Methods to measure the intracellular concentration of unlabeled compounds within cultured cells using liquid chromatography/tandem mass spectrometry

A high-throughput and sensitive liquid chromatography/tandem mass spectrometry assay was established to detect total unlabeled hepatitis C virus inhibitor concentrations in replicon cells. The intracellular concentrations determined by this assay correlated well with concentrations obtained using radiolabeled compound. Some compounds accumulated inside the cells, with concentrations up to 300-fold higher than the input concentration. Confocal microscopic evaluation of two fluorescent-tagged inhibitors confirmed high accumulation inside the cells, sequestered inside vesicles within the cytoplasm. Incubation of cells with compound at 4 °C revealed that nonspecific binding to the outside of the cell membrane and to the cell culture plate occurred for some compounds. Therefore, the total concentration of compound extracted at 37 °C was reduced by the amount that was nonspecifically bound at 4 °C to yield the amount of compound inside the cells. A modification of the protocol was used for compounds with low intracellular concentrations in which cells were harvested with trypsin-EDTA prior to extraction. This eliminated the nonspecific binding to the cell culture plate and decreased the overall background of the assay. This assay was used to understand differences in cellular potency between compounds and the effects of serum proteins on the metabolic stability of compounds during incubation with cells.     

Tissue-specific changes in the hydroxylysine content and cross-links of collagens and alterations in fibril morphology in lysyl hydroxylase 1 knock-out mice

We have generated mice with targeted inactivation of the Plod1 gene for lysyl hydroxylase 1 (LH1). Its human mutations cause Ehlers-Danlos syndrome VIA (EDS VIA) characterized by muscular hypotonia, joint laxity, and kyphoscoliosis. The Plod1(-/-) mice are flaccid and have gait abnormalities. About 15% of them died because of aortic rupture and smooth muscle cells in non-ruptured Plod1(-/-) aortas showed degenerative changes. Collagen fibrils in the Plod1(-/-) aorta and skin had an abnormal morphology. The LH activity level in the Plod1(-/-) skin and aorta samples was 35-45% of that in the wild type. The hydroxylysine content was decreased in all the Plod1(-/-) tissues, ranging from 22% of that in the wild type in the skin to 75 and 86% in the femur and lung. The hydroxylysylpyridinoline crosslinks likewise showed decreases in all the Plod1(-/-) tissues, ranging from 28 and 33% of that in the wild type in the aorta and cornea to 47 and 59% in femur and tendon, while lysylpyridinolines were increased. The hydroxylysines found in the Plod1(-/-) collagens and their cross-links were evidently synthesized by the other two LH isoenzymes. Few data are available on abnormalities in EDS VIA tissues other than the skin. Plod1(-/-) mice offer an in vivo model for systematic analysis of the tissue-specific consequences of the lack of LH1 activity and may also provide a tool for analyzing the roles of connective tissue in muscle function and the complex interactions occurring in the proper assembly of the extracellular matrix.     

Interlaboratory comparison of Taq Nuclease Assays for the quantification of the toxic cyanobacteria Microcystis sp

The application of quantitative real-time PCR has been proposed for the quantification of toxic genotypes of cyanobacteria. We have compared the Taq Nuclease Assay (TNA) in quantifying the toxic cyanobacteria Microcystis sp. via the intergenic spacer region of the phycocyanin operon (PC) and mcyB indicative of the production of the toxic heptapeptide microcystin between three research groups employing three instruments (ABI7300, GeneAmp5700, ABI7500). The estimates of mcyB genotypes were compared using (i) DNA of a mcyB containing strain and a non-mcyB containing strain supplied in different mixtures across a low range of variation (0-10% of mcyB) and across a high range of variation (20-100%), and (ii) DNA from field samples containing Microcystis sp. For all three instruments highly significant linear regression curves between the proportion of the mcyB containing strain and the percentage of mcyB genotypes both within the low range and within the high range of mcyB variation were obtained. The regression curves derived from the three instruments differed in slope and within the high range of mcyB variation mcyB proportions were either underestimated (0-50%) or overestimated (0-72%). For field samples cell numbers estimated via both TNAs as well as mcyB proportions showed significant linear relationships between the instruments. For all instruments a linear relationship between the cell numbers estimated as PC genotypes and the cell numbers estimated as mcyB genotypes was observed. The proportions of mcyB varied from 2 to 28% and did not differ between the instruments. It is concluded that the TNA is able to provide quantitative estimates on mcyB genotype numbers that are reproducible between research groups and is useful to follow variation in mcyB genotype proportion occurring within weeks to months.     

Chronic patients--persons with diabetes frequent attenders in Croatian family practice

Chronic diseases cause high frequency visits and generate the long-term frequent attenders (FAs). The connection between frequent attendance and specific morbidities in the health care systems in transitional Europe has been underestimated. We investigated whether frequent visits of chronic patients in primary care are related to characteristic of chronic disease (diabetes mellitus) and whether this is influenced by the family practice in the transitional health care. We analyzed the number of visits a day time work for 490 persons with diabetes in the period 1997 to 2000. As the cut-off points between frequent attenders and non frequent attenders (NFAs) we used the value of the third quartile (Q3) of visits determined for the sex and age groups in the parallel study in the whole population. The analysis was performed for 23 variables: demographic characteristics of patients, disease characteristic and variables of physician. Logistic regressions were employed to identify the predictors of FAs/NFAs. 56.9% (in 1997) to 62.4% (in 2000) persons with diabetes were FAs, compared to 22.4% to 24.3% FAs patients in the whole population. Logistic regression analysis significantly differentiated the two group of visits with 68% accuracy. 4 variables are significant predictors for FAs/NFAs: diabetes as the main disease (p = 0.0005), diet-only-treatment (p = 0.0062), treatment by secondary care (p = 0.0116), and if glycated hemoglobin test (HbA1c) is determined (p = 0.0272). Understanding the similarities and differences of FAs/NFAs persons with diabetes may be important in improving the care and management of chronic diseases in family medicine in transitional health care systems.     

Sex differences in motor characteristics of elementary school children included/not included in swimming training

The aim of the study was to assess the development of motor abilities in elementary school fifth- to eighth-graders (age 11-14 years) according to sex, age and physical activity. Study sample included 312 subjects divided according to age and sex into four groups: male subjects aged 11-12 (n = 93) and 13-14 years (n = 84); and female subjects aged 11-12 (n = 65) and 13-14 years (n = 70). Then, differences in basic motor abilities between children included (experimental group) and those not included (control group) in swimming training were analyzed. In male fifth- and sixth-graders, experimental group was superior to control group in the variables of trunk repetitive strength, sprint, flexibility and coordination, while in male seventh- and eighth-graders experimental group showed better performance than control group in agility, aerobic endurance and explosive throw and jump strength. In female fifth- and sixth-graders, experimental group proved superior to control group in the variables of explosive strength, coordination, trunk strength and aerobic endurance, whereas in female seventh- and eighth-graders experimental group had better performance in coordination, endurance, explosive strength, speed and flexibility. Discriminative analysis of motor variables between male and female subjects revealed male subjects to be superior in explosive strength, throw strength in particular, coordination and aerobic endurance, whereas female subjects showed better performance in the variables of flexibility and movement frequency, leg movement in particular. Study results showed the formation of appropriate motor system determining achievement of top results in swimming to be influenced by swimming training from age 11 to 14. In male children, motor system was found to integrate coordination/agility, aerobic endurance and explosive strength, whereas in female children it integrated coordination in terms of cortical movement regulation, aerobic endurance, explosive strength and psychomotor speed.     

Relations of the morphological characteristic latent structure and body posture indicators in children aged seven to nine years

With the aim of determining the connection between the indicators of body posture and latent structure of morphological variables in children aged 7 and 8 years, first and second grade of primary school, a set of 17 morphological measures and 12 body posture indicators were longitudinally applied to a sample of 110 boys and 114 girls. The latent structure of morphological variables in both sexes was defined by three factors but at a different order of significance: in boys, the order was longitudinal dimensionality, voluminosity, mass and subcutaneous fat tissue and transverse dimensionality, whereas in girls the order was voluminosity, mass and subcutaneous fat tissue, longitudinal dimensionality and transverse dimensionality. The latent structure of torax body posture indicator was defined by two factors, the status of body posture of the rear part of the thorax, and status of the body posture of the front part of the thorax. The results obtained by canonical correlation analysis between predictive variables, morphological latent structure and criterion variables, latent structure of thorax body posture indicators with two posture indicators of the chest and one of the foot status, showed two important pairs of canonical roots on each measurement, suggesting a significant association between these two sets of parameters.     

Lack of Ephrin Receptor A1 Is a Favorable Independent Prognostic Factor in Clear Cell Renal Cell Carcinoma

The EPH receptor tyrosine kinases and their cell-bound ligands, the ephrins, have been shown to be associated with cancer development and progression. In this study, mRNA and protein expression of the receptors EPHA1 and EPHA2 as well as of their ligand EFNA1 and their prognostic relevance in clear cell renal cell carcinoma was evaluated. Gene expression was measured in 75 cryo-preserved primary tumors and matched non-malignant renal specimens by quantitative PCR. Protein expression was analyzed by immunohistochemistry on tissue microarrays comprising non-malignant, primary tumors and metastatic renal tissues of 241 patients. Gene and protein expression of all three factors was altered in tumor specimens with EPHA1 and EPHA2 being generally diminished in tumors compared to normal renal tissue, whereas EFNA1 was commonly elevated. A positive EPHA1 and EPHA2 protein staining as well as a low EFNA1 protein level were significantly linked to more aggressive tumor features, but only a positive EPHA1 immunoreactivity was significantly associated with poor survival. In subgroup analyses, EPHA1 and EPHA2 protein levels were significantly higher in metastatic than in primary lesions. Patients with EPHA1/EPHA2-positive tumors or with tumors with positive EPHA1 and low EFNA1 immunoreactivity had the shortest survival rates compared to the respective other combinations. In a multivariate model, EPHA1 was an independent prognostic marker for different survival endpoints. In conclusion, an impaired EPH-ephrin signaling could contribute to the pathogenesis and progression of clear cell renal cell carcinoma.     

Prenatal and Childhood Growth,and Hospitalization for Alcohol Use Disorders in Adulthood: The Helsinki Birth Cohort Study

Background

Small birth size - an indicator of a sub-optimal prenatal environment - and variation in growth after birth have been associated with non-communicable diseases in later life. We tested whether birth size or growth in childhood associated with the risk of hospital admission for alcohol use disorders (AUDs) from early to late adulthood.

Methods

The sample comprised 6544 men and 6050 women born between 1934 and 1944 in Helsinki, Finland. Data on anthropometric measures were extracted from medical records and diagnoses of AUD from the Finnish Hospital Discharge Register and Causes of Death Register covering a 40-year period from 1969 to 2008.

Results

Altogether 171 women (2.8%) and 657 men (10.0%) were diagnosed at a hospital with AUD. After adjusting for major confounders, shorter length at birth, shorter height up to two years of age, and lower weight at two years associated with hospitalization for AUD in women. In men, slower growth in height, particularly from 2 to 7 years, and slower weight gain from 7 to 11 years as well as shorter height and lower weight at 7 and 11 years associated with a diagnosis of AUD in men.

Conclusions

Pre- and postnatal growth associates with the risk for AUD later in life differently in women than in men: the fetal period and infancy seem to be the sensitive periods for women, whereas those for men the occur from toddlerhood onwards.     

Identification and Functional Analysis of the S-Layer Protein SplA of Paenibacillus larvae, the Causative Agent of American Foulbrood of Honey Bees

The Gram-positive, spore-forming bacterium Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a globally occurring, deathly epizootic of honey bee brood. AFB outbreaks are predominantly caused by two genotypes of P. larvae, ERIC I and ERIC II, with P. larvae ERIC II being the more virulent genotype on larval level. Recently, comparative proteome analyses have revealed that P. larvae ERIC II but not ERIC I might harbour a functional S-layer protein, named SplA. We here determine the genomic sequence of splA in both genotypes and demonstrate by in vitro self-assembly studies of recombinant and purified SplA protein in combination with electron-microscopy that SplA is a true S-layer protein self-assembling into a square 2D lattice. The existence of a functional S-layer protein is novel for this bacterial species. For elucidating the biological function of P. larvae SplA, a genetic system for disruption of gene expression in this important honey bee pathogen was developed. Subsequent analyses of in vivo biological functions of SplA were based on comparing a wild-type strain of P. larvae ERIC II with the newly constructed splA-knockout mutant of this strain. Differences in cell and colony morphology suggest that SplA is a shape-determining factor. Marked differences between P. larvae ERIC II wild-type and mutant cells with regard to (i) adhesion to primary pupal midgut cells and (ii) larval mortality as measured in exposure bioassays corroborate the assumption that the S-layer of P. larvae ERIC II is an important virulence factor. Since SplA is the first functionally proven virulence factor for this species, our data extend the knowledge of the molecular differences between these two genotypes of P. larvae and contribute to explaining the observed differences in virulence. These results present an immense advancement in our understanding of P. larvae pathogenesis.