Immunoblot analysis of proteins associated with self-assembled monolayer surfaces of defined chemistries

Intact and fragmented proteins, eluted from self-assembled monolayer (SAM) surfaces of alkanethiols of different chemistries (-CH?, -OH, -COOH, -NH?), following exposure to human plasma (HP) or human serum (HS), were examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques. The SAM surfaces were incubated for 1 h with 10% (v/v) sterile-filtered, heat-inactivated (h.i.) HS or 1% (v/v) sterile-filtered h.i. HP preparations [both in phosphate buffered saline (PBS)]. Adsorbed proteins were eluted using 10% SDS/2.3% dithioerythritol for characterization of protein profiles. The type of incubating medium may be an important determinant of adsorbed protein profiles, since some variations were observed in eluates from filtered versus control unfiltered h.i. 10% HS or 1% HP. Albumin and apolipoprotein A1 were consistently detected in both filtered h.i 10% HS and 1% HP eluates from all SAM surfaces and from control tissue culture-treated polystyrene (TCPS). Interestingly, Factor H and Factor I, antithrombin, prothrombin, high molecular weight kininogen (HMWK), and IgG were present in eluates from OH, COOH, and NH? SAM surfaces and in eluates from TCPS but not in eluates from CH? SAM surfaces, following exposure to filtered h.i. 10% HS. These results suggest that CH? SAM surfaces were the least proinflammatory of all SAM surfaces. Overall, similar trends were observed in the profiles of proteins eluted from surfaces exposed to filtered 10% HS or 1% HP. However, the unique profiles of adsorbed proteins on different SAM surface chemistries may be related to their differential interactions with cells, including immune/inflammatory cells.     

Expression of FasL and Fas protein and their soluble form in patients with hypersensitivity pneumonitis

BACKGROUND: Hypersensitivity pneumonitis (HP) is characterized by a lymphocytic alveolitis and loosely formed granulomas in lung biopsy specimens. HP improves or disappears altogether after cessation of antigen exposure. The Fas-Fas ligand (FasL) system is one of the representative systems of apoptosis-signaling receptor molecules, and is involved in various inflammatory diseases. We hypothesized that the Fas-FasL system may be associated with this disorder. METHODS: We examined the expression of FasL and Fas proteins in lung tissues from patients with HP using immunohistochemistry. We also measured the soluble form of FasL (sFasL) and sFas levels in serum and bronchoalveolar lavage fluid (BALF) from patients with HP using enzyme-linked immunosorbent assay (ELISA). Furthermore, we also measured the cytotoxic activity of BALF sFasL in vitro. RESULTS: FasL was detected in infiltrating mononuclear cells, and Fas was detected in infiltrating mononuclear cells, alveolar macrophages, and epithelioid cells in HP, whereas FasL was not detected and Fas was detected in few alveolar macrophages in controls. The levels of sFasL and sFas in BALF, but not in serum, were significantly increased in HP compared with controls. BALF of HP that included high levels of sFasL had no cytotoxic activity for bronchiolar epithelial cells in vitro. CONCLUSIONS: In HP, there is an upregulation of FasL and Fas in lung tissues. Since there is no incidence of apoptosis and no cytotoxic activity for lung epithelial cells in BALF from patients with HP, the increased levels of BALF sFasL and sFas may reflect the activation and sequestration of inflammatory cells rather than apoptosis.     

Searching for hypothetical proteins: theory and practice based upon original data and literature

A large part of mammalian proteomes is represented by hypothetical proteins (HP), i.e. proteins predicted from nucleic acid sequences only and protein sequences with unknown function. Databases are far from being complete and errors are expected. The legion of HP is awaiting experiments to show their existence at the protein level and subsequent bioinformatic handling in order to assign proteins a tentative function is mandatory. Two-dimensional gel-electrophoresis with subsequent mass spectrometrical identification of protein spots is an appropriate tool to search for HP in the high-throughput mode. Spots are identified by MS or by MS/MS measurements (MALDI-TOF, MALDI-TOF-TOF) and subsequent software as e.g. Mascot or ProFound. In many cases proteins can thus be unambiguously identified and characterised; if this is not the case, de novo sequencing or Q-TOF analysis is warranted. If the protein is not identified, the sequence is being sent to databases for BLAST searches to determine identities/similarities or homologies to known proteins. If no significant identity to known structures is observed, the protein sequence is examined for the presence of functional domains (databases PROSITE, PRINTS, InterPro, ProDom, Pfam and SMART), subjected to searches for motifs (ELM) and finally protein-protein interaction databases (InterWeaver, STRING) are consulted or predictions from conformations are performed. We here provide information about hypothetical proteins in terms of protein chemical analysis, independent of antibody availability and specificity and bioinformatic handling to contribute to the extension/completion of protein databases and include original work on HP in the brain to illustrate the processes of HP identification and functional assignment.     

Cloning, characterization and localization of Chinese hamster HP1 isoforms

The Chinese hamster is one of the few mammalian species that are characterized by relatively poor heterochromatin content. It was intriguing to test whether or not the lack of large blocks of heterochromatin in the hamster chromosomes could be correlated with the absence or species-specific differences of the HP1 proteins, the main structural components of heterochromatin. To address this, we attempted to clone HP1 from the Chinese hamster. It is shown here that all three isoforms of HP1 known in mammals are present in hamster, and the amino acid sequences deduced from the cDNAs of the isoforms are 97-100% identical to those of the known mammalian homologues. All three isoforms are localized mainly in heterochromatic regions in the native chromosomes and nuclei. The hamster HP1 alpha gene was cloned, sequenced and mapped to the short arm of hamster chromosome 2.These data indicate that the Chinese hamster has all the HP1 components necessary for the establishment of heterochromatin. The limited amount of heterochromatin in hamster cells may probably be attributed to the unusual satellite DNA content of the hamster genome.     

Effects of hydrostatic pressure on TMJ disc cells

The effects of mechanical stimuli on TMJ disc cells have yet to be investigated. This study examined for the first time the effect of constant and intermittent hydrostatic pressure (HP) on TMJ disc cells. Guided by studies on articular chondrocytes, the chosen amplitude was 10 MPa, the frequency was 1 Hz for intermittent HP, and the duration was 4 h. A one-time application of the HP stimulus was applied in 2-D and 3-D for gene expression studies. A duty cycle of 2 days on, 1 day off for 1 week of HP stimulus was used for biochemical content studies. In monolayer, the intermittent HP regimen increased collagen II expression, while constant HP increased collagen I expression when compared to the non-loaded control. However, the overall expression of collagen I was much higher than collagen II in both constant and intermittent HP. The expression results correlated well with gross morphology, histology, and biochemical content. At Week 1, the intermittent HP group had a lower content of collagen, 7.5 +/- 0.2 microg/construct, than the non-loaded control group, 18.2 +/- 4.0 microg/construct. The constant HP group showed the highest amount of collagen, 24.5 +/- 1.6 microg/construct. These data show that constant HP at 10 MPa for 4 h produces more collagen I than do the non-loaded control or intermittent HP at 10 MPa and 1 Hz.     

Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location

Heterochromatin proteins are thought to play key roles in chromatin structure and gene regulation, yet very few genes have been identified that are regulated by these proteins. We performed large-scale mapping and analysis of in vivo target loci of the proteins HP1, HP1c, and Su(var)3-9 in Drosophila Kc cells, which are of embryonic origin. For each protein, we identified approximately 100-200 target genes among >6000 probed loci. We found that HP1 and Su(var)3-9 bind together to transposable elements and genes that are predominantly pericentric. In addition, Su(var)3-9 binds without HP1 to a distinct set of nonpericentric genes. On chromosome 4, HP1 binds to many genes, mostly independent of Su(var)3-9. The binding pattern of HP1c is largely different from those of HP1 and Su(var)3-9. Target genes of HP1 and Su(var)3-9 show lower expression levels in Kc cells than do nontarget genes, but not if they are located in pericentric regions. Strikingly, in pericentric regions, target genes of Su(var)3-9 and HP1 are predominantly embryo-specific genes, whereas on the chromosome arms Su(var)3-9 is preferentially associated with a set of male-specific genes. These results demonstrate that, depending on chromosomal location, the HP1 and Su(var)3-9 proteins form different complexes that associate with specific sets of developmentally coexpressed genes.     

Dynamic culturing of cartilage tissue: the significance of hydrostatic pressure

Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4?MPa Pulsatile HP; (2) 0.4?MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4?MPa) and physiologic (5?MPa) HP levels. hASCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4?MPa Pulsatile HP; (2) 5?MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and amplitude-dependant manner.     

蛋白质组学在药学研究中的应用

在后基因组时代 ,蛋白质组学逐渐成为科学家研究的热点学科。它从整体水平对蛋白质进行分析 ,并逐渐被应用于医学研究的不同领域。在药学范畴内 ,它针对药物作用靶点的识别与验证 ,药物耐药机制的探索 ,药物毒理学研究 ,分子药理模型的构建等进行研究 ,在药物研究方面已显示出巨大的潜力。     

Can a plant-based vaccine treat hypertension?

Vaccines against hypertension constitute a viable approach to decrease blood pressure. In particular, two vaccines against hypertension (HP) targeting angiotensin II (AgII) have showed promising results and these are currently on evaluation in clinical trials. In parallel, plant-based vaccines have become a biotechnological application that has been assessed in clinical trials for some cases. This report proposes a hypothesis that involves developing a plant-based vaccine against HP. It is hypothesized that a plant-based vaccine having AgII or its AT1 receptor (ATR1) as targets, constitutes a safe, suitable and efficient therapeutic approach for HP. It is known that a number of carrier proteins can be produced in plants retaining its adjuvanticity. Therefore the production in plants of chimeric proteins where either AgII or ATR1 domains are fused to these carriers would be a promising approach to be investigated. Mucosal immunization using plant-derived AgII/ATR1 chimeric proteins would imply several advantages such as low cost and friendly delivery. However due to the lack of a detailed knowledge on the physiological role of AgII at the gastrointestinal tract, the effects of partially blocking the AgII action must be extensively evaluated. An alternative related to this aspect would be the use of transient expression systems where productivity is sufficiently high to allow the purification of the antigen of interest at convenient yields, so that it can constitute a parenteral vaccine. Proving the concept for a plant-based vaccine against HP may have profound implications on the development of a new HP therapy which offers convenient features such as low cost and easier compliance in comparison to pharmacological treatment.     

Antigen characterization of major cork moulds in Suberosis (cork worker's pneumonitis) by immunoblotting

BACKGROUND: We characterized by immunoblotting the antigenicity of the most frequent fungi colonizing cork during its industrial processing, Penicillium glabrum and Chrysonilia sitophila. Penicillium glabrum is the main causative agent of Suberosis, a hypersensitivity pneumonitis of cork workers. Chrysonilia sitophila induces both IgE sensitization and occupational asthma in the wood processing industry. METHODS: Serum-specific IgG, IgG4 and IgE to P. glabrum and C. sitophila from nine cork workers with hypersensitivity pneumonitis (HP) and seven with asthma (four with occupational asthma) were analysed by immunoblotting. RESULTS: Both HP and asthmatic patients' sera showed immunoreactivity to several proteins resolved in the specific immunoblot strips. The frequency of specific IgG recognition to 12-13.5 and 33 kDa proteins of P. glabrum was significantly higher in HP patients. The sera of HP patients had significantly higher specific IgG recognition to 16 and 51-55 kDa proteins of C. sitophila. There was no specific IgE recognition in the sera of HP or asthmatic patients to both fungi. CONCLUSIONS: Different patterns of antibody reactivity to P. glabrum and C. sitophila are seen in cork workers with hypersensitivity pneumonitis or asthma. The 12-13.5 and 33 kDa proteins of P. glabrum and the 16 and 51-55 kDa proteins of C. sitophila may be major antigens in Suberosis.     

Investigation of mechanisms of mammalian hibernation and its possible application in medical treatment

Mammalian hibernation has been reported to increase resistance to various harmful events such as low body temperature, severe ischemia, bacterial infection, irradiation, and muscle disuse, and to prolong the lifespan of the mammal. Therefore, hibernation mechanisms are thought to play a critical role in maintaining healthy organisms. Although the application of this physiological phenomenon to medical fields has strongly been desired, it has been prevented by a poor understanding of the hibernation mechanism. In order to clarify how mammalian hibernation is controlled in organisms, we have looked for a physiological signal of hibernation and found marked changes in cardiac calcium regulation associated with a circannual hibernation. Focusing on these changes, we initially discovered a molecular marker of hibernation, hibernation-specific proteins (HP), of which production in the liver and the blood content are controlled by an endogenous circannual rhythm responsible for hibernation. Our recent studies on HP regulation have revealed that circannual signals for the timing of hibernation are transmitted through the neuroendocrine system and that HP are actively transported into the cerebrospinal fluid (CSF) prior to the onset of hibernation. This suggested that hibernation is controlled by HP in the brain and its regulation system. Based on these results, the future medical application of these results is discussed.     

医学生的医学同理心特点

目的探讨医学生同理心的特点,为有针对性地培养医学生的医患沟通能力提供依据。方法采用杰斐逊医学生同理心量表（JSE—HP）对天津医科大学和天津中医药大学临床专业564名医学生进行问卷调查。通过社会科学统计软件包SPSS17．0建立数据库进行统计分析。采用内部一致性分析各量表信度,描述性统计分析医学生在各变量的分布、t检验比较不同特征医学生的同理心特点,F检验比较不同年级医学生同理心的特点。结果①本研究问卷的Cronbach仅系数在0．608～0．857之间,量表具有较好的信度;②医学生同理心量表（杰斐逊量表）得分在32—138分之间（量表总分范围为20～140）,均值为（103．73±17．87）分,这说明被试医学生的医学同理心高于中等水平;③性别、年级、对所学专业满意程度等人口统计学资料不同的医学生同理心的差异有统计学意义（P〈0．05或〈0．01）。结论①医学生的总体同理心水平略高于中等水平;②性别不同和是否独生子女对医学同理心没有影响,对所学专业满意程度高和学过心理学课程的医学生同理心水平高;③不同年级的医学生同理心变化较大,随着年级的增长,整体呈下降趋势。     

The novel Helicobacter pylori CznABC metal efflux pump is required for cadmium, zinc, and nickel resistance, urease modulation, and gastric colonization

Maintaining metal homeostasis is crucial for the adaptation of Helicobacter pylori to the gastric environment. Iron, copper, and nickel homeostasis has recently been demonstrated to be required for the establishment of H. pylori infection in animal models. Here we demonstrate that the HP0969-0971 gene cluster encoding the Czc-type metal export pump homologs HP0969, HP0970, and the H. pylori-specific protein HP0971 forms part of a novel H. pylori metal resistance determinant, which is required for gastric colonization and for the modulation of urease activity. Insertional mutagenesis of the HP0971, HP0970, or HP0969 genes in H. pylori reference strain 26695 resulted in increased sensitivity to cadmium, zinc, and nickel (czn), suggesting that the encoded proteins constitute a metal-specific export pump. Accordingly, the genes were designated cznC (HP0971), cznB (HP0970), and cznA (HP0969). The CznC and CznA proteins play a predominant role in nickel homeostasis, since only the cznC and cznA mutants but not the cznB mutant displayed an 8- to 10-fold increase in urease activity. Nickel-specific affinity chromatography demonstrated that recombinant versions of CznC and CznB can bind to nickel and that the purified CznB protein interacted with cadmium and zinc, since both metals competitively inhibited nickel binding. Finally, single cznA, cznB, and cznC mutants did not colonize the stomach in a Mongolian gerbil-based animal model. This demonstrates that the metal export functions of H. pylori cznABC are essential for gastric colonization and underlines the extraordinary importance of metal ion homeostasis for the survival of H. pylori in the gastric environment.     

Thioloxidoreductase HP0231 of Helicobacter pylori impacts HopQ-dependent CagA translocation

Thioloxidoreductase HP0231 of Helicobacter pylori plays essential roles in gastric colonization and related gastric pathology. Comparative proteomics and analysis of complexes between HP0231 and its protein substrates suggested that several Hop proteins are its targets. HP0231 is a dimeric oxidoreductase that functions in an oxidizing Dsb (disulfide bonds) pathway of H. pylori. H. pylori HopQ possesses six cysteine residues, which generate three consecutive disulfide bridges. Comparison of the redox state of HopQ in wild-type cells to that in hp0231-mutated cells clearly indicated that HopQ is a substrate of HP0231. HopQ binds CEACAM1, 3, 5 and 6 (carcinoembryonic antigen-related cell adhesion molecules). This interaction enables T4SS-mediated translocation of CagA into host cells and induces host signaling. Site directed mutagenesis of HopQ (changing cysteine residues into serine) and analysis of the functioning of HopQ variants showed that HP0231 influences the delivery of CagA into host cells, in part through its impact on HopQ redox state. Introduction of a C382S mutation into HopQ significantly affects its reaction with CEACAM receptors, which disturbs T4SS functioning and CagA delivery. An additional effect of HP0231 on other adhesins and their redox state, resulting in their functional impairment, cannot be excluded.     

Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable,epigenetic gene silencing: a mammalian cell culture model of gene variegation

Heterochromatin protein 1 (HP1) is a key component of constitutive heterochromatin in Drosophila and is required for stable epigenetic gene silencing classically observed as position effect variegation. Less is known of the family of mammalian HP1 proteins, which may be euchromatic, targeted to expressed loci by repressor-corepressor complexes, and retained there by Lys 9-methylated histone H3 (H3-MeK9). To characterize the physical properties of euchromatic loci bound by HP1, we developed a strategy for regulated recruitment of HP1 to an expressed transgene in mammalian cells by using a synthetic, hormone-regulated KRAB repression domain. We show that its obligate corepressor, KAP1, can coordinate all the machinery required for stable gene silencing. In the presence of hormone, the transgene is rapidly silenced, spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with KAP1, HP1, and the H3 Lys 9-specific methyltransferase, SETDB1, over a highly localized region centered around the promoter. Remarkably, silencing established by a short pulse of hormone is stably maintained for >50 population doublings in the absence of hormone in clonal-cell populations, and the silent transgenes in these clones show promoter hypermethylation. Thus, like variegation in Drosophila, recruitment of mammalian HP1 to a euchromatic promoter can establish a silenced state that is epigenetically heritable.