Adsorption equilibria at interface separating electrolyte solution and phosphatidylcholine-stearylamine liposome membrane

The effect of the pH of an electrolyte solution on the electric surface charge of the liposome membrane was studied. The membrane of vesicles contained egg phosphatidylcholine (PC) with different proportions of stearylamine (ST). The surface charge density of the membrane was determined as a function of pH from electrophoretic mobility measurements. A six equilibria model describing the solution ions adsorption on the PC-ST liposome membrane surface was presented in this paper. The knowledge of the association constants of the -PO(-) and -N(+)CH(3)(3) groups of PC with H(+), OH(-), Na(+), Cl(-) ions: K(A(1)H), K(B(1)OH), K(A(1)Na), K(B(1)Cl), that had been presented earlier, allowed to determine the association constants of the -N(+)H(3) group of ST with OH(-) and Cl(-) ions: K(B(2)OH), K(B(2)Cl). The proposed model has been proved to be correct by comparing the resulting theoretic charge variation curves of the PC-ST liposomal membrane with the experimental data.     

Formation of the 3' end of histone mRNA: getting closer to the end

Nearly all eukaryotic mRNAs end with a poly(A) tail that is added to their 3' end by the ubiquitous cleavage/polyadenylation machinery. The only known exceptions to this rule are metazoan replication-dependent histone mRNAs, which end with a highly conserved stem-loop structure. This distinct 3' end is generated by specialized 3' end processing machinery that cleaves histone pre-mRNAs 4-5 nucleotides downstream of the stem-loop and consists of the U7 small nuclear RNP (snRNP) and number of protein factors. Recently, the U7 snRNP has been shown to contain a unique Sm core that differs from that of the spliceosomal snRNPs, and an essential heat labile processing factor has been identified as symplekin. In addition, cross-linking studies have pinpointed CPSF-73 as the endonuclease, which catalyzes the cleavage reaction. Thus, many of the critical components of the 3' end processing machinery are now identified. Strikingly, this machinery is not as unique as initially thought but contains at least two factors involved in cleavage/polyadenylation, suggesting that the two mechanisms have a common evolutionary origin. The greatest challenge that lies ahead is to determine how all these factors interact with each other to form a catalytically competent processing complex capable of cleaving histone pre-mRNAs.     

Cytotoxic efficacy of a novel dinuclear platinum(II) complex used with anti-MUC1 in human breast cancer cells

Mucin 1 (MUC1) is overexpressed in various cancer cells especially in breast cancer cells. There are known research works on the use of anti-MUC1 antibody with docetaxel in ovarian cancer, but there are no data about combined therapy platinum compounds with anti-MUC1 in breast cancer. The aim of the study was to evaluate the antiproliferative properties of a new dinuclear platinum(II) complex (Pt12) used with anti-MUC1 in human breast cancer cells. The dinuclear platinum(II) complex (Pt12) has been synthesized, and its cytotoxicity with anti-MUC1 has been tested in both MCF-7 and MDA-MB-231 breast cancer cells. In this study, the effects of Pt12 with anti-MUC1 on collagen and DNA biosynthesis in human breast cancer cells were compared to those evoked by cisplatin and cisplatin with anti-MUC1. The mechanism of action of Pt12 with anti-MUC1 was studied employing flow cytometry assessment of annexin V binding assay. It was found that Pt12 with anti-MUC1 was more active inhibitor of DNA and collagen synthesis as well more cytotoxic agent than Pt12 alone and cisplatin with anti-MUC1. Cytotoxicity of Pt12 with anti-MUC1 against breast cancer cells is due to apoptotic cell death as well as necrotic cell death. These results indicate that the use of Pt12 with anti-MUC1 may constitute a novel strategy in the chemotherapy of breast cancer tumors.     

Synthesis,properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH₃-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH₃-analogs and the corresponding analogs containing S instead of BH₃ (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH₃-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m₂^7,2′-OGpp_BH3pG D1 and m₂^7,2′-OGpp_BH3pG D2, respectively, than for in vitro transcribed mRNA capped with m₂^7,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m₂^7,2′-OGpp_BH3pG D1 and m₂^7,2′-OGpp_BH3pG D2 favorable for anticancer immunization.     

Analysis of <Emphasis Type="Italic">COQ2</Emphasis>gene in multiple system atrophy

Background

Loss of function COQ2 mutations results in primary CoQ10 deficiency. Recently, recessive mutations of the COQ2 gene have been identified in two unrelated Japanese families with multiple system atrophy (MSA). It has also been proposed that specific heterozygous variants in the COQ2 gene may confer susceptibility to sporadic MSA. To assess the frequency of COQ2 variants in patients with MSA, we sequenced the entire coding region and investigated all exonic copy number variants of the COQ2 gene in 97 pathologically-confirmed and 58 clinically-diagnosed MSA patients from the United States.

Results

We did not find any homozygous or compound heterozygous pathogenic COQ2 mutations including deletion or multiplication within our series of MSA patients. In two patients, we identified two heterozygous COQ2 variants (p.S54W and c.403?+?10G?>?T) of unknown significance, which were not observed in 360 control subjects. We also identified one heterozygous carrier of a known loss of function p.S146N substitution in a severe MSA-C pathologically-confirmed patient.

Conclusions

The COQ2 p.S146N substitution has been previously reported as a pathogenic mutation in primary CoQ10 deficiency (including infantile multisystem disorder) in a recessive manner. This variant is the third primary CoQ10 deficiency mutation observed in an MSA case (p.R387X and p.R197H). Therefore it is possible that in the heterozygous state it may increase susceptibility to MSA. Further studies, including reassessing family history in patients of primary CoQ10 deficiency for the possible occurrence of MSA, are now warranted to resolve the role of COQ2 variation in MSA.

     

Reliable Single Cell Array CGH for Clinical Samples

Background

Disseminated cancer cells (DCCs) and circulating tumor cells (CTCs) are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape.

Methodology/Principal Findings

Using the Ampli1™ whole genome amplification (WGA) technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points.

Conclusions/Significance

The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations.     

The Effects of Antihypertensive Drugs on Chromium Status, Glucose Metabolism, and Antioxidant and Inflammatory Indices in Spontaneously Hypertensive Rats

The long-term use of hypotensive drugs may cause side effects, including impaired glucose metabolism and mineral status. This study tested the hypothesis that some hypotensive drugs can affect tissular chromium levels and indices of glucose metabolic and antioxidant potential in rats. The experiment was performed on 40 male spontaneously hypertensive rats (SHRs), which were assigned to five groups: control (C), with perindopril (PR), with metoprolol (MT), with indapamide (ID), and with amlodipine (AM). All rats were provided ad libitum standard diet (with or without drugs) and distilled water for 45 days. Glucose and insulin levels, along with total antioxidant status (TAS) and concentrations of TNF-alpha and C-reactive protein, were assayed in serum. Chromium concentrations in the liver and kidney were determined using the flame atomic absorption spectrometry method. Detailed statistical analysis was performed using Statistica for Windows 10.0 (StatSoft, Poland). One-way analysis of variance (ANOVA), followed by a post hoc Tukey test, was used to compare the data between groups. Treatment with indapamide and amlodipine resulted in significantly higher chromium concentrations in the liver and kidney (AM) of the rats, compared with the control group. A markedly higher concentration of glucose was found in the ID group. Treatment with amlodipine significantly increased TAS levels in serum and decreased TNF-alpha concentration in serum of the rats. A significant positive correlation between chromium concentration in tissues and serum TAS level was observed, as was a significant negative correlation between chromium concentration in the kidneys, and TNF-alpha and glucose levels in serum. In conclusion, the administration of amlodipine may lead to an increase in chromium accumulation in the internal organs, which is associated with increased antioxidant status and suppression of the inflammatory response of cells in SHRs.     

Changes of weight and size of European bison testes during postnatal development

The material for this study was obtained from culled European bison (Linnaeus, 1758) originating from both captive breeding and free-ranging populations in Białowieża and Borecka Forests (north-east Poland), and was mostly collected from November until April. Mass difference between the right and left testis of the same individual up to 30% was considered to be physiological asymmetry, which proved typical of the species. Hypoplasia or atrophy of the testes exceeding 30% in size, were found in 37 males (13.6% of the examined animals;n=272). Cryptorchidism was recorded in 20 bison culled in Białowieża Forest at the age over 1 year (10.6% of individuals at the same age;n=188). We registered the weight of testes of 259 males aged 2 months to 20 years. In the examined males, no distinct predominance in size and weight between the right and left testes was observed. The weight of testes was significantly correlated with age (r=0.85,p < 0.0001) and body weight (r=0.93,p < 0.0001). The heaviest testicles belonged to older and sexually mature bulls aged 6–12 years (mean 216 g) and to old ones over 13 years (mean 242 g). Two testis measurements (length and width) were taken from 136 culled males, while two additional measurements (total length with the cauda of epididymis and circumference) from 65 males. The length and width of the testes were moderately correlated with age (r=0.31 andr=0.20,p < 0.01) and highly significantly correlated with testis (r=0.79,p < 0.001) and body weight (r=0.92,p < 0.001).     

Effect of fenitrothion on Spodoptera exigua larval development and ultrastructure of follicle cells

The effect of four sublethal concentrations (from 0.01165 to 0.3025 μg per insect, responding to LC₁₀/10, LC₁₀, LC₃₀ and LC₅₀) of the organophosphorus insecticide, fenitrothion, on Spodoptera exigua (Lepidoptera, Noctuidae) larval development and ultrastructure of follicular cells of adult female moths was tested. The most prominent malformations were: breaks in the cuticle, increased mortality during larval/pupal molting, alterations of nuclear envelope, changed heterochromatin pattern and abnormalities in mitochondrial ultrastructure. Some of the alterations are dose-dependent, but the other ones, like organization of rough endoplasmic reticulum (RER), show non-linear response: cells exposed to lower concentrations (LC₁₀/10 and LC₁₀) of the insecticide possessed clusters of accumulated RER, which were absent within the cells exposed to higher ones (LC₃₀ and LC₅₀). Formation of the eggshell was also markedly postponed within the groups exposed to lower doses. These findings prove that even minute amounts of xenobiotics may cause significant changes within exposed populations.     

U7 snRNA mutations in Drosophila block histone pre-mRNA processing and disrupt oogenesis

Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem-loop structure generated by an endonucleolytic cleavage involving the U7 snRNP, which interacts with histone pre-mRNAs through base-pairing between U7 snRNA and a purine-rich sequence in the pre-mRNA located downstream of the cleavage site. Here we generate null mutations of the single Drosophila U7 gene and demonstrate that U7 snRNA is required in vivo for processing all replication-associated histone pre-mRNAs. Mutation of U7 results in the production of poly A+ histone mRNA in both proliferating and endocycling cells because of read-through to cryptic polyadenylation sites found downstream of each Drosophila histone gene. A similar molecular phenotype also results from mutation of Slbp, which encodes the protein that binds the histone mRNA 3' stem-loop. U7 null mutants develop into sterile males and females, and these females display defects during oogenesis similar to germ line clones of Slbp null cells. In contrast to U7 mutants, Slbp null mutations cause lethality. This may reflect a later onset of the histone pre-mRNA processing defect in U7 mutants compared to Slbp mutants, due to maternal stores of U7 snRNA. A double mutant combination of a viable, hypomorphic Slbp allele and a viable U7 null allele is lethal, and these double mutants express polyadenylated histone mRNAs earlier in development than either single mutant. These data suggest that SLBP and U7 snRNP cooperate in the production of histone mRNA in vivo, and that disruption of histone pre-mRNA processing is detrimental to development.