First BRCA1 and BRCA2 gene testing implemented in the health care system of Stockholm

The aim of the study was to optimize the criteria for the BRCA1 and BRCA2 gene testing and to improve oncogenetic counseling in the Stockholm region. Screening for inherited breast cancer genes is laborious and a majority of tested samples turn out to be negative. The frequencies of mutations in the BRCA1 and BRCA2 genes differ across populations. Between 1997 and 2000, 160 families with breast and/or ovarian cancer were counseled and screened for mutations in the two genes. Twenty-five BRCA1 and two BRCA2 disease-causing mutations were found. Various factors associated with the probability of finding a BRCA1 mutation in the families were estimated. Age of onset in different generations and other malignancies were also studied. Families from our region in which both breast and ovarian cancer occur were likely to carry a BRCA1 mutation (34%). In breast-only cancer families, mutations were found only in those with very early onset. All breast- only cancer families with a mutation had at least one case of onset before 36 years of age and a young median age of onset (<43 years). Other malignancies than breast and ovarian cancers did not segregate in the BRCA1 families and surveillance for other malignancies is not needed, in general. Decreasing age of onset with successive generations was common and must be taken into account when surveillance options are considered.     

Sleep fragmentation in mentally retarded people decreases with increasing daylength in spring

We studied the sleep-wake behavior of mentally retarded people from late winter to early summer at 60 degrees N. During this time the daylength increased 8 h 51 min. The data were collected by observing the sleep-wake status of 293 subjects at 20-min intervals for five randomized 24h periods (= recording days). The intervals during which the individual recording days of the same order (1st, 2nd, etc.) were carried out, were called recording periods. Consequently, there were five recording periods, each containing 293 individual recording days. Even though there was overlap among the recording periods, the median daylength from one period to another increased approximately by 100 min. In the initial statistical analysis, the number of wake-sleep transitions was found to differ significantly among the five recording periods (Friedman test, p < 0.001). The mean ranks in the Friedman test suggested that the number of wake-sleep transitions was highest during the 1st and lowest during the 5th recording period. In further statistical analyses using a program for mixed effects regression analysis (MIXOR 2.0) it was found that the increase in daylength during the study period was associated with a simultaneous decrease of approximately 0.5 wake-sleep transitions in the whole study population (p < 0.001). The decrease in the number of wake-sleep transitions was significant only in the subgroups of subjects with a daylength change of more than 350 min between the 1st and 5th recording days (Wilcoxon tests, p < 0.005). This suggests that after a marked prolongation of the natural photoperiod, the reduction in sleep episodes was more probable than after smaller changes in daylength. It is concluded that the sleep of mentally retarded people living in a rehabilitation center at a northern latitude is more fragmented in winter than in early summer and that the change is related probably to the simultaneous increase in the length of the natural photoperiod. The sleep quality of persons living in institutional settings might be improved by increasing the intensity and/or duration of daily artificial light exposure during the darker seasons.     

Synthesis and characterization of 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine: crystal structures of 4,6-O-butylidene-alpha-D-glucopyranose, 4,6-O-butylidene-beta-D-glucopyranosylamine and 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine

4,6-O-Butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine was synthesized and characterized using analytical, spectral and single-crystal X-ray diffraction methods. 1H and 13C NMR studies showed the presence of the beta-anomer, which has also been confirmed by the crystal structure. The molecular structure of this compound showed the presence of the tridentate ONO ligation-core. Both precursors, 4,6-O-butylidene-alpha-D-glucopyranose and 4,6-O-butylidene-beta-D-glucopyranosylamine were characterized using single crystal X-ray diffraction. The alpha-anomeric nature of the former and beta-anomeric nature of the latter were proposed based on 1H NMR studies and were confirmed by determining the crystal structures. In addition, the crystal structure of 4,6-O-butylidene-beta-D-glucopyranosylamine revealed the C-1-N-glycosylation. In all the three molecules, the saccharide unit exhibits a 4C(1) chair conformation. In the lattice, the molecules are connected by hydrogen-bond interactions. The conformation of 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine is stabilized via an O-H...N intramolecular interaction, and each molecule in the lattice interacts with three neighboring molecules through hydrogen bonds of the type O-H...O and C-H...O.     

Surface display of foreign epitopes on the Lactobacillus brevis S-layer

So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.     

Spectrum of styrene-induced DNA adducts: the relationship to other biomarkers and prospects in human biomonitoring

Styrene is an important industrial chemical that has shown genotoxicity in many toxicology assays. This is believed to be related to the DNA-binding properties of styrene-7,8-oxide (SO), a major metabolite of styrene. In this review, we have summarized knowledge on various aspects of styrene genotoxicity, especially in order to understand the formation and removal of primary DNA lesions, and the usefulness of biomarkers for risk assessment. Biological significances of specific DNA adducts and their role in the cascade of genotoxic events are discussed. Links between markers of external and internal exposure are evaluated, as well as metabolic aspects leading to the formation of DNA adducts and influencing biomarkers of biological effect. Finally, we suggest a design of a population study, which may contribute to our understanding genotoxic events in the exposure either to single xenobiotic or complex mixture.     

False positive non-synonymous polymorphisms of G-protein coupled receptor genes

Polymorphisms of G-protein coupled receptor (GPCR) genes are associated with disease risk and modification, and the response to receptor-directed therapy. Genomic sequencing ( approximately 1700 automated runs) from as many as 120 chromosomes from 60 multiethnic individuals was performed to confirm non-synonymous coding polymorphisms reported in the dbSNP database from 25 randomly selected GPCR genes. These polymorphisms were in regions of the receptors responsible for structural integrity, ligand binding, G-protein coupling and phosphoregulation. However, most of these putative polymorphisms could not be confirmed (false positive rate of 68%). Based on these results, we suggest that the variability of the superfamily is not well defined, and we caution against exclusive reliance on databases for selection of candidate GPCR polymorphisms for disease association and pharmacogenetic studies.     

A genome-wide strategy for the identification of essential genes in Staphylococcus aureus

To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.     

Expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in human uterine leiomyomas and myometrium during the menstrual cycle and after menopause

To investigate the expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak proteins in human uterine leiomyomas and homologous myometrium during the menstrual cycle and after menopause.The expression of Bcl-2, Bcl-x, Mcl-1, Bax and Bak in leiomyomas (n=24) and myometrial samples (n=22) from women with leiomyomas was measured by immunohistochemistry and Western blot. Measured by immunohistochemistry, a significant difference between leiomyomas and myometrium was observed only for the Bax protein, in tissues obtained from women in the secretory phase of the menstrual cycle. The Bcl-2 staining was more abundant in leiomyomas than in myometrium only in tissues obtained in the proliferative phase of the cycle. Bcl-2 was more abundant in leiomyomas from women of fertile age than in leiomyomas from menopausal women. No significant differences were observed for the Bcl-x or Bak proteins, whereas the Mcl-1 protein was significantly less abundant in secretory phase leiomyomas than in leiomyomas from menopausal women. Western blot analysis based on pools of tissue extracts from the different groups essentially confirmed the data obtained by immunohistochemistry. Bcl-2 family proteins are expressed in leiomyomas and myometrium in different phases related to and influenced by gonadal steroids. These proteins are suggested to interact with each other in the regulation of programmed cell death, apoptosis, but their specific role in growth control of uterine leiomyomas remains to be investigated.     

Recognition of T*G mismatched base pairs in DNA by stacked imidazole-containing polyamides: surface plasmon resonance and circular dichroism studies

An imidazole-containing polyamide trimer, f-ImImIm, where f is a formamido group, was recently found using NMR methods to recognize T·G mismatched base pairs. In order to characterize in detail the T·G recognition affinity and specificity of imidazole-containing polyamides, f-ImIm, f-ImImIm and f-PyImIm were synthesized. The kinetics and thermodynamics for the polyamides binding to Watson–Crick and mismatched (containing one or two T·G, A·G or G·G mismatched base pairs) hairpin oligonucleotides were determined by surface plasmon resonance and circular dichroism (CD) methods. f-ImImIm binds significantly more strongly to the T·G mismatch-containing oligonucleotides than to the sequences with other mismatched or with Watson–Crick base pairs. Compared with the Watson–Crick CCGG sequence, f-ImImIm associates more slowly with DNAs containing T·G mismatches in place of one or two C·G base pairs and, more importantly, the dissociation rate from the T·G oligonucleotides is very slow (small k_d). These results clearly demonstrate the binding selectivity and enhanced affinity of side-by-side imidazole/imidazole pairings for T·G mismatches and show that the affinity and specificity increase arise from much lower k_d values with the T·G mismatched duplexes. CD titration studies of f-ImImIm complexes with T·G mismatched sequences produce strong induced bands at ~330 nm with clear isodichroic points, in support of a single minor groove complex. CD DNA bands suggest that the complexes remain in the B conformation.     

High pressure effects on cellular expression profile and mRNA stability. A cDNA array analysis

Hydrostatic pressure has a profound effect on cartilage tissue and chondrocyte metabolism. Depending on the type and magnitude of pressure various responses can occur in the cells. The mechanisms of mechanotransduction at cellular level and the events leading to specific changes in gene expression are still poorly understood. We have previously shown that induction of stress response in immortalized chondrocytes exposed to high static hydrostatic pressure increases the stability of heat shock protein 70 mRNA. In this study, our aim was to examine the effect of high pressure on gene expression profile and to study whether stabilization of mRNA molecules is a general phenomenon under this condition. For this purpose a cDNA array analysis was used to compare mRNA expression profile in pressurized vs. non-pressurized human chondrosarcoma cells (HCS 2/8). mRNA stability was analyzed using actinomycin-treated and nontreated samples collected after pressure treatment. A number of immediate-early genes, and genes regulating cell cycle and growth were up-regulated due to high pressure. Decrease in osteonectin, fibronectin, and collagen types VI and XVI mRNAs was observed. Also bikunin, cdc37 homologue and Tiam1, genes linked with hyaluronan metabolism, were down-regulated. In general, stability of down-regulated mRNA species appeared to increase. However, no increase in mRNA above control level due to stabilization was noticed in the genes available in the array. On the other hand, mRNAs of certain immediate-early genes, like c-jun, jun-B and c-myc, became destabilized under pressure treatment. Increased accumulation of mRNA on account of stabilization under high pressure conditions seems to be a tightly regulated, specific phenomenon.