Detection of substance P and its mRNA in human blast cells in childhood lymphoblastic leukaemia using immunocytochemistry and in situ hybridisation

The study focused on determining the expression of substance P (SP) in neoplastic cells of childhood acute lymphoblastic leukaemia (ALL) at the levels of its mRNA and the protein production. The study group comprised 44 children treated for ALL in the Department of Paediatric Haematology and Oncology, Karol Marcinkowski University of Medical Sciences in Poznań, in the years 1999-2001. Bone marrow smears were obtained by needle biopsy. Expression of SP was examined by immunocytochemistry with specific antibody against human SP and by in situ hybridisation with anti-mRNA 5'-biotinylated probe. The results of the study demonstrated that SP could be detected in the cytoplasm of lymphoblasts (mean percentage of 81.8% for immunocytochemical and 84.5% for in situ hybridisation technique) in leukaemias of the common and T-cell types. SP was absent from blasts in B-cell leukaemia and from normal haematopoietic, cells in children of the control group. The results show that lymphoblasts of common and T-cell origin acquire the capability to synthesise SP after their neoplastic transformation in childhood acute leukaemia. SP may be involved in auto- and paracrine mechanisms capable of inducing hyperplasia of the neoplastic cells.     

Genomic imprinting controls matrix attachment regions in the Igf2 gene

Genomic imprinting at the Igf2/H19 locus originates from allele-specific DNA methylation, which modifies the affinity of some proteins for their target sequences. Here, we show that AT-rich DNA sequences located in the vicinity of previously characterized differentially methylated regions (DMRs) of the imprinted Igf2 gene are conserved between mouse and human. These sequences have all the characteristics of matrix attachment regions (MARs), which are known as versatile regulatory elements involved in chromatin structure and gene expression. Combining allele-specific nuclear matrix binding assays and real-time PCR quantification, we show that retention of two of these Igf2 MARs (MAR0 and MAR2) in the nuclear matrix fraction depends on the tissue and is specific to the paternal allele. Furthermore, on this allele, the Igf2 MAR2 is functionally linked to the neighboring DMR2 while, on the maternal allele, it is controlled by the imprinting-control region. Our work clearly demonstrates that genomic imprinting controls matrix attachment regions in the Igf2 gene.     

Quantitative analysis of flavonoids in the flowers and leaves of Ficaria verna Huds

The quantitative determination of flavonoid compounds in flowers and leaves of Ficaria verna Huds. (Ranunculaceae) was carried out in different growing seasons of the plant, using Christ-Müller's method (Polish Pharmacopoeia, 1999) and high performance liquid chromatography (HPLC) analysis after acid hydrolysis. The flavonoid content was much higher in flowers than in leaves.     

On the NMR structure determination of a 44n RNA pseudoknot: Assignment strategies and derivation of torsion angle restraints

The complete T- and pseudoknotted acceptor arm of the tRNA-like structure of turnip yellow mosaic virus (TYMV) genomic RNA has been studied by NMR spectroscopy. Resonance assignment and the gathering of restraints of the 44-mer are impeded by spectral complexity as well as by line broadening. The latter is caused by local dynamical effects in the pseudoknot domain in the molecule. These specific problems could be solved by using different field strengths and selectively ¹³C/¹⁵ labeled samples. Experiments for assigning the sugar spin systems were adjusted to satisfy the requirements of this system. Furthermore, the quality of the structure could be improved by determining the backbone torsion angles , and , using new approaches that were tailored for use in large RNA molecules.     

High Viral Loads of Epstein-Barr Virus DNA in Peripheral Blood of Patients with Chronic Lymphocytic Leukemia Associated with Unfavorable Prognosis

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus that infects more than 90% of the world population. The potential involvement of EBV in the clinical course of chronic lymphocytic leukemia (CLL) remains unexplained. The aim of this study was to determine whether EBV-DNA load in the peripheral blood mononuclear cells (PBMCs) of CLL patients may influence heterogeneity in the course of the disease. The study included peripheral blood samples from 115 previously untreated patients with CLL (54 women and 61 men) and 40 healthy controls (16 women and 24 men). We analyzed the association between the EBV-DNA load in PBMCs and the stage of the disease, adverse prognostic factors, and clinical outcome. Detectable numbers of EBV-DNA copies in PBMCs were found in 62 out of 115 CLL patients (53.91%). The EBV-DNA copy number/μg DNA was significantly higher in patients who required early implementation of treatment, presented with lymphocyte count doubling time <12 months, displayed CD38-positive or ZAP-70-positive phenotype, and with the del(11q22.3) cytogenetic abnormality. Furthermore, the EBV-DNA copy number/μg DNA showed significant positive correlation with the concentrations of lactate dehydrogenase (LDH) and beta-2-microglobulin. We have shown that in CLL patients, higher EBV-DNA copy number predicted shorter survival and shorter time to disease progression, and it was associated with other established unfavorable prognostic factors. This suggests that EBV may negatively affect the outcome of CLL.     

Changes in Ovaries and Uterus after Aglepristone Administration in the Third Week of Luteal Phase of Non-Pregnant Bitches

Objective

The mechanism of aglepristone action in the placentation time in the bitch remains unclear. The aim of this study was to describe the mechanism by which aglepristone influences ovaries and uterus and to measure the levels of steroid sex hormones in non-pregnant bitches.

Materials and Methods

Fourteen bitches assigned to a study (n=9) and control (n=5) group were given aglepristone and saline solution, respectively, on the 19th and 20th day after LH peak. On the 26th day after LH peak an ovariohysterectomy was performed. Blood samples were screened for estradiol and progesterone concentrations. Ovaries and uterine horns and bodies were isolated for histological and morphometrical diagnosis and immunohistochemistry analysis of α-estrogen and progesterone receptor expression.

Results

A decrease of progesterone (p<0.01) and no differences in total estrogen level in the study group were observed. There were no significant differences either in the histomorphometry or α-estrogen and progesterone receptors expression in ovaries. Increase in expression of progesterone receptors in endometrium without surface epithelium of horns (p<0.05), endometrial surface epithelium (p<0.05), myometrium of uterine body (p<0.01) and estrogen receptors in endometrium without surface epithelium of horns (p<0.05) was observed. Elevated estrogen receptors probably increased sensitivity of tissues to estrogens in the bloodstream and led to notable inflammation, haemorrhages, and hyperplasia in endometrium with mononuclear immune cell infiltration. The myometrium of horns and endometrium of uterine body of study bitches were significantly thicker than in the control group (p<0.05 and p<0.01). Furthermore myometrium of uterine body was thicker than myometrium of horns (p<0.001) and expression of progesterone receptors was higher in uterine body (p<0.01). No differences were observed between endometrium of horns and body within groups.

Conclusion

To the knowledge of the authors this is the first study, which describes the inflammatory effect developing in uterus in response to aglepristone administration, and attempts to elucidate its mechanisms.     

Anti-HmuY Antibodies Specifically Recognize Porphyromonas gingivalis HmuY Protein but Not Homologous Proteins in Other Periodontopathogens

Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.     

The Effect of Different Types of Musculoskeletal Injuries on Blood Concentration of Serum Amyloid A in Thoroughbred Racehorses

BackgroundTraining-induced muscle, skeletal and joint trauma may result in acute phase response reflected by the changes in the blood concentration of serum amyloid A (SAA) in racehorses. It remains yet unclear if such systemic reaction could be triggered by sport injuries and what is the impact of different types of musculoskeletal trauma on SAA concentrations in racehorses. This study aimed to determine changes in the SAA blood concentration in racehorses with different types of injuries of musculoskeletal system.ResultsMean SAA concentration within the first 4 days of the injury of muscle and tendon was significantly higher than in bone fractures, dorsal metacarpal disease, joint trauma or in the healthy horses (p<0,001). There were no significant differences between the other groups.ConclusionsStrain injuries of muscle and tendons can cause a moderate increase in SAA blood concentration in racehorses, reflecting the occurrence of the acute phase response. Similar reaction is not observed in the stress-related bone injuries.