Linkage analysis and allelic imbalance in human breast cancer kindreds using microsatellite markers from the short arm of chromosome 3

Eight Icelandic breast cancer kindreds were subjected to linkage analyses with respect to 28 microsatellite loci dispersed along the short arm of chromosome 3. Breast tumors derived from these kindreds were concurrently scored for allelic imbalance with ten of the markers. Linkage to most markers could be excluded on the basis of negative LOD scores and haplotype analyses, although some moderately positive LOD scores resulted. A high frequency of imbalance in the familial tumors was seen with two of the markers in comparison with results obtained from sporadic material. The highest frequency (68%) of imbalance was detected with the marker D3S1217, which is located on 3p14.2-p14.1. Imbalance at the D3S1211 locus, which is more telomeric (3p24.2-p22), was not significantly elevated in the familial tumors. We suggest that the genetic defect responsible for breast cancer susceptibility in these families either promotes instability in the 3p14.2-p14.1 region or enhances the selective advantage of such changes.     

Abnormal calcium metabolism in normocalcaemic sarcoidosis.

In studies of calcium metabolism in 13 unselected patients with untreated sarcoidosis all were normocalcaemic but five had hypercalcuria. All had normal renal function. Calcium absorption was indexed by a double isotope test. 45Ca hyperabsorption occurred in six patients. Ten kinetic studies were carried out with 47Ca and in six bone turnover was increased. 45Ca absorption correlated well with the calculated bone uptake rate of calcium, and with urine calcium excretion. These results suggest that in sarcoidosis abnormalities in calcium metabolism are fairly common although they rarely result in sustained hypercalcaemia.     

Simulation of Lake Victoria Circulation Patterns Using the Regional Ocean Modeling System (ROMS)

Lake Victoria provides important ecosystem services including transport, water for domestic and industrial uses and fisheries to about 33 million inhabitants in three East African countries. The lake plays an important role in modulating regional climate. Its thermodynamics and hydrodynamics are also influenced by prevailing climatic and weather conditions on diel, seasonal and annual scales. However, information on water temperature and circulation in the lake is limited in space and time. We use a Regional Oceanographic Model System (ROMS) to simulate these processes from 1^st January 2000 to 31^st December 2014. The model is based on real bathymetry, river runoff and atmospheric forcing data using the bulk flux algorithm. Simulations show that the water column exhibits annual cycles of thermo-stratification (September–May) and mixing (June–August). Surface water currents take different patterns ranging from a lake-wide northward flow to gyres that vary in size and number. An under flow exists that leads to the formation of upwelling and downwelling regions. Current velocities are highest at the center of the lake and on the western inshore waters indicating enhanced water circulation in those areas. However, there is little exchange of water between the major gulfs (especially Nyanza) and the open lake, a factor that could be responsible for the different water quality reported in those regions. Findings of the present study enhance understanding of the physical processes (temperature and currents) that have an effect on diel, seasonal, and annual variations in stratification, vertical mixing, inshore—offshore exchanges and fluxes of nutrients that ultimately influence the biotic distribution and trophic structure. For instance information on areas/timing of upwelling and vertical mixing obtained from this study will help predict locations/seasons of high primary production and ultimately fisheries productivity in Lake Victoria.     

Site-specific incorporation of nitroxide spin-labels into 2'-positions of nucleic acids

A protocol is described for the incorporation of nitroxide spin-labels into specific 2'-sites within nucleic acids. This labeling strategy facilitates the investigation of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) spectroscopy and macromolecular complex formation using paramagnetic relaxation enhancement NMR spectroscopy. A spin-labeling reagent, 4-isocyanato TEMPO, which can be prepared in one facile step or obtained commercially, is used for postsynthetic modification of site-specifically 2'-amino-modified nucleic acids. This spin-labeling protocol has been applied primarily to RNA, but is also applicable to DNA. Subsequently, EPR spectroscopic analysis of the spin-labeled nucleic acids allows for the measurements of distances, solvent accessibilities and conformation dynamics. Using the spin-labeling strategy described here, spin-labeled samples can be prepared in 2-4 d.     

Therapeutic approaches for prion and Alzheimer's diseases

Alzheimer's and prion diseases belong to a category of conformational neurodegenerative disorders [Prusiner SB (2001) N Eng J Med344, 1516-1526; Sadowski M & Wisniewski T (2007) Curr Pharm Des 13, 1943-1954; Beekes M (2007) FEBS J 274, 575]. Treatments capable of arresting or at least effectively modifying the course of disease do not yet exist for either one of these diseases. Alzheimer's disease is the major cause of dementia in the elderly and has become an ever greater problem with the aging of Western societies. Unlike Alzheimer's disease, prion diseases are relatively rare. Each year only approximately 300 people in the USA and approximately 100 people in the UK succumb to various forms of prion diseases [Beekes M (2007) FEBS J 274, 575; Sigurdsson EM & Wisniewski T (2005) Exp Rev Vaccines 4, 607-610]. Nevertheless, these disorders have received great scientific and public interest due to the fact that they can be transmissible among humans and in certain conditions from animals to humans. The emergence of variant Creutzfeld-Jakob disease demonstrated the transmissibility of the bovine spongiform encephalopathy to humans [Beekes M (2007) FEBS J 274, 575]. Therefore, the spread of bovine spongiform encephalopathy across Europe and the recently identified cases in North America have put a large human population at risk of prion infection. It is estimated that at least several thousand Britons are asymptomatic carriers of prion infections and may develop variant Creutzfeld-Jakob disease in the future [Hilton DA (2006) J Pathol 208, 134-141]. This delayed emergence of human cases following the near elimination of bovine spongiform encephalopathy in the UK may occur because prion disease have a very prolonged incubation period, ranging from months to decades, which depends on the amount of inoculum, the route of infection and the genetic predisposition of the infected subject [Hilton DA (2006) J Pathol 208, 134-141]. Therefore, there is a great need for effective therapies for both Alzheimer's disease and prion diseases.     

Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.     

Sequence context effect on the structure of nitrous acid induced DNA interstrand cross-links

In the preceding paper in this journal, we described the solution structure of the nitrous acid cross-linked dodecamer duplex [d(GCATCCGGATGC)]2 (the cross-linked guanines are underlined). The structure revealed that the cross-linked guanines form a nearly planar covalently linked 'G:G base pair', with the complementary partner cytidines flipped out of the helix. Here we explore the flanking sequence context effect on the structure of nitrous acid cross-links in [d(CG)]2 and the factors allowing the extrahelical cytidines to adopt such fixed positions in the minor groove. We have used NMR spectroscopy to determine the solution structure of a second cross-linked dodecamer duplex, [d(CGCTACGTAGCG)]2, which shows that the identity of the flanking base pairs significantly alters the stacking patterns and phosphate backbone conformations. The cross-linked guanines are now stacked well on adenines preceding the extrahelical cytidines, illustrating the importance of purine- purine base stacking. Observation of an imino proton resonance at 15.6 p.p.m. provides evidence for hydrogen bonding between the two cross-linked guanines. Preliminary structural studies on the cross-linked duplex [d(CGCGACGTCGCG)]2 show that the extrahelical cytidines are very mobile in this sequence context. We suggest that favorable van der Waals interactions between the cytidine and the adenine 2 bp away from the cross-link localize the cytidines in the previous cross-linked structures.     

Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA

Whole genome amplification (WGA) procedures such as primer extension preamplification (PEP) or multiple displacement amplification (MDA) have the potential to provide an unlimited source of DNA for large-scale genetic studies. We have performed a quantitative evaluation of PEP and MDA for genotyping single nucleotide polymorphisms (SNPs) using multiplex, four-color fluorescent minisequencing in a microarray format. Forty-five SNPs were genotyped and the WGA methods were evaluated with respect to genotyping success, signal-to-noise ratios, power of genotype discrimination, yield and imbalanced amplification of alleles in the MDA product. Both PEP and MDA products provided genotyping results with a high concordance to genomic DNA. For PEP products the power of genotype discrimination was lower than for MDA due to a 2-fold lower signal-to-noise ratio. MDA products were indistinguishable from genomic DNA in all aspects studied. To obtain faithful representation of the SNP alleles at least 0.3 ng DNA should be used per MDA reaction. We conclude that the use of WGA, and MDA in particular, is a highly promising procedure for producing DNA in sufficient amounts even for genome wide SNP mapping studies.     

Determination of DNA minor groove width in distamycin-DNA complexes by solid-state NMR

We have performed solid-state ³¹P-¹⁹F REDOR nuclear magnetic resonance (NMR) experiments to monitor changes in minor groove width of the oligonucleotide d(CGCAAA^2′FUTGGC)·d(GCCAAT(pS)TT GCG) (A₃T₂) upon binding of the drug distamycin A at different stoichiometries. In the hydrated solid-state sample, the minor groove width for the unbound DNA, measured as the ^2′FU7–pS19 inter-label distance, was 9.4 ± 0.7 Å, comparable to that found for similar A:T-rich DNAs. Binding of a single drug molecule is observed to cause a 2.4 Å decrease in groove width. Subsequent addition of a second drug molecule results in a larger conformational change, expanding this minor groove width to 13.6 Å, consistent with the results of a previous solution NMR study of the 2:1 complex. These ³¹P-¹⁹F REDOR results demonstrate the ability of solid-state NMR to measure distances of 7–14 Å in DNA–drug complexes and provide the first example of a direct spectroscopic measurement of minor groove width in nucleic acids.