Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.     

Unusual growth pattern in the Frasnian alveolitids (Tabulata) from the Holy Cross Mountains (Poland)

Abstract: Growth periodicity is a phenomenon occurring in fossil and modern corals. The most apparent feature is growth banding, and environmental changes are broadly accepted as controls on this phenomenon. If environment controls the growth, then all corallites within a colony should repeat the same growth pattern, as individuals are clones and must have shared the same environment. A study on several species of Alveolitidae (Anthozoa, Tabulata) from the Late Devonian (Early Frasnian) of the Holy Cross Mountains (Poland) shows that the growth pattern varies between neighbouring individuals within the same corallum. This contradicts observations of closely related Favositida as demonstrated on Pachyfavosites sp. from the Givetian of Avesnois, France, where neighbouring individuals repeat the same pattern. Therefore, environmental control on growth rhythm in Alveolitidae can be excluded; the causes of differences between individuals remain unknown.     

Confocal laser scanning microscopy, a new in vivo diagnostic tool for schistosomiasis

Background

The gold standard for the diagnosis of schistosomiasis is the detection of the parasite''s characteristic eggs in urine, stool, or rectal and bladder biopsy specimens. Direct detection of eggs is difficult and not always possible in patients with low egg-shedding rates. Confocal laser scanning microscopy (CLSM) permits non-invasive cell imaging in vivo and is an established way of obtaining high-resolution images and 3-dimensional reconstructions. Recently, CLSM was shown to be a suitable method to visualize Schistosoma mansoni eggs within the mucosa of dissected mouse gut. In this case, we evaluated the suitability of CLSM to detect eggs of Schistosoma haematobium in a patient with urinary schistosomiasis and low egg-shedding rates.

Methodology/Principal Findings

The confocal laser scanning microscope used in this study was based on a scanning laser system for imaging the retina of a living eye, the Heidelberg Retina Tomograph II, in combination with a lens system (image modality). Standard light cystoscopy was performed using a rigid cystoscope under general anaesthesia. The CLSM endoscope was then passed through the working channel of the rigid cystoscope. The mucosal tissue of the bladder was scanned using CLSM. Schistoma haematobium eggs appeared as bright structures, with the characteristic egg shape and typical terminal spine.

Conclusion/Significance

We were able to detect schistosomal eggs in the urothelium of a patient with urinary schistosomiasis. Thus, CLSM may be a suitable tool for the diagnosis of schistosomiasis in humans, especially in cases where standard diagnostic tools are not suitable.     

Analysis of O6-methylguanine-DNA methyltransferase methylation status in sporadic colon polyps

Background/aimThe aim of our study was to check how MGMT methylation status together with known factors influenced the risk of colon cancer development.Materials and methodsWe examined patients with colon polyps. Information concerning gender, age, lifestyle, diet, anthropometry and medical information, including cancer and family history of cancer, was analyzed. Polymorphism variety of MGMT gene was investigated in another study. Genetic analysis for MGMT methylation assessment was performed for polyp tissue samples from 143 patients.ResultsPositive methylation MGMT status was found in 55 patients. There was no correlation between gender and MGMT methylation status (p = 0.43). We did not find correlation between patients younger and older than 60 (p = 0.87). There was no correlation between smoking and MGMT methylation status (p = 0.36). We did not find correlation between BMI and MGMT methylation status (p = 0.86). We did not find correlation between MGMT methylation status and colon cancer in familial history (p = 0.45).ConclusionOur study showed no correlations between methylation status of MGMT polymorphisms and clinical features like age, gender, polyp localization, smoking status, or obesity. It has been shown previously that MGMT methylation status may show nonspecific methylation in colon polyps. Gene methylation status in adenoma tissues has also been associated by other authors with the adenoma's size, histology, and degree of atypia. In our study, we evaluated the gene methylation status in colon polyps and found no association with adenoma characteristics. The present study showed no correlation for MGMT methylation in polyps in different regions of colon.     

Thermal and resonance neutrons generated by various electron and X-ray therapeutic beams from medical linacs installed in polish oncological centers

BackgroundHigh-energy photon and electron therapeutic beams generated in medical linear accelerators can cause the electronuclear and photonuclear reactions in which neutrons with a broad energy spectrum are produced. A low-energy component of this neutron radiation induces simple capture reactions from which various radioisotopes originate and in which the radioactivity of a linac head and various objects in the treatment room appear.AimThe aim of this paper is to present the results of the thermal/resonance neutron fluence measurements during therapeutic beam emission and exemplary spectra of gamma radiation emitted by medical linac components activated in neutron reactions for four X-ray beams and for four electron beams generated by various manufacturers’ accelerators installed in typical concrete bunkers in Polish oncological centers.Materials and methodsThe measurements of neutron fluence were performed with the use of the induced activity method, whereas the spectra of gamma radiation from decays of the resulting radioisotopes were measured by means of a portable high-purity germanium detector set for field spectroscopy.ResultsThe fluence of thermal neutrons as well as resonance neutrons connected with the emission of a 20 MV X-ray beam is ～10⁶ neutrons/cm² per 1 Gy of a dose in water at a reference depth. It is about one order of magnitude greater than that for the 15 MV X-ray beams and about two orders of magnitude greater than for the 18–22 MeV electron beams regardless of the type of an accelerator.ConclusionThe thermal as well as resonance neutron fluence depends strongly on the type and the nominal potential of a therapeutic beam. It is greater for X-ray beams than for electrons. The accelerator accessories and other large objects should not be stored in a treatment room during high-energy therapeutic beam emission to avoid their activation caused by thermal and resonance neutrons. Half-lives of the radioisotopes originating from the simple capture reaction (n,γ) (from minutes to hours) are long enough to accumulate radioactivity of components of the accelerator head. The radiation emitted by induced radioisotopes causes the additional doses to staff operating the accelerators.     

Expression of ghrelin receptor (GHSR-1a) in rat epididymal spermatozoa and the effects of its activation

In this study we demonstrated the expression of the ghrelin receptor GHSR-1a in rat spermatids and epididymal spermatozoa, as well as some effects of ghrelin on the spermatozoa in vitro. For the demonstration of GHSR-1a the immunocytochemical, immunofluorescence and Western blotting techniques were applied using three different types of antibodies. The response of spermatozoa to ghrelin was tested in a series of in vitro experiments and their effects were evaluated using confocal microscopy and flow cytometry. GHSR-1a protein was found as expressed in the Golgi and acrosomes of spermatids and acrosome regions or the head cell membrane of epididymal spermatozoa. The GHSR-1a expression in spermatozoa was also confirmed by Western blot. No differences were found in percentage of spermatozoa showing annexin-V binding and expression of active form caspase-3 between control and ghrelin-treated spermatozoa. This result may indicate no pro-apoptotic effects of ghrelin neither at 10^?9 nor 10^?6 mol/L concentration. Ghrelin (10^?6 mol/L) increased free intracellular calcium ion concentration in the rat spermatozoa. Moreover, stimulation with 10^?6 mol/L ghrelin increased, while 10^?4 mol/L ghrelin decreased the number of spermatozoa showing progressive motility. In conclusion, the expression of the GHSR-1a receptor in spermatozoa, as well as ghrelin influences on sperm motility and intracellular calcium ion concentration suggest that such biological effects of ghrelin may be produced under in vivo conditions.     

CYP17 and CYP19 genetic variants are not associated with age at natural menopause in Polish women

The aim of the study was to investigate associations between two common polymorphisms of CYP17 and CYP19, encoding key enzymes of estrogen biosynthesis, and age at menopause in Polish women. One hundred fifty women after menopause (49.5 ± 3.8 years), with no previous history of hormone replacement therapy took part in the study. The genetic control group consisted of 150 newborns from the same population. We investigated an association between the age at menopause and the single nucleotide polymorphism T → C in the 5′ untranslated region (promoter) of the CYP17 gene (c.-34T>C; rs743572 – MspA1) or the number of tetranucleotide repeats [TTTA]_n (rs60271534) including deletion/insertion (D/I) of a 3 bp sequence in intron 4 of the CYP19 gene. CYP17 polymorphism was analyzed by PCR-RFLP and CYP19 by PCR and capillary electrophoresis. In the case of CYP17 polymorphism, 28.7% and 36.7% wild homozygous (TT), 50.7% and 46.0% heterozygous (TC), as well as 20.6% and 17.3% mutated homozygous (CC) types were identified in the subjects and controls, respectively. The frequency of mutated alleles (C) was 46.0% vs. 40.3% (p = 0.19). In the case of CYP19 polymorphism, 34.0% and 32.0% of homozygotes (1_1), 50.7% and 51.3% of heterozygotes (1_2), 15.3% and 16.7% of homozygotes (2_2) were identified in the subjects and controls, respectively. No association between the studied CYP17 or CYP19 polymorphisms and age at menopause was found in Polish women.     

Genetic distinctness of parthenogenetic forms of European Polydrusus weevils of the subgenus Scythodrusus

Abstract Among eight species of Polydrusus weevils which belong to subgenus Scythodrusus, at least two possess parthenogenetic forms: P. (S.) inustus and P. (S.) pilifer. Both of these species consist of dioecious populations in the Caspian area and of parthenogenetic populations in Eastern Europe (P. (S.) inustus), the Caucasus region (both species) and Middle Asia (P. (S.) pilifer). The origin of parthenogenesis in this subgenus is unresolved; however some data suggest that the parthenogenetic forms are of hybrid ancestry. The genetic distinctness of parthenogenetic Scythodrusus was assessed on the basis of COII, ITS2, EF1‐α and Wolbachiawsp, 16S ribosomal DNA, ftsZ and hcpA sequence comparisons. Both taxa turned out to be monophyletic for all markers, which is an evidence against hybridization of their dioecious ancestors. On the other hand, a high frequency of heterozygous P. (S.) inustus females suggests an origin resulting from hybridization between genetically distinct dioecious representatives of this species. Very similar strains of Wolbachia supergroup A were found in both species, indicating that they have been either inherited from a common ancestor or were transmitted between parthenogenetic Scythodrusus weevils and probably spread randomly across their ranges.     

Thiocyanate concentration in saliva of cystic fibrosis patients

Thiocyanates (SCN-) are ubiquitous in nature. There are indispensable part of host defense system that act as a substrate for lactoperoxidase (LPO). In our study we present initial data on SCN- concentration in saliva of CF patients in comparison to healthy non-smokers and healthy smokers. 5 ml of saliva was collected from each subject to a sterile tube and thiocyanate concentration was measured in each sample. The results of the measurements are presented on Fig. 1. Mean concentration of SCN- in saliva of CF patients was 0.031 +/- 0.0052 g/l, in healthy non-smokers 0.039 +/- 0.0048 g/l and in healthy smokers 0.048 +/- 0.0161 g/l. The differences between each group were statistically significant. Studies on larger group of patients and probably on different material (BALF or induced sputum) should present interesting data complementing the in vitro studies.     

Human cathepsin D

A literature survey was performed of human cathepsin D gene, cathepsin D biosynthesis, posttranslatory modifications, transport within the cell, substrate specificity and catalytic effect. Methods used to determine the activity and level of this proteinase as well as its role in the biochemistry and pathobiochemistry of cells, tissues and organs were considered.