Interaction patterns of methoprene-tolerant and germ cell-expressed Drosophila JH receptors suggest significant differences in their functioning.

Methoprene-tolerant (Met) and germ cell-expressed (Gce) proteins were shown to be juvenile hormone (JH) receptors of Drosophila melanogaster with partially redundant functions. We raised the question of where the functional differentiation of paralogs comes from. Therefore, we tested Met and Gce interaction patterns with selected partners. In this study, we showed the ability of Gce and its C-terminus (GceC) to interact with 14-3-3 in the absence of JH. In contrast, Met or Met C-terminus (MetC) interactions with 14-3-3 were not observed. We also performed a detailed structural analysis of Met/Gce interactions with the nuclear receptor fushi tarazu factor-1 (Ftz-F1) ligand-binding domain. We showed that GceC comprising an Ftz-F1-binding site and full-length protein interacts with Ftz-F1. In contrast to Gce, only MetC (not full-length Met) can interact with Ftz-F1 in the absence of JH. We propose that the described differences result from the distinct tertiary structure and accessibility of binding sites in the full-length Met/Gce. Moreover, we hypothesize that each interacting partner can force disordered MetC and GceC to change the structure in a partner-specific manner. The observed interactions seem to determine the subcellular localization of Met/Gce by forcing their translocation between the nucleus and the cytoplasm, which may affect the activity of the proteins. The presented differences between Met and Gce can be crucial for their functional differentiation during D. melanogaster development and indicate Gce as a more universal and more active paralog. It is consistent with the theory indicating gce as an ancestor gene.

Molecular detection of Toxoplasma gondii in natural surface water bodies in Poland.

The aim of this study was molecular detection of Toxoplasma gondii in 36 natural surface water bodies in Poland, including preliminary genotype identification and determination of co-occurrence of this parasite with other protozoa that have been detected in previous studies. The examined DNA samples were obtained before to detect Cryptosporidium, Giardia and free-living amoebae. Nested polymerase chain reaction (PCR) based on B1 gene and sequencing was performed for both confirmation of parasite presence in water and genotype identification. T. gondii DNA was detected in 19.4% (7/36) water bodies, while in the case of other studies, T. gondii prevalence ranged between 0% and over 56%. These differences may be caused by natural variations in T. gondii occurrence as well as different sample volumes and methods of sample processing or DNA isolation and detection. Two cases of double contamination were reported: T. gondii with Cryptosporidium parvum and T. gondii with potentially pathogenic Acanthamoeba T4 genotype, thus there is a possibility of mixed infection in humans after occasional contact with water. Obtained T. gondii strains were genetically identical or closely similar (99.8%) to RH strain representing genotype I, however, further examinations involving more loci will be conducted to identify the genotype.

Assessment of molecular methods as a tool for detecting pathogenic protozoa isolated from water bodies.

Several species belong to the Cryptosporidium and Giardia genus, the main parasitic protozoa occurring in water, but only some of them are infectious to humans. We investigated the occurrence of Cryptosporidium and Giardia and identified their species in the water samples collected from natural water bodies in north-western Poland. A total of 600 samples from water bodies used for bathing, sewage discharge, as drinking water sources and watering places for animals were screened. The samples were collected during a 3-year period in each of the four seasons and filtered using Filta-Max (IDEXX Laboratories, USA). Genomic DNA was extracted from all samples and used as a target sequence for polymerase chain reaction (PCR) and TaqMan real-time PCR, as well as for reverse line blotting (RLB) methods. PCR methods seem to be more sensitive to detect Giardia and Cryptosporidium DNA in water samples than RLB methods. All PCR products were sequenced and three were identified as C. parvum and four as G. intestinalis. The overall prevalence of C. parvum (0.5%) and G. intestinalis (0.6%) in the samples suggests that the risk of Cryptosporidium and Giardia infections in north-western Poland is minimal.

Recovery of DNA of Giardia intestinalis cysts from surface water concentrates measured with PCR and real time PCR.

The most important restriction for the detection in water samples is the low concentration of Giardia intestinalis cysts, additional difficulty is the presence of PCR inhibitors. We have carried out trials in order to assess the sensitivity of semi-nested PCR and TaqMan real time PCR on the basis of DNA extracted from G. intestinalis cysts coming from spiked environmental and distilled water samples, filtrated with the use of Filta-Max® equipment (1623 Method). Removal of inhibitors was carried out with addition of BSA in different concentrations. During the filtration and concentration of water samples, losses of cysts have been recorded. Moreover, addition of BSA to the PCR and real time PCR mix increases the sensitivity of reaction. The optimal concentration of BSA for semi­nested PCR was 15 and 20 ng/µl, whereas for real time PCR 5 ng/µl.

Comparison of efficiency of various DNA extraction methods from cysts of Giardia intestinalis measured by PCR and TaqMan real time PCR.

The aim of the presented study was to work out an effective method of extraction of DNA from Giardia intestinalis cysts as well as a sensitive and specific method for detection of DNA of this protozoan using a polymerase chain reaction (PCR). Twelve protocols for DNA extraction have been compared. Purification and extraction of DNA were preceded by additional actions in order to destroy the cysts' wall. The highest effectiveness of DNA extraction was obtained in case of alternating application of freezing the samples in liquid nitrogen and their incubation in water bath in the temperature of 100 degrees C, and then the extraction with the QIAamp DNA Tissue Mini Kit (QIAGEN)--T kit--with an all night long incubation with proteinase K in 56 degrees C. Effectiveness of DNA extraction with the use of each kit after extraction with each treatment was measured by nested PCR product of beta-giardin gene fragment and C(T) values of real time PCR of the SSU rRNA gene of G. intestinalis. The detection limit, defined as the lowest number detected in 100% cases, was 100 cysts per 200 microl when effectiveness was evaluated with nested PCR and 50 oocysts with real time PCR after extraction DNA with T kit. Results of our comparative studies have shown that all stages preceding the molecular detection of G. intestinalis DNA are equally important, and materially influence on the final effect and this version of method seems to be very useful for the sensitive detection of DNA of G. intestinalis.

Interleukin-18 promoter polymorphism in patients with atopic asthma.

Allergic asthma is a chronic inflammatory disease in which interleukin-18 (IL-18) plays an important role. However, there are controversial reports on IL-18 promoter polymorphism as an independent marker of asthma susceptibility. The aim of the present study was to examine the IL-18 promoter polymorphism in patients with allergic asthma. Two hundred and thirty-one patients with allergic asthma from a Polish population diagnosed according to the National Heart, Lung, and Blood Institute (NHLBI)/WHO guidelines were examined. An allele-specific polymerase chain reaction was used to analyse polymorphisms at positions -137 and -607 in the promoter region of the IL-18 gene. Neither in the -607 C>A nor in the -137 G>C promoter polymorphism were there any differences observed between the total group of asthmatic patients and the controls in the frequencies of genotypes, alleles, diplotypes or haplotypes. In patients with severe asthma, the -607 CC and -137 GG genotypes were observed significantly more frequently (P = 0.03 for both), whereas in patients with mild and moderate asthma, the -137 CC genotype was more prevalent than in the former group. The strongest difference between mild to moderate and severe asthma was observed in -137 allele frequencies (P = 0.006). The results of the present study suggest that the -137 G allele and the C-G/C-G diplotype seem to be involved in the pathogenesis of the severe form of asthma.

Mitochondrial diversity of early-branching metazoa is revealed by the complete mt genome of a haplosclerid demosponge.

The first mitochondrial (mt) genomes of demosponges have recently been sequenced and appear to be markedly different from published eumetazoan mt genomes. Here we show that the mt genome of the haplosclerid demosponge Amphimedon queenslandica has features that it shares with both demosponges and eumetazoans. Although the A. queenslandica mt genome has typical demosponge features, including size, long noncoding regions, and bacterialike rRNA genes, it lacks atp9, which is found in the other demosponges sequenced to date. We found strong evidence of a recent transposon-mediated transfer of atp9 to the nuclear genome. In addition, A. queenslandica bears an incomplete tRNA set, unusual amino acid deletion patterns, and a putative control region. Furthermore, the arrangement of mt rRNA genes differs from that of other demosponges. These genes evolve at significantly higher rates than observed in other demosponges, similar to previously observed nuclear rRNA gene rates in other haplosclerid demosponges.

Crystal structures of beta-galactosidase from Penicillium sp. and its complex with galactose.

Beta-galactosidases catalyze the hydrolysis of beta(1-3) and beta(1-4) galactosyl bonds in oligosaccharides as well as the inverse reaction of enzymatic condensation and transglycosylation. Here we report the crystallographic structures of Penicillium sp. beta-galactosidase and its complex with galactose solved by the SIRAS quick cryo-soaking technique at 1.90 A and 2.10 A resolution, respectively. The amino acid sequence of this 120 kDa protein was first assigned putatively on the basis of inspection of the experimental electron density maps and then determined by nucleotide sequence analysis. Primary structure alignments reveal that Penicillium sp. beta-galactosidase belongs to family 35 of glycosyl hydrolases (GHF-35). This model is the first 3D structure for a member of GHF-35. Five distinct domains which comprise the structure are assembled in a way previously unobserved for beta-galactosidases. Superposition of this complex with other beta-galactosidase complexes from several hydrolase families allowed the identification of residue Glu200 as the proton donor and residue Glu299 as the nucleophile involved in catalysis. Penicillium sp. beta-galactosidase is a glycoprotein containing seven N-linked oligosaccharide chains and is the only structure of a glycosylated beta-galactosidase described to date.

FGFs control the patterning of the inner ear but are not able to induce the full ear program.

FGF2 or FGF8 applied ectopically, close to the developing otic placode enhances transcription of a subset of ear marker genes such as Nkx5-1, SOHo1 and Pax2. Other ear expressed genes (Dlx5 and BMP4) are not up-regulated by FGFs. Ectopic FGFs lead to an increase in size of the vestibulo-cochlear ganglion. This phenotypic change is due to an increased recruitment of epithelial cells to the neuronal fate rather than to an enhanced proliferation. We also observed an induction of additional, vesicle-like structures upon ectopic FGF treatment, but this induction never led to enrolment of a full ear program. We further demonstrate that FGF8 is expressed in two separate, short waves, first at the otic placode stage and later at the vesicle stage. Both activities correspond to critical morphogenetic events in ear development. We propose that FGF8 is an important regulator of otocyst patterning.

Five Nkx5 genes show differential expression patterns in anlagen of sensory organs in medaka: insight into the evolution of the gene family.

We report the identification and characterisation of five different Nkx5-related genes in medaka fish (Oryzias latipes). They constitute homologues of genes previously isolated in higher vertebrates, Nkx5--1, Nkx5--2, Hmx1/Nkx5--3 and SOHo-1, and were named accordingly: OlNkx5--1.1, OlNkx5--2, OlNkx5--3 and OlSOHo. For the Nkx5--1 gene a new, second homologue, OlNkx5--1.2, was isolated. In medaka, Nkx5 and SOHo genes are differentially expressed in three developing sensory organs: eye, ear and lateral line and later in defined brain regions. Phylogenetic analyses of the entire Nkx5 family revealed that four paralogous Nkx5 groups, Nkx5--1, Nkx5--2, Hmx1/Nkx5--3/GH6 and SOHo, are present in vertebrates. Only some of the Nkx5 family members have been identified in singular vertebrate species so far. Here we present, for the first time, the isolation of representatives of each Nkx5 subgroup in one species, the medaka fish. Based on similarities in sequence and expression patterns, and genomic organisation we propose a model of the evolutionary history of the Nkx5 family. The model predicts that the four vertebrate Nkx5 genes arose by a tandem duplication, followed by chromosomal duplication. The two Nkx5--1 genes identified so far exclusively in medaka most probably result from an additional genome duplication in the fish lineage.

Inner ear and lateral line expression of a zebrafish Nkx5-1 gene and its downregulation in the ears of FGF8 mutant, ace.

An orthologue of the mouse homeobox gene Nkx5-1 was cloned and characterized in the zebrafish. As in the mouse and chick, the zebrafish Nkx5-1 gene is expressed in the ear placode and vesicle and in cells forming the vestibulo-acoustic ganglion. In addition, a novel expression domain, the lateral line, appears in the zebrafish, supporting a common precursor hypothesis for these two organs. In the FGF8 zebrafish mutant ace, expression of Nkx5-1 in the otic structures is diminished. The most significant reduction of zfNkx5-1 expression was observed in cells of the vestibulo-acoustic ganglion.

Immunogold labelling of PCNA and Ki-67 antigen at the ultrastructural level in laryngeal squamous cell carcinoma and its correlation with lymph node metastasis and histological grade.

Streptavidin-gold was used for the immunolocalization of PCNA and Ki-67 antigen at the ultrastructural level with a postembedding technique in biopsies of 15 patients with laryngeal squamous cell carcinoma. Positive immunoelectron staining was obtained in 9 cases for PCNA (60%) and in 8 cases for Ki-67 (53%). PCNA was predominantly found in heterochromatin of the nucleus of laryngeal carcinoma cells in a granular pattern. Positivity for PCNA was not found in nucleoli. In 4 cases, positive staining was observed both in nucleus and cytoplasm. In the cytoplasm, it was found to be present on the endoplasmic reticulum and on ribosomes throughout the cytoplasm. Ki-67 antigen was localized in the nucleus where it was associated with heterochromatin and euchromatin. It was also observed in nucleoli in all cases. Cytoplasmic localization of Ki-67 antigen was similar to that of PCNA. All 8 cases that were positive for Ki-67 were also positive for PCNA. Control incubations did not result in labelling with steptavidin-gold particles for both antigens. A significant correlation between PCNA and Ki-67 expression in association with pathological characteristics such as nodal status and histological grade was not found. Our data indicate that Ki-67 antigen staining correlates with PCNA labelling, whereas a relationship between proliferation markers and tumour progression was not found.

[Biotransformation of phenazone and sulfadimidine as markers of liver metabolism of drug and xenobiotic oxidation and acetylation in women with breast cancer]. / Biotransformacja fenazou i sulfadimidyny jako wskazników watrobowego metabolizmu leków i ksenobiotyków droga utleniania i acetylacji u kobiet chorych na raka sutka.

The aim of our study was to determine phenazone pharmacokinetics as an index of hepatic microsomal enzymes activity and acetylation phenotype in women with breast cancer. Phenazone test was made in 41 women with breast cancer and in 25 healthy women as a control group. Acetylation phenotype was measured in 40 women with breast cancer and in 25 healthy women as a control group. The plasma concentration of phenazone was estimated by the spectrophotometric method of Brodie and associates. Acetylation phenotype was determined by the Bratton-Marshall method in Varley's modification. The mean phenazone half-life time (t0,5), shortened significantly and the mean elimination rate constant (K) increased in women compared with a group of healthy persons. Mean clearance rate was increased in women with breast cancer too, compared with a group of healthy women. Acceleration of phenazone elimination may suggest that in patients with breast cancer elimination of the other drugs metabolized by the pathway similar to phenazone also may be changed. This should be considered in selection of their dosage. We have observed the predominance of rapid acetylators in women with breast cancer comparing with healthy persons. This difference was not statistically significant. Our results of acetylation phenotype in women with breast cancer suggests that rapid acetylation may be a factor of susceptibility to breast cancer.

Microtubules are responsible for the asymmetrical distribution of organelles in the oocytes of the hymenopteran, Chrysis ignita.

Previous morphological investigations have shown that the distribution of cellular organelles and reserve materials in hymenopteran oocytes is uneven and often gradient-like. It has been suggested that the microtubular cytoskeleton is responsible for creating and/or maintaining the distribution gradients. To test this hypothesis, adult females of the hymenopteran, Chrysis ignita were treated with a microtubule-assemble inhibitor, colchicine. The experiment resulted in a number of abnormalities observed in the egg chambers: the oocytes were devoid of organelle-free periplasm and oosome; cellular organelles and reserve materials were distributed randomly within the oocyte; nurse cells organization was significantly changed. These observations have confirmed a key role of microtubules in forming and/or maintaining the asymmetry of ooplasm in Chrysis ignita.

[The time necessary for establishing a morphological diagnosis in patients with inoperable lung cancer]. / O sroke, neobkhodimom dlia ustanovleniia morfologicheskogo diagnoza u bol'nykh s neoperabel'nym rakom legkogo.

The authors analyze the time period necessary for morphological diagnosis in 100 consecutive patients with inoperable lung cancer admitted for treatment to the Department of pulmonology and tuberculosis of the Szczecin Medical Academy from 1989 through 1990. The following methods were used: cytological examination of sputum, bronchoscopy, fiber bronchoscopy, transthoracic biopsy by a thin needle, biopsy of the metastatic focus and cytological examination of the pleural cavity fluid. The causes of inoperability were: tumour histobiology variants (anaplastic small-cell cancer), cardiovascular failure and technical in operability. The diagnosis was established in all patients averagely within 16 days of hospital stay. More frequently the diagnostic material was obtained by means of bronchoscopy, then cytological examination of the sputum and transthoracic biopsy. In some patients without symptoms it is expedient to carry out diagnostic procedures in outpatient condition which is of economic importance.

Phosphorus fractions of crayfish haemolymph, serum and haemocyanin.

Different amounts of haemolymph proteins participate in clot formation in Astacus leptodactylus and Orconectes limosus (20% and 10%, respectively), although in both species the content of proteins in haemolymph is similar. In both species the content of total phosphorus in the clot was similar (0.33-0.49%, w/w) but it was about 6 times lower in serum of O. limosus than in serum of A. leptodactylus (2.2 and 14.8 mg/100 ml, respectively). An even greater difference in phosphorus content was found in the protein precipitated from serum. In both species lipid phosphorus was predominant (77% of total haemolymph phosphorus in A. leptodactylus and 52% in O. limosus). Phospholipids were found mainly in serum. Only traces of phosphorus (0.005%) and small amounts of fatty acids were found in purified haemocyanin preparations.

Effect of starvation on the content of free amino acids in plasma of different breeds of hen.

'Leghorn', 'Cornish' and 'White Rock' hens were subjected to starvation. Free amino acids were determined in blood samples taken after 48, 72 and 96 h of starvation. A progressive decrease in concentration of the majority of amino acids was found. Changes in amino acid concentrations during starvation were dependent on the breed of hen.