Random Motion of Chromatin Is Influenced by Lamin A Interconnections

Using fluorescence correlation spectroscopy in single-plane illumination microscopy, we investigated the dynamics of chromatin in interphase mouse adult fibroblast cell nuclei under the influence of the intermediate filament protein lamin A. We find that 1) lamin A-eGFP and histone H2A-mRFP show significant comobility, indicating that their motions are clearly interconnected in the nucleus, and 2) that the random motion of histones H2A within the chromatin network is subdiffusive, i.e., the effective diffusion coefficient decreases for slow timescales. Knocking out lamin A changes the diffusion back to normal. Thus, lamin A influences the dynamics of the entire chromatin network. Our conclusion is that lamin A plays a central role in determining the viscoelasticity of the chromatin network and helping to maintain local ordering of interphase chromosomes.     

Elucidation of insulin assembly at acidic and neutral pH: Characterization of low molecular weight oligomers

Deficiency in insulin secretion and function that characterize type 2 diabetes often requires administration of extraneous insulin, leading to injection‐site amyloidosis. Insulin aggregation at neutral pH is not well understood. Although oligomer formation is believed to play an important role, insulin oligomers have not been fully characterized yet. Here, we elucidate similarities and differences between in vitro insulin aggregation at acidic and neutral pH for a range of insulin concentrations (2.5–100 μM) by using kinetic thioflavin T fluorescence, circular dichroism, atomic force and electron microscopy imaging. Importantly, we characterize the size distribution of insulin oligomers at different assembly stages by the application of covalent cross‐linking and gel electrophoresis. Our results show that at the earliest assembly stage, oligomers comprise up to 40% and 70% of soluble insulin at acidic and neutral pH, respectively. While the highest oligomer order increases with insulin concentration at acidic pH, the opposite tendency is observed at neutral pH, where oligomers up to heptamers are formed in 10 μM insulin. These findings suggest that oligomers may be on‐ and off‐pathway assemblies for insulin at acidic and neutral pH, respectively. Agitation, which is required to induce insulin aggregation at neutral pH, is shown to increase fibril formation rate and fibrillar mass both by an order of magnitude. Insulin incubated under agitated conditions at neutral pH rapidly aggregates into large micrometer‐sized aggregates, which may be of physiological relevance and provides insight into injection‐site amyloidosis and toxic pulmonary aggregates induced by administration of extraneous insulin.     

Preclinical Evaluation of Novel Triphenylphosphonium Salts with Broad-Spectrum Activity

Background

Recently, there has been a surge of interest in developing compounds selectively targeting mitochondria for the treatment of neoplasms. The critical role of mitochondria in cellular metabolism and respiration supports this therapeutic rationale. Dysfunction in the processes of energy production and metabolism contributes to attenuation of response to pro-apoptotic stimuli and increased ROS production both of which are implicated in the initiation and progression of most human cancers.

Methodology/Principal Findings

A high-throughput MTT-based screen of over 10,000 drug-like small molecules for anti-proliferative activity identified the phosphonium salts TP187, 197 and 421 as having IC₅₀ concentrations in the submicromolar range. TP treatment induced cell cycle arrest independent of p53 status, as determined by analysis of DNA content in propidium iodide stained cells. In a mouse model of human breast cancer, TP-treated mice showed significantly decreased tumor growth compared to vehicle or paclitaxel treated mice. No toxicities or organ damage were observed following TP treatment. Immunohistochemical staining of tissue sections from TP187-treated tumors demonstrated a decrease in cellular proliferation and increased caspase-3 cleavage. The fluorescent properties of analog TP421 were exploited to assess subcellular uptake of TP compounds, demonstrating mitochondrial localization. Following mitochondrial uptake cells exhibited decreased oxygen consumption and concomittant increase in mitochondrial superoxide production. Proteomics analysis of results from a 600 target antibody microarray demonstrated that TP compounds significantly affected signaling pathways relevant to growth and proliferation.

Conclusions/Significance

Through our continued interest in designing compounds targeting cancer-cell metabolism, the Warburg effect, and mitochondria we recently discovered a series of novel, small-molecule compounds containing a triphenylphosphine moiety that show remarkable activity in a panel of cancer cell lines as well as in a mouse model of human breast cancer. The mechanism of action includes mitochondrial localization causing decreased oxygen consumption, increased superoxide production and attenuated growth factor signaling.     

NMR spectroscopy-based metabolomic study of serum in sulfur mustard exposed patients with lung disease

Sulfur mustard (SM) is a vesication chemical warfare agent for which there is currently no antidote. Despite years of research, there is no common consensus about the pathophysiological basis of chronic pulmonary disease caused by this chemical warfare agent. In this study, we combined chemometric techniques with nuclear magnetic resonance (NMR) spectroscopy to explore the metabolic profile of sera from SM-exposed patients. A total of 29 serum samples obtained from 17 SM-injured patients, and 12 healthy controls were analyzed by Random Forest. Increased concentrations of seven amino acids, glycerol, dimethylamine, ketone bodies, lactate, acetate, citrulline and creatine together with the decreased very low-density lipoproteins (VLDL) levels were observed in patients compared with control subjects. Our study reveals the metabolic profile of sera from SM-injured patients and indicates that NMR-based methods can distinguish these patients from healthy controls.     

Evaluation of functional RAGE gene polymorphisms in childhood acute lymphoblastic leukemia—A case-control study from Iran

We examined the possible relationship between three RAGE polymorphisms, ?429C/T, ?374 T/A, and 63-bp deletion, and susceptibility to childhood acute lymphoblastic leukemia (ALL) in an Iranian population. This study included 75 ALL patients and 115 healthy subjects. Genotyping was performed using HEXA-ARMS-polymerase chain reaction. We found no significant association among RAGE gene polymorphisms and the risk for ALL at genotype, allelic and haplotype levels (P > 0.05). The hemoglobin levels were higher in patients with RAGE ?374 TT than in the TA carriers (P = 0.019). Our results demonstrated that the RAGE gene variations were not associated with risk of pediatrics ALL.     

Taking organelles apart, putting them back together and creating new ones: lessons from the endoplasmic reticulum

The endoplasmic reticulum (ER) is a highly dynamic organelle. It is composed of four subcompartments including nuclear envelope (NE), rough ER (rER), smooth ER (sER) and transitional ER (tER). The subcompartments are interconnected, can fragment and dissociate and are able to reassemble again. They coordinate with cell function by way of protein regulators in the surrounding cytosol. The activity of the many associated molecular machines of the ER as well as the fluid nature of the limiting membrane of the ER contribute extensively to the dynamics of the ER. This review examines the properties of the ER that permit its isolation and purification and the physiological conditions that permit reconstitution both in vitro and in vivo in normal and in disease conditions.     

RANTES gene mRNA expression and its -403 G/A promoter polymorphism in coronary artery disease

Objective

Increased RANTES expression has been described to have a role in atherosclerosis plaque formation. Functional polymorphisms within RANTES promoter region have shown association with increased risk of coronary atherosclerosis (CAD). The aim of this study was to examine the RANTES mRNA expression in patients with CAD compared to patients without CAD and its association with RANTES − 403 G/A polymorphism in an Iranian population.

Methods

The study was performed on 319 patients who underwent coronary artery angiography and patients with > 50% stenosis in vessels considered as case groups (CAD+) N = 191 and normal vessels group as control (CAD−) N = 128. In each group 20 patients were examined for RANTES mRNA expression.RANTES mRNA expression was examined using quantitative real-time PCR. Genotyping of − 403 polymorphism was performed using PCR-RFLP technique.

Results

We found that RANTES mRNA expression was increased to 1.37 fold in CAD patients compared to the controls but the difference was not statistically significant. Also comparing the RANTES mRNA expression in patients with different RANTES − 403 G/A polymorphism showed that in patients carrying AA genotype RANTES mRNA expression was increased to 1.74 fold compared to patients carrying GG genotype and to 1.51 fold compared to patients carrying GA genotype. No significant difference for allele and genotype frequencies of RANTES − 403 polymorphism was found between cases and controls.

Conclusion

More studies on larger number of samples are required to further evaluate role of RANTES in pathogenesis of CAD.     

Common Familial Mediterranean Fever gene mutations in a Turkish cohort

Familial Mediterranean Fever (FMF) is an autosomal recessive autoinflammatory disorder with the responsible gene of MEFV which primarily affects Jewish, Armenian, Turkish and Arab populations. The FMF gene (MEFV) has recently been cloned to chromosome 16p, which encodes pyrin. In the present study, we enrolled 2,067 unrelated patients with the suspicion of FMF in Middle Anatolia between the years 2006–2009 and identified the 12 MEFV mutations. DNA was amplified by PCR and subjected to reverse hybridization for the detection of MEFV gene mutations. Among the 2,067 patients, 866 (41.9%) were males and 1,201 (58.1%) were females. The mutations were homozygous in 176 (16.85%) patients, compound heterozygous in 314 (30.1%) patients, heterozygous in 546 (52.25%) patients and the other forms of mutations were found in 8 patients (0.76%). No mutation was detected in 1,023 (49.5%) patients. The most frequent mutations were M694V, M680I (G/C), E148Q and V726A. We could not find any significant differences between the two common mutations according to the gender. The high incidence of MEFV gene mutations in the Turkish population indicated that newborn screening may be discussed in the future. Because of the ethnic origin of Anatolia, larger serial analyses are necessary to investigate the rate and coexistence of these mutations.