[Blocking PAK1 kinase activity promotes the differentiation of acute megakaryocytic leukemia cells and induces their apoptosis].

Objective: To investigate the effect of blocking P21 activated kinase 1 (PAK1) activity on the proliferation, differentiation, and apoptosis of acute megakaryocytic leukemia (AMKL) cell lines (CHRF and CMK) . Methods: Cell counts were used to detect the effects of PAK1 inhibitors (IPA-3 and G5555) on AMKL cell proliferation inhibition and colony formation, and flow cytometry was used to detect its effects on AMKL cell cycle. The effect of PAK1 inhibitor on the expression of cyclin D1 and apoptosis-related protein Cleaved caspase 3 was detected using Western blot, while interference with the protein expression level of PAK1 in AMKL cells was assessed using lentivirus-mediated shRNA transfection technology. Flow cytometry was used to detect the effects of knockdown of PAK1 kinase activity on the ability of polyploid DNA formation and cell apoptosis in AMKL cells. Results: PAK1 inhibitors inhibited the proliferation of AMKL cells in a dose-dependent manner and reduced the ability of cell colony formation, and the difference was statistically significant when compared with the control group (P<0.05) . Moreover, they also reduced the percentage of AMKL cells in S phase, and Western blot detection showed that the expression levels of phosphorylated PAK1 and cyclin D1 decreased significantly. Finally, PAK1 inhibitors induced AMKL cell apoptosis by up-regulating Cleaved caspase 3 and showed different abilities to increase the content of polyploid DNA in megakaryocytes. Only high concentrations of IPA-3 and low doses of G5555 increased the number of polyploid megakaryocytes, while knockdown of PAK1 kinase activity promoted AMKL cell differentiation and increased the apoptosis rate. Conclusion: PAK1 inhibitor significantly arrests AMKL cell growth and promotes cell apoptosis. Knocking down the expression of PAK1 promotes the formation of polyploid DNA and induces AMKL cell apoptosis. The above findings indicate that inhibiting the activity of PAK1 may control AMKL effectively.

FENDRR reduces tumor invasiveness in prostate cancer PC-3 cells by targeting CSNK1E.

OBJECTIVE: Prostate cancer, one of the most common malignant tumors in urology, now has become a malignant disease that seriously threatens the health of men in China. Although there are a large number of clinical studies on the treatment of patients with prostate cancer, many patients have entered the advanced stage of diagnosis, and little is known about its pathogenesis. MATERIALS AND METHODS: We identified a series of ncRNA and TF by differential expression analysis, co-expression analysis, enrichment analysis, connectivity analysis, and hypergeometric test strategies for prostate cancer expression genomes. RESULTS: 53 modules related to prostate cancer PC-3 cells were obtained, involving module focusing of 4448 genes. Based on these modules, we predicted that miR-26a-5p, miR-130a-3p, miR-519d-3p, etc. have important regulatory effects on prostate cancer PC-3 cells. At the same time, a series of transcription factors (relating to RELA, SOX10, TP53, and TWIST2, etc.) were obtained and may play a key regulatory role in prostate cancer PC-3 cell-related modules. CONCLUSIONS: These results suggest that FENDRR in prostate cancer may reduce tumor invasion in prostate cancer PC-3 cells by targeting CSNK1E, which may have favourable effort to better understand the underlying pathogenesis of prostate cancer and provide a tough theoretical basis for further studying prostate cancer.

Induction of IL-10 producing CD4+ T cells with regulatory activities by stimulation with IL-10 gene-modified bone marrow derived dendritic cells.

Dendritic cells (DCs) can induce both tolergenic as well as effective immune responses in the lung. Pulmonary DCs producing interleukin (IL)-10 mediated tolerance induced by respiratory exposure to antigen. IL-10 is an important immunosuppressive cytokine, which inhibits maturation and function of DC. To assess whether IL-10 producing DCs can exert the tolergenic effect through the differentiation of regulatory T cells, bone marrow derived DCs were genetically modified by IL-10 expressing adenovirus. IL-10 gene modified DCs (Ad-IL-10-DC) displayed a characteristic phenotype of immature DCs. Here we showed that in vitro repetitive stimulation of naïve DO11.10 CD4(+) T cells with Ad-IL-10-DCs resulted in a development of IL-10 producing T-cell regulatory cells. These T cells could not proliferate well but also lost their ability to produce interferon-gamma upon restimulation with irradiated splenocytes and ovalbumin peptide. Furthermore, in co-culture experiments these T cells inhibited the antigen-driven proliferation of naïve CD4+ T cells in a dose-dependent manner. Our findings demonstrated that IL-10 producing DCs had the potential to induce the differentiation of Tr1-like cells and suggested their therapeutic use.

Desmoglein-1-specific T lymphocytes from patients with endemic pemphigus foliaceus (fogo selvagem).

Fogo selvagem (FS), the endemic form of pemphigus foliaceus, is a cutaneous autoimmune disease characterized by subcorneal blistering of the epidermis and the production of autoantibodies against the desmosomal antigen desmoglein-1 (Dsg1). Previously, we showed that mice injected with autoantibodies from FS patients develop a skin disease that reproduces the clinical, histological, and immunological features of FS, indicating that autoantibodies play an essential role in the development of this disease. The purpose of this study was to characterize the autoimmune T-cell response associated with FS. We provide here the first evidence, to our knowledge, that the great majority of FS patients have circulating T lymphocytes that specifically proliferate in response to the extracellular domain of Dsg1. Long-term T cells developed from these patients also responded to Dsg1, and this antigen-specific response was shown to be restricted to HLA-DR molecules. These Dsg1-reactive FS T cells exhibited a CD4-positive memory T-cell phenotype and produced a T helper 2-like cytokine profile. These findings represent the initial steps in defining the role of T cells in FS autoimmunity.

Inhibition of acetyl CoA carboxylase by GTP gamma S.

The effect of nonhydrolyzable guanine nucleotides on mammalian acetyl CoA carboxylase (ACC) activity was examined. Using porous rat adipocytes and crude fat cell homogenates to study metabolic pathway flux, GMPPNP and/or GTP gamma S inhibited [14C]fatty acid formation by up to 95% when either [6-14C]glucose-6-phosphate or [1-14C]acetyl CoA was used as substrate. If [2-14C]malonyl CoA initiated flux, however, no inhibition was apparent. These pathway flux studies suggested that ACC was the locus of inhibition, and that the mechanism might involve a disruption of guanine nucleotide hydrolysis by the nonhydrolyzable analogues. Using partially and avidin-sepharose-purified ACC preparations from rat fat, liver and mammary tissue, citrate-stimulated ACC activity was inhibited by 25-75% with 50 microM GTP gamma S. Related compounds and nucleotides had absent-to-minimal effects on ACC. ATP gamma S was inhibitory (10-30% at 5-15 microM), but always to a lesser degree than equimolar GTP gamma S. Filter binding assays with [alpha-32P]GTP or [35S]GTP gamma S were negative, but low-level GTPase activity was detected. Using photoaffinity labelling techniques, [alpha-32P]GTP was found to bind ACC and not pyruvate carboxylase. The hypothesis that citrate-responsive ACC activity may be modulated by an intrinsic or associated GTP binding site is explored. Since ACC forms polymers, as does the cytoskeletal protein beta-tubulin, amino acid sequence comparisons between ACC and atypical GTP binding domain of beta tubulin are presented.

Epitopes targeted by bullous pemphigoid T lymphocytes and autoantibodies map to the same sites on the bullous pemphigoid 180 ectodomain.

Bullous pemphigoid is a blistering skin disease characterized by autoantibodies directed against the NC16A domain of bullous pemphigoid 180 (collagen XVII), a transmembrane protein of epidermal basal cells. Passive transfer studies in mice have shown that antibodies that bind to this immunodominant region of bullous pemphigoid 180 are capable of inducing a skin disease that closely mimics bullous pemphigoid, supporting the hypothesis that epitopes within NC16A are involved in the pathogenesis of bullous pemphigoid. In this study, we examined the autoimmune T cell response in bullous pemphigoid patients. T cells from eight of 12 bullous pemphigoid patients, all of whom had circulating anti-bullous pemphigoid 180 autoantibodies, showed a specific proliferative response to recombinant forms of NC16A. T cell lines and clones developed from four of these patients recognize the same NC16A peptides as those targeted by autoantibodies from the corresponding individuals. These NC16A-responding T lymphocytes express alpha/beta T cell receptors and CD4 memory T cell surface markers and exhibited a Th1/Th2 mixed cytokine profile that may support the production of antibodies. This new information will aid in defining the key steps involved in the development of the autoimmune response in bullous pemphigoid.

Risk factors for age-related maculopathy: the Visual Impairment Project.

OBJECTIVE: To describe the risk factors and associated population attributable risk for age-related maculopathy (ARM) and age-related macular degeneration (AMD) in Australians aged 40 years and older. METHODS: Residents were recruited from 9 randomly selected urban clusters and 4 randomly selected rural clusters in Victoria, Australia. At locally established test sites, the following information was collected: visual acuity, medical and health history, lifetime sunlight exposure, dietary intake, and fundus photographs. Age-related maculopathy and AMD were graded from the fundus photographs using an international classification and grading system. Backwards logistic regression was used to identify the independent risk factors for ARM and AMD. RESULTS: The participation rate was 83% (n = 3271) among the urban residents and 92% (n = 1473) among the rural residents. Gradable fundus photographs of either eye were available for 4345 (92%) of the 4744 participants. There were 656 cases of ARM, giving a weighted prevalence of 15.1% (95% confidence limit [CL], 13.8, 16.4); and there were 30 cases of AMD, giving a weighted prevalence of 0.69% (95% CL, 0.33, 1.03). In multiple logistic regression, the risk factors for AMD were as follows: age (odds ratio [OR], 1.23; 95% CL, 1.17, 1.29), smoked cigarettes for longer than 40 years (OR, 2.39; 95% CL, 1.02, 5.57), and ever taken angiotensin-converting enzyme inhibitors (OR, 3.26; 95% CL, 1.33, 8.01). The magnitude of all of these risk factors was slightly less for ARM, and having ever taken blood cholesterol-lowering medications was also significant (OR, 1.67; 95% CL, 1.12, 2.47; P =.001). CONCLUSION: Smoking is the only modifiable risk factor for ARM and AMD, among the many environmental and systemic factors that were assessed.

Leptin does not affect adipocyte glucose metabolism: studies in fresh and cultured adipocytes.

Leptin, the 16-kd hormone produced by white fat cells, regulates energy homeostasis, satiety, and multiple sites in the neuroendocrine system. Leptin receptors have been identified in the central nervous system (CNS) and are widespread in peripheral tissues, including fat. Given the association between insulin resistance and obesity, it is important to establish whether leptin has additional effects on peripheral insulin action and glucose metabolism. This study examined whether leptin has a direct autocrine/paracrine action on glucose metabolism in both freshly isolated and 24-hour cultured rat fat cells. Freshly isolated rat adipocytes were incubated for 30 minutes with 200 ng/mL recombinant murine leptin. Thereafter, basal and insulin-stimulated (10(-8) mol/L) glucose transport, glycolysis-Krebs oxidation and lipogenesis ([6-14C]glucose conversion to [14C]O2 and to [14C]triglyceride), and lipolysis were measured. Upon leptin exposure, no statistical differences were detected in glucose transport or metabolism. Increasing the leptin concentration to 1 to 2 microg/mL or prolonging the duration of preincubation with the fat cells to 60 minutes before the metabolic assays did not alter the results. Finally, using two disparate fat cell culture methods with differing substrate additions (pyruvate and high or low glucose concentrations), there was no effect of 24-hour exposure to leptin (200 ng/mL) on basal and insulin-stimulated glucose transport or lipogenesis. We conclude that leptin does not modulate basal or insulin-stimulated glucose metabolism in isolated and cultured fat cells in vitro. However, in vivo, higher pericellular leptin concentrations, as well as other cellular or soluble serum factors, may exist that might lead to a physiologically relevant autocrine action of leptin.

The epidemiology of cataract in Australia.

PURPOSE: To describe the prevalence and risk factors for cataract in an Australian population aged 40 years and older. METHODS: Participants were recruited by a household census and stratified, random cluster sampling to represent residents of Victoria, Australia, aged 40 years and older. The following information was collected: initial visual acuity and best-corrected visual acuity, demographic details, health history, dietary intake of antioxidants, lifetime ocular ultraviolet B exposure, and clinical eye examination, including lens photography. Cortical opacities were measured in sixteenths. Cortical cataract was defined as opacity greater than or equal to 4/16 of pupil circumference. Nuclear opacities were graded according to the Wilmer cataract grading scheme, and cataract was defined as greater than or equal to nuclear standard 2.0 of four standards. The height and width of any posterior subcapsular opacity was measured and recorded. Posterior subcapsular cataract was defined as posterior subcapsular opacity greater than or equal to 1 mm2. The worse eye was selected for analysis. Backward stepwise logistic regression was used to quantify independent risk factors for cataract. RESULTS: A total of 3,271 (83% of eligible) of the urban residents, 403 (90% of eligible) nursing home residents, and 1,473 (92% of eligible) rural residents participated. The urban residents ranged in age from 40 to 98 years (mean, 59 years), and 1,511 (46%) were men. The nursing home residents ranged in age from 46 to 101 years (mean, 82 years), and 85 (21%) were men. The rural residents ranged in age from 40 to 103 years (mean, 60 years), and 701 (47.5%) were men. The overall weighted rate of cortical cataract was 11.3% (95% confidence limits, 9.68%, 13.0%) excluding cataract surgery and 12.1% (95% confidence limits, 10.5%, 13.8%) including cataract surgery. The risk factors for cortical cataract that remained in the multivariate logistic regression model were age, female gender, diabetes duration greater than 5 years, gout duration greater than 10 years, arthritis diagnosis, myopia, use of oral beta-blockers, and increased average annual ocular ultraviolet B exposure. Overall, 12.6% (95% confidence limits, 9.61%, 15.7%) of Victorians aged 40 years and older had nuclear cataract including previous cataract surgery, and 11.6% (95% confidence limits, 8.61%, 14.7%) had nuclear cataract excluding previous cataract surgery. In the urban and rural cohorts, age, female gender, rural residence, brown irides, diabetes diagnosed 5 or more years earlier, myopia, age-related maculopathy, having smoked for greater than 30 years, and an interaction between ocular ultraviolet B exposure and vitamin E were all risk factors for nuclear cataract. The rate of posterior subcapsular cataract excluding previous cataract surgery was 4.08% (95% confidence limits, 3.01%, 5.14%), whereas the overall rate of posterior subcapsular cataract including previous cataract surgery was 4.93% (95% confidence limits, 3.68%, 6.17%) . The independent risk factors for posterior subcapsular cataract in the urban and rural cohorts that remained were age in years, rural location, use of thiazide diuretics, vitamin E intake, and myopia. CONCLUSIONS: The expected increase in the prevalence of cataract with the aging of the population highlights the need to plan appropriate medical services and public health interventions for primary and secondary prevention. Many of the identified risk factors for cataract in the population have the potential for being modified through public health interventions.

Epidemiology of pterygium in Victoria, Australia.

AIM: To describe the prevalence of and risk factors for pterygium in a population based sample of residents of the Australian state of Victoria who were aged 40 years and older. METHODS: The strata comprised nine randomly selected clusters from the Melbourne statistical division, 14 nursing homes randomly selected from the nursing homes within a 5 kilometre radius of the nine Melbourne clusters, and four randomly selected clusters from rural Victoria. Pterygium was measured in millimetres from the tip to the middle of the base. During an interview, people were queried about previous ocular surgery, including surgical removal of pterygium, and their lifetime exposure to sunlight. RESULTS: 5147 people participated. They ranged in age from 40 to 101 years and 2850 (55.4%) were female. Only one person in the Melbourne cohort reported previous pterygium surgery, and seven rural residents reported previous surgery; this information was unavailable for the nursing home residents. Pterygium was present upon clinical examination in 39 (1.2%) of the 3229 Melbourne residents who had the clinical examination, six (1. 7%) of the nursing home residents, and 96 (6.7%) of the rural residents. The overall weighted population rate in the population was 2.83% (95% CL 2.35, 3.31). The independent risk factors for pterygium were found to be age (OR=1.23, 95% CL=1.06, 1.44), male sex (OR=2.02, 95% CL=1.35, 3.03), rural residence (OR=5.28, 95% CL=3. 56, 7.84), and lifetime ocular sun exposure (OR=1.63, 95% CL=1.18, 2. 25). The attributable risk of sunlight and pterygium was 43.6% (95% CL=42.7, 44.6). The result was the same when ocular UV-B exposure was substituted in the model for broad band sun exposure. CONCLUSION: Pterygium is a significant public health problem in rural areas, primarily as a result of ocular sun exposure.

Prevalence of amblyopia and associated refractive errors in an adult population in Victoria, Australia.

The study aimed to describe the prevalence of amblyopia and associated refractive errors among an adult Australian population. The Visual Impairment Project (VIP) is a population-based study of age-related eye disease in the state of Victoria, Australia. Data were collected through standardised interviews and orthoptic and ophthalmic dilated examinations. Amblyopia was defined as best-corrected visual acuity of 6/9 or worse in the absence of any pathological cause. The participants were 3,265 urban residents and 1,456 rural residents of the VIP ranging in age from 40-92 years (mean = 59 years; 53% female). The prevalence of unilateral amblyopia was 3.06% (95% C.I. 2.59, 3.53). Amblyopia was not found to be statistically different by age group (p=0.096), gender (p=0.675), or place of birth (p=0.14). Anisometropia was statistically more common (p<0.001) in amblyopic cases (51.1%) compared to the normal population (9.7%), and 54% of amblyopic eyes had visual acuity of worse than 6/12. Amblyopia is a significant cause of unilateral reduced visual acuity in a population aged 40 years and older. Anisometropia was more prevalent and the degree of anisometropia was greater in the amblyopic group compared with the normal population. Oblique astigmatism was more prevalent in the amblyopic group compared with the normal population.

[Effect of amygdaloid stimulation on receptive field of "on" LGB neurons in rabbit].

With the aim of study of the effect of stimulating amygdaloid body (basal) on the responsive characteristics of the receptive field (RF) of on-response LGB neurons, electrophysiological experiments were performed on 37 New Zealand rabbits anaethetized with urethane and immobilized by flaxedil. Of the 117 recorded neurons, 47 showed changes in inresponse to light stimulation (facilitation or inhibition) and/or changes in RF configuration, although stimulation of amygdaloid body alone resulted in no visible change of the neuronal activities. The above results suggested that amygdaloid may modulate ascending visual information proccessing at thalami level by changing various combinations of the response characteristics of the on-response LGB neurons.

Polymeric forms of carbon in dense lithium carbide.

The immense interest in carbon nanomaterials continues to stimulate intense research activities aimed at realizing carbon nanowires, since linear chains of carbon atoms are expected to display novel and technologically relevant optical, electrical and mechanical properties. Although various allotropes of carbon (e.g., diamond, nanotubes, graphene, etc) are among the best-known materials, it remains challenging to stabilize carbon in the one-dimensional form because of the difficulty of suitably saturating the dangling bonds of carbon. Here, we show through first-principles calculations that ordered polymeric carbon chains can be stabilized in solid Li(2)C(2) under moderate pressure. This pressure-induced phase (above 5 GPa) consists of parallel arrays of twofold zigzag carbon chains embedded in lithium cages, which display a metallic character due to the formation of partially occupied carbon lone-pair states in sp(2)-like hybrids. It is found that this phase remains the most favorable one in a wide range of pressures. At extreme pressure (larger than 215 GPa) a structural and electronic phase transition towards an insulating single-bonded threefold-coordinated carbon network is predicted.

Spin transition in a four-coordinate iron oxide.

Spin transition has attracted the interest of researchers in various fields since the early 1930s, with thousands of examples now recognized, including those in minerals and biomolecules. However, so far the metal centres in which it has been found to occur are almost always octahedral six-coordinate 3d(4) to 3d(7) metals, such as Fe(II). A five-coordinate centre is only rarely seen. Here we report that under pressure SrFe(II)O(2), which features a four-fold square-planar coordination, exhibits a transition from high spin (S = 2) to intermediate spin (S = 1). This is accompanied by a transition from an antiferromagnetic insulating state to a ferromagnetic so-called half-metallic state: only half of the spin-down (d(xz),d(yz)) states are filled. These results highlight the square-planar coordinated iron oxides as a new class of magnetic and electric materials.

Electronic and structural origin of ultraincompressibility of 5d transition-metal diborides MB(2) (M=W, Re, Os).

First-principles theory was used to investigate the roles of bond topology and covalency in the phase stability and elastic strength of 5d transition-metal diborides, focusing on elements (M=W, Re, Os) that have among the lowest compressibilities of all metals. Among the phases studied, the ReB(2)-type structure exhibits the largest incompressibility (c axis), comparable to that of diamond. This ReB(2) structure is predicted to be the ground-state phase for WB(2) and a pressure-induced phase (above 2.5 GPa) for OsB(2). Both strong covalency and a zigzag topology of interconnected bonds underlie these ultraincompressibilities. Interestingly, the Vickers hardness of WB(2) is estimated to be similar to that of superhard ReB(2).

Vacancy mechanism of high oxygen solubility and nucleation of stable oxygen-enriched clusters in Fe.

First-principles studies identify a vacancy mechanism underlying the unusually high O solubility and nucleation of stable O-enriched nanoclusters in defect-containing Fe. Oxygen, confined as an interstitial, shows an exceptionally high affinity for vacancies, an effect enhanced by spin polarization. If vacancies preexist, the O-vacancy pair formation energy essentially vanishes, allowing the O concentration to approach that of the vacancies. This O-vacancy mechanism enables the nucleation of O-enriched nanoclusters, that attract solutes with high O affinities (Ti and Y) and strengthen Fe-based alloys.

Adenovirus expressing interleukin-1 receptor antagonist alleviates allergic airway inflammation in a murine model of asthma.

Interleukin-1 (IL-1) is a proinflammatory cytokine and IL-1 receptor antagonist (IL-1ra) is a natural inhibitor that binds to IL-1 receptor type I without inducing signal transduction. It is suggested that IL-1 is required for allergen-specific T helper type 2 cell activation and the development of airway hyper-responsiveness (AHR), but the immunologic effect of exogenous IL-1ra in allergic asthma remains unclear. To examine the effect of IL-1ra on airway inflammation and immunoeffector cells in allergic asthma, recombinant adenovirus expressing human IL-1ra (Ad-hIL-1ra) was delivered intranasally into ovalbumin (OVA)-immunized mice. Single intranasal administration of Ad-hIL-1ra before airway antigen challenge in OVA-immunized mice significantly decreased the severity of AHR and reduced pulmonary infiltration of eosinophils and neutrophils. Suppression of IL-5 and eotaxin with concomitant enhancement of interferon gamma in bronchoalveolar lavage fluid was also noted in OVA-immunized mice by administration of Ad-hIL-1ra. In addition, histological studies showed that Ad-hIL-1ra was able to decrease OVA-induced peribronchial inflammation. Taken together, our results indicated that administration of Ad-hIL-1ra may have therapeutic potential for the immunomodulatory treatment of allergic asthma.

Identification of a locus within the hydrogenase gene cluster involved in intracellular nickel metabolism in Bradyrhizobium japonicum.

A 0.6-kb fragment of DNA involved in intracellular Ni metabolism was isolated and cloned from a cosmid containing 23.2 kb of hydrogenase-related genes of Bradyrhizobium japonicum. This locus is located 8.3 kb upstream of the hydrogenase structural genes. The hydrogenase activity of a mutant with a gene-directed mutation at this locus (strain JHK7) showed dependency on nickel provided during hydrogenase derepression. The hydrogenase activity was only 20% of that in the wild-type strain, JH, at a concentration of 0.5 microM NiCl2. The hydrogenase activity in JH reached its maximum at 3 microM NiCl2, whereas the mutant (JHK7) reached wild-type levels of hydrogenase activity when derepressed in 50 microM NiCl2. Studies with the hup-lacZ transcriptional fusion plasmid pSY7 in JHK7 showed that the mutant JHK7 expressed less promoter activity under low-nickel conditions than did strain JH. The mutant accumulated less nickel during a 45-h hydrogenase derepression period than did the wild type. However, both JHK7 and the JH wild-type strain had the same short-term Ni transport rates, and the KmS for Ni of both strains were about 62 microM. When incubated under non-hydrogenase-derepression conditions, the mutant accumulated Ni at the same rate as strain JH. However, this stored source of nickel was unable to restore hydrogenase expression ability of the mutant to wild-type levels during derepression without nickel. The results indicate that the locus identified in B. japonicum is not involved in nickel-specific transport; indeed, it was not at all homologous to the "nickel transporter" hoxN gene of Alcaligenes eutrophus.(ABSTRACT TRUNCATED AT 250 WORDS)