plays a key role in forming cytoplasmic inclusions in neuronal cells. Our data thus suggest that the process that disrupts the dimeric structure, such as the proteolytic cleavage of TDP-43 within the RRM2 that removes the N-terminal dimerization domain, may produce unassembled truncated RRM2 fragments with abnormally exposed β-strands, which can oligomerize into high-order inclusions. [ABSTRACT FROM AUTHOR]

Amyloid fibrils of α-synuclein are the main constituent of Lewy bodies deposited in substantial nigra of Parkinson's disease brains. α-Synuclein is an intrinsically disordered protein lacking compact secondary and tertiary structures. To enhance the understanding of its structure and function relationship, we utilized temperature treatment to study α-synuclein conformational changes and the subsequent effects. We found that after 1 hr of high temperature pretreatment, >80°C, asynuclein fibrillization was significantly inhibited. However, the temperature melting coupled with circular dichroism spectra showed that α-synuclein was fully reversible and the NMR studies showed no observable structural changes of α-synuclein after 95°C treatment. By using cross-linking and analytical ultracentrifugation, rare amount of pre-existing α-synuclein oligomers were found to decrease after the high temperature treatment. In addition, a small portion of C-terminal truncation of α-synuclein also occurred. The reduction of pre-existing oligomers of α-synuclein may contribute to less seeding effect that retards the kinetics of amyloid fibrillization. Overall, our results showed that the pre-existing oligomeric species is a key factor contributing to α-synuclein fibrillization. Our results facilitate the understanding of α-synuclein fibrillization. [ABSTRACT FROM AUTHOR]

Purpose: Our objective was to develop a system to simultaneously and quantitatively measure the expression levels of the insulin-like growth factor (IGF) family proteins in numerous samples and to apply this approach to profile the IGF family proteins levels in cancer and adjacent tissues from patients with hepatocellular carcinoma (HCC). Experimental Design: Antibodies against ten IGF family proteins (IGF-1, IGF-1R, IGF-2, IGF-2R, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, and Insulin) were immobilized on the surface of a glass slide in an array format to create an IGF signaling antibody array. Tissue lysates prepared from patient's liver cancer tissues and adjacent tissues were then applied to the arrays. The proteins captured by antibodies on the arrays were then incubated with a cocktail of biotinylated detection antibodies and visualized with a fluorescence detection system. By comparison with standard protein amount, the exact protein concentrations in the samples can be determined. The expression levels of the ten IGF family proteins in 25 pairs of HCC and adjacent tissues were quantitatively measured using this novel antibody array technology. The differential expression levels between cancer tissues and adjacent tissues were statistically analyzed. Results: A novel IGF signaling antibody array was developed which allows the researcher to simultaneously detect ten proteins involved in IGF signal pathway with high sensitivity and specificity. Using this approach, we found that the levels of IGF-2R and IGFBP-2 in HCC tissues were higher than those in adjacent tissues. Conclusion: Our IGF signaling antibody array which can detect the expression of ten IGF family members with high sensitivity and specificity will undoubtedly prove a powerful tool for drug and biomarker discovery. [ABSTRACT FROM AUTHOR]

Amyloid precursor protein (APP) mutations associated with familial Alzheimer's disease (AD) usually lead to increases in amyloid β-protein (Aβ) levels or aggregation. Here, we identified a novel APP mutation, located within the Aβ sequence (AbD7H), in a Taiwanese family with early onset AD and explored the pathogenicity of this mutation. Cellular and biochemical analysis reveal that this mutation increased Ab production, Aβ42/40 ratio and prolonged Aβ42 oligomer state with higher neurotoxicity. Because the D7H mutant Ab has an additional metal ion-coordinating residue, histidine, we speculate that this mutation may promote susceptibility of Ab to ion. When co-incubated with Zn2+ or Cu2+, AβD7H aggregated into low molecular weight oligomers. Together, the D7H mutation could contribute to AD pathology through a "double punch" effect on elevating both Aβ production and oligomerization. Although the pathogenic nature of this mutation needs further confirmation, our findings suggest that the Aβ N-terminal region potentially modulates APP processing and Aβ aggregation, and further provides a genetic indication of the importance of Zn2+ and Cu2+ in the etiology of AD. [ABSTRACT FROM AUTHOR]

VPS4B, an AAA ATPase (ATPase associated with various cellular activities), participates in vesicular trafficking and autophagosome maturation in mammalian cells. In solid tumors, hypoxia is a common feature and an indicator of poor treatment outcome. Our studies demonstrate that exogenous or endogenous (assessed with anchorage-independent three-dimensional multicellular spheroid culture) hypoxia induces VPS4B downregulation by the ubiquitin-proteasome system. Inhibition of VPS4B function by short hairpin VPS4 (sh-VPS4B) or expression of dominant negative VPS4B(E235Q) promotes anchorage-independent breast cancer cell growth and resistance to gefitinib, U0126, and genotoxicity. Biochemically, hyperactivation of epidermal growth factor receptor (EGFR), a receptor tyrosine kinase essential for cell proliferation and survival, accompanied by increased EGFR accumulation and altered intracellular compartmentalization, is observed in cells with compromised VPS4B. Furthermore, enhanced FOS/JUN induction and AP-1 promoter activation are noted in EGF-treated cells with VPS4B knockdown. However, VPS4B depletion does not affect EGFRvIII stability or its associated signaling. An inverse correlation between VPS4B expression and EGFR abundance is observed in breast tumors, and high-grade or recurrent breast carcinomas exhibit lower VPS4B expression. Together, our findings highlight a potentially critical role of VPS4B downregulation or chronic-hypoxia-induced VPS4B degradation in promoting tumor progression, unveiling a nongenomic mechanism for EGFR overproduction in human breast cancer. [ABSTRACT FROM AUTHOR]

Amyloid formation is initiated by protein misfolding, followed by self-association to ultimately form amyloid fibrils. The discovery of toxic prefibrillar oligomers in many amyloidosis underscores the importance of understanding the folding mechanism prior to such aggregation. Here, we investigated the folding properties of the natively unfolded amyloid-β (Aβ) peptide and the familial variants (A21G, E22Q, E22G, E22K, and D23N) in Alzheimer's disease (AD). In combinations of native electrophoresis, analytical ultra-centrifugation, fluorescence emission, and far-UV circular dichroism, we showed that all Aβ40 variants are predominantly monomeric with similar residual secondary structures, but distinct hydrophobic-exposed protein surfaces. Guanidine hydrochloride (GdnHCl) denaturation in the absence and presence of trifluoroethanol (TFE) showed that Aβ variants adopt an apparent 2-state equilibrium model with different stabilities, in which wild type is less stable than A21G but more stable than D23N and E22 mutants. By correlating the folding stability with the nucleation phase in fibrillization, we found the more stable the variant, the slower the nucleation, except for D23N. Besides, the unfolding of Aβ conformation leads to reduced formation of mature fibrils, but an increase in nonfibrillar, amorphous type of aggregates. Overall, we demonstrated that folding stability of Aβ is an important determinant of the nucleation kinetics. [ABSTRACT FROM AUTHOR]

Abnormally high concentrations of Zn2+, Cu2+, and Fe3+ are present along with amyloid-β (Aβ) in the senile plaques in Alzheimer disease, where Al3+ is also detected. Aβ aggregation is the key pathogenic event in Alzheimer disease, where Aβ oligomers are the major culprits. The fundamental mechanism of these metal ions on Aβ remains elusive. Here, we employ 4,4′-Bis(1-anilinonaphthalene 8-sulfonate) and tyrosine fluorescence, CD, stopped flow fluorescence, guanidine hydrochloride denaturation, and photo-induced cross-linking to elucidate the effect of Zn2+, Cu2+, Fe3+, and Al3+ on Aβ at the early stage of the aggregation. Furthermore, thioflavin T assay, dot blotting, and transmission electron microscopy are utilized to examine Aβ aggregation. Our results show that Al3+ and Zn2+, but not Cu2+ and Fe3+, induce larger hydrophobic exposures of Aβ conformation, resulting in its significant destabilization at the early stage. The metal ion binding induces Aβ conformational changes with micromolar binding affinities and millisecond binding kinetics. Cu2+ and Zn2+ induce similar assembly of transiently appearing Aβ oligomers at the early state. During the aggregation, we found that Zn2+ exclusively promotes the annular protofibril formation without undergoing a nucleation process, whereas Cu2+ and Fe3+ inhibit fibril formation by prolonging the nucleation phases. Al3+ also inhibits fibril formation; however, the annular oligomers co-exist in the aggregation pathway. In conclusion, Zn2+, Cu2+, Fe3+, and Al3+ adopt distinct folding and aggregation mechanisms to affect Aβ, where Aβ destabilization promotes annular protofibril formation. Our study facilitates the understanding of annular Aβ oligomer formation upon metal ion binding. [ABSTRACT FROM AUTHOR]

Background: Dendritic cells (DCs) are professional antigen presenting cells that initiate specific immune responses against tumor cells. Transcription factor RBP-J-mediated Notch signaling regulates DC genesis, but whether this pathway regulates DC function in anti-tumor immunity remains unclear. In the present work we attempted to identify the role of Notch signaling in DC-mediated anti-tumor immune response. Results: When DCs were co-inoculated together with tumor cells, while the control DCs repressed tumor growth, the RBP-J deficient DCs had lost tumor repression activity. This was most likely due to that DCs with the conditionally ablated RBP-J were unable to evoke anti-tumor immune responses in the solid tumors. Indeed, tumors containing the RBP-J deficient DCs had fewer infiltrating T-cells, B-cells and NK-cells. Similarly, the draining lymph nodes of the tumors with RBP-J-/- DCs were smaller in size, and contained fewer cells of the T, B and NK lineages, as compared with the controls. At the molecular level, the RBP-J deficient DCs expressed lower MHC II, CD80, CD86, and CCR7, resulting in inefficient DC migration and T-cell activation in vitro and in vivo. T-cells stimulated by the RBP-J deficient DCs did not possess efficient cytotoxicity against tumor cells, in contrast to the control DCs. Conclusion: The RBP-J-mediated Notch signaling is essential for DC-dependent anti-tumor immune responses. The deficiency of RBP-J impairs the DC-based anti-tumor immunity through affecting series of processes including maturation, migration, antigen presentation and T-cell activation. The Notch signaling pathway might be a target for the establishment of the DC-based anti-tumor immunotherapies. [ABSTRACT FROM AUTHOR]

Background: Outbreaks of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae), which is a very important forest pest in Taiwan, have occurred every five to 10 years. This moth has expanded its range of host plants to include more than 65 species of broadleaf trees. LyxyMNPV (L. xylina multiple nucleopolyhedrovirus) is highly virulent to the casuarina moth and has been investigated as a possible biopesticide for controlling this moth. LdMNPV-like virus has also been isolated from Lymantria xylina larvae but LyxyMNPV was more virulent than LdMNPV-like virus both in NTU-LY and IPLB-LD-652Y cell lines. To better understand LyxyMNPV, the nucleotide sequence of the LyxyMNPV DNA genome was determined and analysed. Results: The genome of LyxyMNPV consists of 156,344 bases, has a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified as homologous to those reported in the LdMNPV genome. Two genes (Lyxy49 and Lyxy123) were homologous to other baculoviruses, and four unique LyxyMNPV ORFs (Lyxy11, Lyxy19, Lyxy130 and Lyxy131) were identified in the LyxyMNPV genome, including a gag-like gene that was not reported in baculoviruses. LdMNPV contains 23 ORFs that are absent in LyxyMNPV. Readily identifiable homologues of the gene host range factor-1 (hrf-1), which appears to be involved in the susceptibility of L. dispar to NPV infection, were not present in LyxyMNPV. Additionally, two putative odv-e27 homologues were identified in LyxyMNPV. The LyxyMNPV genome encoded 14 bro genes compared with 16 in LdMNPV, which occupied more than 8% of the LyxyMNPV genome. Thirteen homologous regions (hrs) were identified containing 48 repeated sequences composed of 30-bp imperfect palindromes. However, they differed in the relative positions, number of repeats and orientation in the genome compared to LdMNPV. Conclusion: The gene parity plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that LyxyMNPV is a Group II NPV that is most closely related to LdMNPV but with a highly distinct genomic organisation. [ABSTRACT FROM AUTHOR]

Recent in vitro and in vivo studies suggest that destabilized proteins with defective folding induce aggregation and toxicity in protein-misfolding diseases. One such unstable protein state is called amyloid oligomer, a precursor of fully aggregated forms of amyloid. Detection of various amyloid oligomers with A11, an anti-amyloid oligomer conformation-specific antibody, revealed that the amyloid oligomer represents a generic conformation and suggested that toxic β-aggregation processes possess a common mechanism. By using A11 antibody as a probe in combination with mass spectrometric analysis, we identified GroEL in bacterial lysates as a protein that may potentially have an amyloid oligomer conformation. Surprisingly, A11 reacted not only with purified GroEL but also with several purified heat shock proteins, including human Hsp27, 40, 70, 90; yeast Hsp104; and bovine Hsc70. The native folds of A11-reactive proteins in purified samples were characterized by their anti-β-aggregation activity in terms of both functionality and in contrast to the β-aggregation promoting activity of misfolded pathogenic amyloid oligomers. The conformation-dependent binding of A11 with natively folded Hsp27 was supported by the concurrent loss of A11 reactivity and anti-β-aggregation activity of heat-treated Hsp27 samples. Moreover, we observed consistent anti-β-aggregation activity not only by chaperones containing an amyloid oligomer conformation but also by several A11-immunoreactive non-chaperone proteins. From these results, we suggest that the amyloid oligomer conformation is present in a group of natively folded proteins. The inhibitory effects of A11 antibody on both GroEL/ES-assisted luciferase refolding and Hsp70-mediated decelerated nucleation of Ab aggregation suggested that the A11-binding sites on these chaperones might be functionally important. Finally, we employed a computational approach to uncover possible A11-binding sites on these targets. Since the β-sheet edge was a common structural motif having the most similar physicochemical properties in the A11-reactive proteins we analyzed, we propose that the β-sheet edge in some natively folded amyloid oligomers is designed positively to prevent b aggregation. [ABSTRACT FROM AUTHOR]