Conserved DNA Motifs,Including the CENP-B Box-like,Are Possible Promoters of Satellite DNA Array Rearrangements in Nematodes

Tandemly arrayed non-coding sequences or satellite DNAs (satDNAs) are rapidly evolving segments of eukaryotic genomes, including the centromere, and may raise a genetic barrier that leads to speciation. However, determinants and mechanisms of satDNA sequence dynamics are only partially understood. Sequence analyses of a library of five satDNAs common to the root-knot nematodes Meloidogyne chitwoodi and M. fallax together with a satDNA, which is specific for M. chitwoodi only revealed low sequence identity (32–64%) among them. However, despite sequence differences, two conserved motifs were recovered. One of them turned out to be highly similar to the CENP-B box of human alpha satDNA, identical in 10–12 out of 17 nucleotides. In addition, organization of nematode satDNAs was comparable to that found in alpha satDNA of human and primates, characterized by monomers concurrently arranged in simple and higher-order repeat (HOR) arrays. In contrast to alpha satDNA, phylogenetic clustering of nematode satDNA monomers extracted either from simple or from HOR array indicated frequent shuffling between these two organizational forms. Comparison of homogeneous simple arrays and complex HORs composed of different satDNAs, enabled, for the first time, the identification of conserved motifs as obligatory components of monomer junctions. This observation highlights the role of short motifs in rearrangements, even among highly divergent sequences. Two mechanisms are proposed to be involved in this process, i.e., putative transposition-related cut-and-paste insertions and/or illegitimate recombination. Possibility for involvement of the nematode CENP-B box-like sequence in the transposition-related mechanism and together with previously established similarity of the human CENP-B protein and pogo-like transposases implicate a novel role of the CENP-B box and related sequence motifs in addition to the known function in centromere protein binding.     

Protein degradation in extracts of exponential and stationary phase Vibrio cells

The degradation of the foreign protein [14C]methyl apohaemoglobin ([14C-me]globin) was stimulated by ATP in cell-free extracts from exponential phase and shaken and standing stationary phase Vibrio cells. A marked stimulation by ATP of the degradation of [14C-me]globin was observed with exponential phase cell extracts which were preincubated for 30 min at 30 degrees C. Maximum stimulation was obtained with 3 mM-ATP and optimum degradation was at pH 8.0-8.5. Preincubation of extracts from both types of stationary phase cells did not affect the degree of ATP stimulation. The amount of ATP stimulation of [14C-me]globin degradation by exponential phase extracts decreased markedly when the cells were starved in a growth limiting minimal medium before preparation of the cell extracts. In the exponential and both types of stationary phase extracts most of the activity was located in the cytoplasmic fractions. Although the periplasmic preparations contained a minor portion of the total activity, this activity showed a greater percentage stimulation by ATP. In the absence of ATP the specific proteolytic activities of the extracts from exponential and both types of stationary phase cells were similar. The proteolytic activities in all the cell extracts were inhibited to the same extent by phenylmethylsulphonyl fluoride, but the exponential and both types of stationary phase cell extracts were inhibited to different extents by EDTA and p-hydroxymercuribenzoate. The results suggest that the proteolytic systems responsible for the degradation of abnormal proteins are different in exponential and stationary phase Vibrio cells.     

Germination response of Arabian desert species to gibberellic acid and potassium nitrate seed treatment

Abstract

Desert plant species commonly use seed dormancy to prevent germination during unfavorable environmental conditions and thus increase the probability of seedling survival. Seed dormancy presents a challenge for restoration ecology, particularly in desert species for which our knowledge of dormancy regulation is limited. In the present study the effect of gibberellic acid (GA₃) and potassium nitrate (KNO₃) on seed dormancy release was investigated on eight Arabian desert species. Both treatments significantly enhanced the germination of most species tested. GA₃ was more effective than KNO₃ in enhancing germination percentage, reducing mean germination time and synchronizing the germination in most of the studied species. Light requirement during germination was species-specific, but in general the presence of light promoted germination more effectively when combined with KNO₃ and GA₃. The wide variation in dormancy and germination requirements among the tested species is indicative of distinct germination niches, which might assist their co-existence in similar habitat/environmental conditions. Seed pre-treatments that optimize germination in this habitat must therefore be assessed for individual species to improve the outcomes of ecological restoration.     

Angiopoietin decoy secreted at tumor site impairs tumor growth and metastases by inducing local inflammation and altering neoangiogenesis

The extracellular domain of the receptor tyrosine kinase Tie2/TEK (exTEK) has been used as an angiopoietin decoy to study the role of angiopoietins in the tumor–host interactions, using a syngeneic model of experimental metastases and subcutaneous tumor. Soluble exTEK secreted by transfected tumor cells inhibited HUVECs from forming tubes in Matrigel. ExTEK-transfected C26 colon carcinoma and TS/A mammary tumor cells displayed reduced growth rate when injected subcutaneously, and reduced ability to form experimental metastases when injected intravenously. Immunohistochemical analysis of tumors and metastases showed increased leukocytes infiltration and signs of inflammation in exTEK-secreting compared to parental tumor, as well as impairment in neo-vessel growth and organization. However, while neoangiogenesis eventually rescued in the subcutis, it failed to organize in the experimental metastases of exTEK-secreting tumor, contributing to the hampering of metastatic growth and to increased mice survival. The reactive infiltrate of C26TEK contained a different percentage of leukocytes and was responsible for the tumor inhibition. In fact, leukopenia induced by -irradiation of recipient mice or injection into interferon gamma (IFN-) gene knockout (GKO) mice resulted in reduced mouse survival and an increased number of lung metastases. On the other hand, interleukin (IL)-12 treatment prolonged the survival of mice bearing subcutaneous C26TEK but not of those bearing lung metastases, suggesting that IL-12 could exert further antiangiogenic effects at the site where the tumor can restore neoangiogenesis. These results show in vivo that reduced angiopoietin availability at the tumor site induces a local inflammatory response and impairment of neoangiogenesis which act synergistically to limit tumor growth and metastasis.Abbreviations AEC amino-ethylcarbazole - ELISA enzyme-linked immunosorbent assay - HRP horseradish peroxidase - HUVEC human umbilical vascular endothelial cell - i.v. intravenous - s.c. subcutaneous - TBS Tris-HCl buffered solution     

The results after transrectal prostate biopsy with 12 biopsy cores taken

This report describes the clinical value of transrectal prostate biopsy during which 12 biopsy cores are taken in comparison to the classical sextant method. There were 106 patients included in the study, who had transrectal prostate biopsy (TRB) due to abnormal finding after digitorectal examination (DRE) and/or values of PSA > 4 ng/ml in the period from 4 October 2001 till 14 August 2002. There were 117 biopsies with 12 biopsy cores taken, 6 cores from each lobe. Prostate cancer was confirmed in 49 patients (46%). Out of total number of confirmed cancer cases, initial biopsy detected 94%. There were three patients who had suspicious DRE finding, with PSA value of < 4 ng/ml, but cancer was not detected in any of them. In the patient group with PSA value between 4-10 ng/ml, cancer was detected in 26% of them and in the group with PSA value > 10 ng/ml cancer was detected in 58%. The most common Gleason score in the case of cancer was 7 (43%). During the biopsy procedure, 3 patients experienced strong vasovagal reactions, meaning that out of 117 biopsies incidence of complications was 2.6%. Few days after the biopsy, two patients developed urogenital tract infections (1.7%) and right after the procedure, there was one case of strong hematuria (0.8%) and strong rectal bleeding (0,8%) that needed hospitalization. Our results regarding the incidence of complications do not differ much from the results in the literature. According to data in the literature regarding sextant biopsy, 15-34% of cancer cases remain undiagnosed at initial biopsy. The method of 12 biopsy cores fails to diagnose only 6% of all cancers, but it is important to note that in the mentioned period, re-biopsy was indicated only in 11 from 60 patients with negative biopsies.     

The chemical composition of the meat of fat dormice (<Emphasis Type="Italic">Glis glis</Emphasis> L.)

The fat dormouse (Glis glis) is a traditional game species in the Republic of Croatia. Although today the fat dormouse is not frequently caught as game, it is still a source of animal protein in human nutrition in certain rural areas of Croatia. In this paper the chemical analysis of fat dormouse meat is presented. The average values for the quantity of water, fat, protein and ash in dormouse meat are comparable with the chemical composition of the meat of rabbits and brown hares, except for the important fact that rabbit and hare meat contains a greater quantity of fat on average. According to our results, the meat of fat dormice can be categorised as dietary food, characterised by a small percentage of fat (mean 2.83%) and a high amount of protein (mean 21.01%).     

Metabolic Changes in <Emphasis Type="Italic">Clostridium absonum</Emphasis> ATCC 27555 Accompanying Induction of Epimerization of a Primary Bile Acid

Some parameters of fermentation have been determined for Clostridium absonum in a chemostat by using a chemically defined medium with glucose as the sole source of carbon and energy. Steady-state continuous cultures were achieved at two dilution rates (D). Trends of the carbon flow were determined by comparison of ratios between the specific rates of formation of the three products of metabolism (lactate, acetate, butyrate). Chenodeoxycholate induced the 7- and 7-hydroxysteroid dehydrogenases of C. absonum. In the presence of this inducer, the growth yield and the carbon recovery decreased, the carbon flow distribution was altered favoring acetate production, and a deficit in the reoxydation of nucleotidic cofactors was observed. In the presence of chenodeoxycholate, C. absonum would favor the production of energy at the expense of the reoxidation of nucleotidic cofactors so as to ensure its growth, and the epimerization of chenodeoxycholate to ursodeoxycholate.     

Comparative morphology of incisor enamel and dentin in humans and fat dormice (Glis glis)

The structure of teeth in all living beings is genetically predetermined, although it can change under external physiological and pathological factors. The author's hypothesis was to indicate evolutional shifts resulting from genetic, functional and other differences. A comparative study about certain characteristics of incisors in humans and myomorpha, the fat dormouse (Glis glis) being their representative as well, comprised measurements of enamel and dentin thickness in individual incisor segments, evaluation of external enamel index, and also assessment of histological structure of enamel and dentin. The study results involving dormice showed the enamel to be thicker in lower than in the upper teeth, quite contrary to enamel thickness in humans. In the upper incisors in dormice the enamel is the thickest in the medial layer of the crown, and in the cervical portion of the crown in the lower incisors. The thickness of dentin in dormice is greater in the oral than in the vestibular side. These findings significantly differ from those reported in reference literature, but they are based on the function of teeth in dormice. Histological characteristics of hard dental tissues in dormice are similar to those in humans, with exception of uniserial structure of enamel and appearance of dentinoenamel junction.     

Affinity labeling of highly hydrophobic integral membrane proteins for proteome-wide analysis

The ability to identify and quantitate integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and facilitate derivatization of these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified, of which 40 were integral membrane proteins containing from one to nine mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g., ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g., pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.     

Impact of ‘Stretch’ Targets for Cardiovascular Disease Management within a Local Pay-for-Performance Programme

Pay-for-performance programs are often aimed to improve the management of chronic diseases. We evaluate the impact of a local pay for performance programme (QOF+), which rewarded financially more ambitious quality targets (‘stretch targets’) than those used nationally in the Quality and Outcomes Framework (QOF). We focus on targets for intermediate outcomes in patients with cardiovascular disease and diabetes. A difference-in-difference approach is used to compare practice level achievements before and after the introduction of the local pay for performance program. In addition, we analysed patient-level data on exception reporting and intermediate outcomes utilizing an interrupted time series analysis. The local pay for performance program led to significantly higher target achievements (hypertension: p-value <0.001, coronary heart disease: p-values <0.001, diabetes: p-values <0.061, stroke: p-values <0.003). However, the increase was driven by higher rates of exception reporting (hypertension: p-value <0.001, coronary heart disease: p-values <0.03, diabetes: p-values <0.05) in patients with all conditions except for stroke. Exception reporting allows practitioners to exclude patients from target calculations if certain criteria are met, e.g. informed dissent of the patient for treatment. There were no statistically significant improvements in mean blood pressure, cholesterol or HbA1c levels. Thus, achievement of higher payment thresholds in the local pay for performance scheme was mainly attributed to increased exception reporting by practices with no discernable improvements in overall clinical quality. Hence, active monitoring of exception reporting should be considered when setting more ambitious quality targets. More generally, the study suggests a trade-off between additional incentive for better care and monitoring costs.