Molecular cladistic markers in New World monkey phylogeny (Platyrrhini,Primates)

Transpositions of primate-specific Alu elements were applied as molecular cladistic markers in a phylogenetic analysis of South American primates. Seventy-four human and platyrrhine loci containing intronic Alu elements were PCR screened in various New World monkeys and the human outgroup to detect the presence of orthologous retrotransposons informative of New World monkey phylogeny. Six loci revealed size polymorphism in the amplification pattern, indicating a shared derived character state due to the presence of orthologous Alu elements confirmed by subsequent sequencing. Three markers corroborate (1) New World monkey monophyly and one marker supports each of the following callitrichine relationships: (2) Callithrix and Cebuella are more closely related to each other than to any other callitrichine, (3) the callitrichines form a monophyletic clade including Callimico, and (4) the next living relatives to the callitrichines are Cebus, Saimiri, and Aotus.     

Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes

Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.     

Involvement of the endoplasmic reticulum in peroxisome formation

The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived.     

Disruption of the Fanconi anemia-BRCA pathway in cisplatin-sensitive ovarian tumors

Ovarian tumor cells are often genomically unstable and hypersensitive to cisplatin. To understand the molecular basis for this phenotype, we examined the integrity of the Fanconi anemia-BRCA (FANC-BRCA) pathway in those cells. This pathway regulates cisplatin sensitivity and is governed by the coordinate activity of six genes associated with Fanconi anemia (FANCA, FANCC, FANCD2, FANCE, FANCF and FANCG) as well as BRCA1 and BRCA2 (FANCD1). Here we show that the FANC-BRCA pathway is disrupted in a subset of ovarian tumor lines. Mono-ubiquitination of FANCD2, a measure of the function of this pathway, and cisplatin resistance were restored by functional complementation with FANCF, a gene that is upstream in this pathway. FANCF inactivation in ovarian tumors resulted from methylation of its CpG island, and acquired cisplatin resistance correlated with demethylation of FANCF. We propose a model for ovarian tumor progression in which the initial methylation of FANCF is followed by FANCF demethylation and ultimately results in cisplatin resistance.     

Molecular monitoring in malaria vaccine trials

Molecular techniques offer new approaches for malaria field trials, particularly PCR techniques, which facilitate accurate diagnosis of Plasmodium infections and increase the power of estimates of vaccine effects on malaria prevalence or incidence. Molecular methods also help to assess selective effects of vaccines. Longitudinal genotyping data can be used to initiate novel analyses of parasite dynamics, including estimates of incidence of infection with individual parasite clones and duration of infections. In addition, high-throughput methods can be used to apply these techniques routinely in randomized controlled trials, as well as programme-based evaluations of malaria control.     

Evaluation of five different cDNA labeling methods for microarrays using spike controls

Background

Several different cDNA labeling methods have been developed for microarray based gene expression analysis. We have examined the accuracy and reproducibility of such five commercially available methods in detection of predetermined ratio values from target spike mRNAs (A. thaliana) in a background of total RNA. The five different labeling methods were: direct labeling (CyScribe), indirect labeling (FairPlay? – aminoallyl), two protocols with dendrimer technology (3DNA^® Array 50? and 3DNA^® submicro?), and hapten-antibody enzymatic labeling (Micromax? TSA?). Ten spike controls were mixed to give expected Cy5/Cy3 ratios in the range 0.125 to 6.0. The amounts of total RNA used in the labeling reactions ranged from 5 – 50 μg.

Results

The 3DNA array 50 and CyScribe labeling methods performed best with respect to relative deviation from the expected values (16% and 17% respectively). These two methods also displayed the best overall accuracy and reproducibility. The FairPlay method had the lowest total experimental variation (22%), but the estimated values were consistently higher than the expected values (36%). TSA had both the largest experimental variation and the largest deviation from the expected values (45% and 48% respectively).

Conclusion

We demonstrate the usefulness of spike controls in validation and comparison of cDNA labeling methods for microarray experiments.

     

Surface pretreatments for medical application of adhesion

Medical implants and prostheses (artificial hips, tendono- and ligament plasties) usually are multi-component systems that may be machined from one of three material classes: metals, plastics and ceramics. Typically, the body-sided bonding element is bone.     

Strain and strain rate parametric imaging. A new method for post processing to 3-/4-dimensional images from three standard apical planes. Preliminary data on feasibility, artefact and regional dyssynergy visualisation

Background

We describe a method for 3-/4D reconstruction of tissue Doppler data from three standard apical planes, post processing to derived data of strain rate/strain and parametric colour imaging of the data. The data can be displayed as M-mode arrays from all six walls, Bull's eye projection and a 3D surface figure that can be scrolled and rotated. Numerical data and waveforms can be re-extracted.

Methods

Feasibility was tested by Strain Rate Imaging in 6 normal subjects and 6 patients with acute myocardial infarction. Reverberation artefacts and dyssynergy was identified by colour images. End systolic strain, peak systolic and mid systolic strain rate were measured.

Results

Infarcts were visualised in all patients by colour imaging of mid systolic strain rate, end systolic strain and post systolic shortening by strain rate. Reverberation artefacts were visible in 3 of 6 normals, and 2 of 6 patients, and were identified both on bull's eye and M-mode display, but influenced quantitative measurement. Peak systolic strain rate was in controls minimum -1.11, maximum -0.89 and in patients minimum -1.66, maximum 0.02 (p = 0.04). Mid systolic strain rate and end systolic strain did not separate the groups significantly.

Conclusion

3-/4D reconstruction and colour display is feasible, allowing quick visual identification of infarcts and artefacts, as well as extension of area of post systolic shortening. Strain rate is better suited to colour parametric display than strain.     

Hormone replacement therapy in rheumatoid arthritis is associated with lower serum levels of soluble IL-6 receptor and higher insulin-like growth factor 1

Hormone replacement therapy (HRT) modulates the imbalance in bone remodeling, thereby decreasing bone loss. Sex hormones are known to influence rheumatic diseases. The aim of this study was to investigate the effects of HRT on the serum levels of hormones and cytokines regulating bone turnover in 88 postmenopausal women with active rheumatoid arthritis (RA) randomly allocated to receive HRT plus calcium and vitamin D3 or calcium and vitamin D3 alone for 2 years. An increase in estradiol (E2) correlated strongly with improvement of bone mineral density in the hip (P < 0.001) and lumbar spine (P < 0.001). Both baseline levels and changes during the study of IL-6 and erythrocyte sedimentation rate were correlated positively (P < 0.001). HRT for 2 years resulted in an increase of the bone anabolic factor, insulin-like growth factor 1 (IGF-1) (P < 0.05) and a decrease of serum levels of soluble IL-6 receptor (sIL-6R) (P < 0.05), which is known to enhance the biological activity of IL-6, an osteoclast-stimulating and proinflammatory cytokine. Baseline levels of IL-6 and IGF-1 were inversely associated (P < 0.05), and elevation of IGF-1 was connected with decrease in erythrocyte sedimentation rate (P < 0.05) after 2 years. Interestingly, increase in serum levels of E2 was associated with reduction of sIL-6R (P < 0.05) and reduction of sIL-6R was correlated with improved bone mineral density in the lumbar spine (P < 0.05). The latter association was however not significant after adjusting for the effect of E2 (P = 0.075). The influences of IGF-1 and the IL-6/sIL-6R pathways suggest possible mechanisms whereby HRT may exert beneficial effects in RA. However, to confirm this hypothesis future and larger studies are needed.