Intercalary heterochromatin in Drosophila melanogaster polytene chromosomes and the problem of genetic silencing

The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.     

A nucleotide phosphatase activity in the nucleotide binding domain of an orphan resistance protein from rice

Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.     

Panel of SEREX-defined antigens for breast cancer autoantibodies profile detection

Content: Identification of panel of SEREX-defined antigens for breast cancer autoantibodies profile detection.

Objective: To create panel of antigens that can differentiate breast cancer patients and healthy individuals.

Methods: SEREX (serological analysis of cDNA expression libraries) method, ELISA (enzyme-linked immunosorbent assay), qPCR (quantitative polymerase chain reaction).

Results: In large-scale screening of 16 SEREX-antigens by sera of breast cancer patients and healthy donors, a combination of six antigens (RAD50, PARD3, SPP1, SAP30BP, NY-BR-62 and NY-CO-58) was identified, which can differentiate breast cancer patients and healthy donors with 70% sensitivity and 91% specificity. Elevated mRNA expression of SPP1 gene was revealed in breast tumors (2–7-fold) that correlated with SPP1 antigen immunoreactivity in autologous patients’ sera.

Conclusions: The new panel of six SEREX-antigens was proposed, which enables creation of serological assay for breast cancer diagnostics and/or prognosis.     

Bacteriolytic activity of human interleukin-2

In this paper we report the discovery of bacteriolytic activity of an immune system cytokine mediator, interleukin-2. Bacteriolytic activity of interleukin-2 was compared with a well-known bacteriolytic enzyme — chicken egg white lysozyme — by monitoring the lysis of the Gram-negative bacterium Escherichia coli, the Gram-positive coccus Micrococcus luteus, and the Gram-positive spore-forming bacillus Bacillus subtilis. It was found that interleukin-2 has greater specificity to the Gram-negative bacterium E. coli than does lysozyme. In contrast to chicken egg white lysozyme, interleukin-2 does not lyse the Gram-positive coccus M. luteus and the Gram-positive spore-forming bacillus B. subtilis. These results give a new understanding of the biological functions of interleukin-2, a regulatory protein that plays a role in oncological and infectious diseases.     

Phosphoproteome of resting human platelets

Beside their main physiological function in hemostasis, platelets are also highly involved in pathological processes, such as atherothrombosis and inflammation. During hemostasis, binding of adhesive substrates to tyrosine-kinase-linked adhesion receptors and/or soluble agonists to G-protein coupled receptors leads to a cascade of intracellular signaling processes based on substrate (de)phosphorylation. The same mechanisms are involved in platelet activation at sites of atherosclerotic plaque rupture, contributing to vessel occlusion and consequently to pathologic states, such as myocardial infarction, stroke, or peripheral artery disease. To gain a deeper insight into platelet function, we analyzed the phosphoproteome of resting platelets and identified 564 phosphorylation sites from more than 270 proteins, of which many have not been described in platelets before. Among those were several unknown potential protein kinase A (PKA) and protein kinase G (PKG) substrates. Because platelet inhibition is tightly regulated especially by PKA and PKG activity, these proteins may represent important new targets for cardiovascular research. Thus, our finding that GPIbalpha is phosphorylated at Ser603 in resting platelets may represent a novel mechanism for the regulation of one of the most important platelet receptor (GPIb-IX-V) mediated signaling pathways by PKA/PKG.     

Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals

Splice sites (SSs)—short nucleotide sequences flanking introns—are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints.     

Aurora Kinase A Is Not Involved in CPEB1 Phosphorylation and cyclin B1 mRNA Polyadenylation during Meiotic Maturation of Porcine Oocytes

Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.     

A robust method of determination of high concentrations of peptides and proteins

In this paper, we pioneer application of a unique method of protein determination by coloring peptide bonds for analysis of a variety of biomolecules with different grades of purity (e.g., oligopeptides, membrane, and glycol proteins). We demonstrated that the calibration curve for all studied molecules is universal and linear within 0.1 to 1.2 mg protein content range. The assay thus can be used to analyze peptides without preliminary dilutions and calibration in up to 1 g/ml solutions of peptides, which is crucial for many biotechnological processes, such as development of coatings, scaffolds, and biocompatible materials.     

A 10-year experience in the treatment of intraabdominal cerebrospinal fluid pseudocysts

The aim of this retrospective study was to assess a ten-year experience in the treatment of rare complications of ventriculoperitoneal shunting--intraabdominal cerebrospinal fluid pseudocysts. At this time there are no data about incidence, clinical course and treatment of these complications in Croatia. Cerebrospinal fluid (CSF) abdominal pseudocyst is an uncommon but important complication of ventriculoperitoneal shunts. Retrospective data were obtained from 5 children with abdominal CSF pseudocysts, treated between 1996 and 2007. The incidence of intraabdominal CSF pseudocysts in our study is 2.9%. All patients were girls ranged in age from 4 to 12 years old (mean 8.8 years). In most cases etiology of hydrocephalus was congenital, idiopathic. Abdominal pain and distension were the most frequent clinical finding (4/5). Although infection has been reported as responsible for pseudocyst formation, we did not found it in our series. Laparotomy with cyst wall excision and catheter replacement was performed in 2/5 cases, and only cyst fluid aspiration with catheter replacement in 3/5 cases. Recurrence of the abdominal cyst was observed in one girl who was in terminal stadium of anaplastic ependymoma. It is our opinion that only catheter replacement and cyst fluid evacuation, as one of the treatment modalities, may be successful, even in large CSF intraperitoneal pseudocysts.