Proteomics-based identification of cancer-associated proteins in chronic lymphocytic leukaemia

BACKGROUND: Chronic lymphocytic leukaemia (CLL) is a neoplasm of B-cells characterized by variable prognosis. Exploring the proteome of CLL cells may provide insights into the disease. Therefore, eleven proteomics experiments were conducted on eleven primary CLL samples. RESULTS: We reported a CLL proteome consisting of 919 proteins (false discovery rate (FDR) 1%) whose identification was based on the sequencing of two or more distinct peptides (FDR of peptide sequencing 1%). Mass spectrometry-based protein identification was validated for four different proteins using Western blotting and specific antibodies in different CLL samples. Small sizes of nucleolin (~57 kDa and ~68 kDa) showed a potential association with good prognosis CLL cells (n = 8, p < 0.01). Compared with normal B-cells, CLL cells over-expressed thyroid hormone receptor-associated protein 3 (THRAP3; n = 9; p = 0.00007), which is implicated in cell proliferation; and heterochromatin protein 1-binding protein 3 (HP1BP3; n = 10; p = 0.0002), which promotes cell survival and tumourogenesis. A smaller form of HP1BP3, which may correspond to HP1BP3 isoform-2, was specifically identified in normal B-cells (n = 10; p = 0.0001). HP1BP3 and THRAP3 predicted poor prognosis of CLL (p 0.05). Consistently, THRAP3 and HP1BP3 were found to be associated with cancer-related pathways (p 0.05). CONCLUSIONS: Our findings add to the known proteome of CLL and confirm the prognostic importance of two novel cancer-associated proteins in this disease.

Total synthesis of D-glycero-D-mannno-heptose 1β, 7-bisphosphate with 3-O-amyl amine linker and its monophosphate derivative / 中国天然药物

D-Glycero-D-mannno-heptose 1β, 7-bisphosphate (HBPβ) is an important intermediate for constructing the core structure of Gram-negative bacterial lipopolysaccharides and was reported as a pathogen-associated molecular pattern (PAMP) that regulates immune responses. HBPβ with 3-O-amyl amine linker and its monophosphate derivative D-glycero-D-mannno-heptose 7-phosphate (HP) with 1α-amyl amine linker have been synthesized as candidates for immunity study of HBPβ. The O3-amyl amine linker of heptose was installed by dibutyltin oxide-mediated regioselective alkylation under fine-tuned protecting condition. The stereoselective installation of 1β-phosphate ester was achieved by NIS-mediated phosphorylation at low temperature. The strategy for installation of 3-O-amyl amine linker onto HBP derivative can be expanded to the syntheses of other conjugation-ready carbohydrates bearing anomeric phosphoester.

HP1γ promotes the development of ovarian cancer and is modulated by miR-30b / 实用医学杂志

Objective To investigate the effect of HP1γ on the development of ovarian cancer. Methods RT-qPCR was used to deteched the level of HP1γin ovarian cancer samples. The proliferation of A2780 and SKOV3 cell line was detected by CCK8 assay. RNAi was used to knock down the expression of HP1γ mRNA. Results The level of HP1γwas higher in ovarian cancer than that in the normal tissue. Mir-30b may inhibit the proliferation of ovarian cancer cell. Conclusion HP1γpromotes the proliferation and progress of ovarian cancer cells.

Interactome Analysis Reveals that Heterochromatin Protein 1gamma (HP1gamma) Is Associated with the DNA Damage Response Pathway / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Heterochromatin protein 1gamma (HP1gamma) interacts with chromosomes by binding to lysine 9-methylated histone H3 or DNA/RNA. HP1gamma is involved in various biological processes. The purpose of this study is to gain an understanding of how HP1gamma functions in these processes by identifying HP1gamma-binding proteins using mass spectrometry. MATERIALS AND METHODS: We performed affinity purification of HP1gamma-binding proteins using G1/S phase or prometaphase HEK293T cell lysates that transiently express mock or FLAG-HP1gamma. Coomassie staining was performed for HP1gamma-binding complexes, using cell lysates prepared by affinity chromatography FLAG-agarose beads, and the bands were digested and then analyzed using a mass spectrometry. RESULTS: We identified 99 HP1gamma-binding proteins with diverse cellular functions, including spliceosome, regulation of the actin cytoskeleton, tight junction, pathogenic Escherichia coli infection, mammalian target of rapamycin signaling pathway, nucleotide excision repair, DNA replication, homologous recombination, and mismatch repair. CONCLUSION: Our results suggested that HP1gamma is functionally active in DNA damage response via protein-protein interaction.

Effect of staphylococcal protein A on pro-inflammatory cytokines expression in mononuclear macrophage / 重庆医学

Objective To study the expression and regulation of pro‐inflammatory cytokinesTNF‐α,IL‐1 ,IL‐6 in mononucle‐ar macrophages stimulated with staphylococcal protein A (SpA) .Methods THP‐1 was incubated with PMA and induced into mononuclear macrophages .Then the macrophages were incubated with varying concentrations of SpA under different time points . The effect of SpA on macrophage proliferation was measured by MTT method .The levels of inflammatory cytokines ,TNF‐α,IL‐1 and IL‐6 from the cultured cell media were measured by ELISA respectively .The levels of mRNA expression corresponding to TNF‐α,IL‐1 and IL‐6 were detected by RT‐PCR from the macrophages stimulated with SpA .All statistical analyses were performed by SPSS17 .0 software .Results The MTT result indicated that SpA had a positive effect on the proliferation of THP‐1 cells in a dosage depended manner .The addition of SpA could enhance the mRNA expression of TNF‐α,IL‐1 and IL‐6 in the stimulated mac‐rophages .It also showed a specific dose‐effect and time‐effect correlation .The macrophages secreted inflammatory cytokines and its corresponding mRNA reached its peak levels at 12 h post stimulation .Compared with the control group ,the expression and release of TNF‐α,IL‐1 and IL‐6 in macrophages from the experimental group was increased with statistical significance(P<0 .01) .Conclu‐sion SpA can promote the secretion and expression of early pro‐inflammatory cytokines ,such as TNF‐α,IL‐1 and IL‐6 in macro‐phages .Therefore ,SpA plays a very important role in the initiation and development of the staphylococcus aureus sepsis .

A hypomorphic Cbx3 allele causes prenatal growth restriction and perinatal energy homeostasis defects.

Mammals have three HP1 protein isotypes HP1β (CBX1), HP1γ (CBX3) and HP1α (CBX5) that are encoded by the corresponding genes Cbx1, Cbx3 and Cbx5. Recent work has shown that reduction of CBX3 protein in homozygotes for a hypomorphic allele (Cbx3hypo) causes a severe postnatal mortality with around 99% of the homozygotes dying before weaning. It is not known what the causes of the postnatal mortality are. Here we show that Cbx3hypo/hypo conceptuses are significantly reduced in size and the placentas exhibit a haplo-insufficiency. Late gestation Cbx3hypo/hypo placentas have reduced mRNA transcripts for genes involved in growth regulation, amino acid and glucose transport. Blood vessels within the Cbx3hypo/hypo placental labyrinth are narrower than wild-type. Newborn Cbx3hypo/hypo pups are hypoglycemic, the livers are depleted of glycogen reserves and there is almost complete loss of stored lipid in brown adipose tissue (BAT). There is a 10-fold reduction in expression of the BAT-specific Ucp1 gene, whose product is responsible for nonshivering themogenesis. We suggest that it is the small size of the Cbx3hypo/hypo neonates, a likely consequence of placental growth and transport defects, combined with a possible inability to thermoregulate that causes the severe postnatal mortality.

Compostos fenólicos e capacidade antioxidante em frutos de tomateiros mutantes fotomorfogenéticos / Phenolic compounds and antioxidant capacity in fruits of photomorphogenic mutant tomato

Compostos fenólicos e capacidade antioxidante são mecanismos de defesa das plantas aos danos do estresse oxidativo. Os compostos fenólicos são sintetizados pela via dos fenilpropanoides, cuja enzima chave, fenilalanina amônia liase, é influenciada pela luz e ação de fotorreceptores, como o fitocromo. O objetivo do presente trabalho é avaliar a concentração de compostos fenólicos e a capacidade antioxidante de frutos de microtomateiro selvagem, cultivar "Micro-Tom" (MT), e seus mutantes fotomorfogenéticos high pigment 1 (hp1), super-responsivo a eventos mediados por luz e aurea (au), deficiente quantitativo em fitocromos. Vinte frutos maduros de cada genótipo (MT, hp1 e au) foram utilizados para as análises, realizadas em triplicata. Para quantificação dos compostos fenólicos totais, foi utilizado o método de Folin-Ciocalteu e a capacidade antioxidante foi realizada pelos métodos Ferric Reducing Antioxidant Power (FRAP) e 2,2-diphenyl-1-picrylhydrazyl (DPPH). Os frutos do mutante hp1 apresentaram maiores conteúdos de compostos fenólicos totais e também maior capacidade antioxidante em relação à cultivar selvagem ("MT") e ao mutante au, o qual não diferiu significativamente da cultivar "MT".

Phenolic compounds and antioxidant capacity are defense mechanisms of plants against the oxidative stress damage. Phenolic compounds are synthesized through the phenylpropanoid pathway, where the enzyme phenylalanine-ammonia-lyase plays a key role and it is influenced by light and photoreceptors such as phytochromes. The present research aims to evaluate the phenolic compounds content and antioxidant capacity of the wild "Micro-Tom" (MT) cultivar tomato fruits and its photomorphogenic mutant tomato plants high pigment 1 (hp1), super responsive to events mediated by light, and aurea (au), quantitative phytochrome deficient. Twenty mature fruits of each genotype ("MT", hp1, au) were used in triplicate for analyses. To quantify the total phenolic compounds the Folin-Ciocalteu method was used and the antioxidant capacity was analyzed by Ferric Reducing Antioxidant Power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. The hp1 mutant presented the highest total phenolic compounds content and higher antioxidant capacity than wild cultivar ("MT") and au mutant, which did not differ significantly from "MT" cultivar.

The expression of pimonidazole hydrochloride(HP-1)in rat condylar cartilage in the early growth stage / 实用口腔医学杂志

Objective:To investigate hypoxia location in condylar cartilage in the early growth stage of rats.Methods:40 Sprague-Dawley rats were breastfed from 1 4 d to 21 d of age.1 0 rats were sacrificed at 1 2,24,48 and 96 h respectively after initiation of normal food at 21 d of age.The rats were administered pimonidazole hydrochloride (HP-1 )at a dose of 60 mg/kg by intraperitoneal injection 2 h before sacrifice.The expression of HP-1 in the whole condylar cartilage was detected by immunohistochemical staining.Results:HP-1 was mainly expressed in the chondrocytes of the fibrous and proliferative layer of cartilage,primarily concentrated in the weight-bearing area of joint-anterior aspect of the condyle and posterior aspect of the articular eminence at all time points.The highest expres-sion was observed at 24 h after initiation of normal food (P <0.01 ).Conclusion:In the early growth stage of rats,dietary loading may directly induce hypoxia in uper layer of condylar cartilage,the hyoxia level may change with time of dietary loading.

Photosynthate partitioning and morphoanatomical aspects of photomorphogenic mutants of tomato / Partição de fotoassimilados e aspectos morfoanatômicos em tomateiros mutantes fotomorfogenéticos

The aim of this study was to analyze photosynthate partitioning in tomato photomorphogenic mutants at the ends of the vegetative (40 days after emergence [DAE]) and reproductive (69 DAE) stages and to determine its interaction with morphoanatomical aspects. The mutants aurea (au), phytochrome-deficient, high pigment-1 (hp1), light-exaggerated response, were studied along with the non-mutant Micro-Tom (MT) cultivar. The plants were analyzed at 40 and 68 DAE to identify photosynthate source organs and tissues as well as the target organs of remobilized photosynthate during the reproductive stage. The plants were evaluated for their internal and external morphology as well as the percentage of dry mass of their organs. Photosynthate allocation in the hp1 mutant occurred primarily in the roots and leaves, and allocation in the au mutant occurred primarily in fruits. The au mutant showed a high capacity for photosynthate remobilization to fruit during the reproductive stage, and the predominant sources of these remobilized photosynthates were the leaf spongy parenchyma, the root vascular cylinder and the marrow stem.

O objetivo deste estudo foi analisar a partição de fotoassimilados em tomateiros mutantes fotomorfogenéticos ao final da fase vegetativa, aos 40 dias após a emergência (DAE), e ao final da fase reprodutiva, aos 69 DAE, e sua interação com aspectos morfoanatômicos. Foram estudados os mutantes aurea (au), deficiente em fitocromo, e hp1, o qual expressa resposta exagerada à luz, e o tomateiro selvagem cultivar Micro-Tom (MT). As plantas foram analisadas 40 dias após a emergência (DAE) e 68 DAE, tentando identificar os órgãos e tecidos dos fotoassimilados remobilizados e seus órgãos de destino durante o estádio reprodutivo. As plantas foram avaliadas quanto à sua morfologia interna e externa e percentagem de massa seca entre os órgãos. A alocação de fotoassimilados no mutante hp1 ocorreu prioritariamente em raízes e folhas comparativamente aos demais órgãos, e no mutante au ocorreu prioritariamente em frutos comparativamente aos demais órgãos. O mutante au deteve alta capacidade de remobilização de fotoassimilados durante sua fase reprodutiva para os frutos e os fotoassimilados remobilizados tiveram origem preponderante do parênquima lacunoso foliar, do cilindro vascular radicular e da medula caulinar.

The Heterochromatin-1 Phosphorylation Contributes to TPA-Induced AP-1 Expression

Activator protein-1 (AP-1) is an inducible transcription factor that contributes to the generation of chronic inflammation in response to oxidative and electrophilic stress. Previous studies have demonstrated that the PI3K/Akt1 pathway plays an important role in the transcriptional regulation of AP-1 expression. Although the histone post-translational modifications (PTMs) are assumed to affect the AP-1 transcriptional regulation by the PI3K/Akt pathway, the detailed mechanisms are completely unknown. In the present study, we show that heterochromatin 1 gamma (HP1gamma) plays a negative role in TPA-induced c-Jun and c-Fos expression. We show that TPA-induced Akt1 directly phosphorylates HP1gamma, abrogates its suppressive function and increases the interaction between histone H3 and 14-3-3epsilon. Collectively, these our data illustrate that the activation of PI3K/Akt pathway may play a permissive role in the recruitment of histone readers or other coactivators on the chromatin, thereby affecting the degree of AP-1 transcription.

Interplay between Epigenetics and Genetics in Cancer

Genomic instability, which occurs through both genetic mechanisms (underlying inheritable phenotypic variations caused by DNA sequence-dependent alterations, such as mutation, deletion, insertion, inversion, translocation, and chromosomal aneuploidy) and epigenomic aberrations (underlying inheritable phenotypic variations caused by DNA sequence-independent alterations caused by a change of chromatin structure, such as DNA methylation and histone modifications), is known to promote tumorigenesis and tumor progression. Mechanisms involve both genomic instability and epigenomic aberrations that lose or gain the function of genes that impinge on tumor suppression/prevention or oncogenesis. Growing evidence points to an epigenome-wide disruption that involves large-scale DNA hypomethylation but specific hypermethylation of tumor suppressor genes, large blocks of aberrant histone modifications, and abnormal miRNA expression profile. Emerging molecular details regarding the modulation of these epigenetic events in cancer are used to illustrate the alterations of epigenetic molecules, and their consequent malfunctions could contribute to cancer biology. More recently, intriguing evidence supporting that genetic and epigenetic mechanisms are not separate events in cancer has been emerging; they intertwine and take advantage of each other during tumorigenesis. In addition, we discuss the collusion between epigenetics and genetics mediated by heterochromatin protein 1, a major component of heterochromatin, in order to maintain genome integrity.