Two-parameter data acquisition system for rapid slit-scan analysis of mammalian chromosomes

A data acquisition system is described for recording two independent signals simultaneously from a laser-based flow cytometer for rapid slit-scan chromosome analysis. High-aperture microscope optics allow recording of fluorescence distributions along the longest axis of metaphase chromosomes with a spatial resolution better than 1 micron. Fluorescence and small angle forward light scatter as well as dual-wavelength fluorescence signals from Indian muntjac chromosomes stained with propidium iodide (PI) or acridine orange (AO) have been recorded simultaneously. While maintaining the multi-user operation of the computer, photomultiplier signals are digitized at a rate of 400 signals per second, stored temporarily in high-speed cache memories, and transferred subsequently to a minicomputer for further storage. Extensive software packages for data acquisition, analysis, and display of the results are described. Data acquisition is generally done in list mode, allowing complete reconstruction of individual signals (profiles) at any time. The distribution of stained constituents along the chromosomes can be displayed. Furthermore, histograms of various parameters of the input signals may be generated.     

Seasonality of Rotifers and Temperature in Lough Neagh,N. Ireland

Long runs of seasonal rotifer population data allow analysis of seasonal occurrence using mathematical tools. The application of Fourier analysis to a 15 year dataset describes seasonality in simple mathematical terms. This facilitates comparison of population expression with potential population driving variables and provides a basic modelling tool. Results show that annual patterns of occurrence and density have linkages with annual maximum and minimum environmental temperature, although the exact relationships are not clear.     

Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.     

Aminopyridinecarboxamide-based inhibitors: Structure–activity relationship

Series of aminopyridinecarboxamide-based inhibitors were synthesized and tested against human recombinant IKK-2 and in IL-1β stimulated synovial fibroblasts. The 2-amino-5-chloropyridine-4-carboxamides were identified as the most potent inhibitors with improved cellular activity.     

Chromosome-specific DNA repeat probes.

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with alpha-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.     

Takayasu's arteritis is associated with HLA-B*52, but not with HLA-B*51, in Turkey

Introduction

HLA-B*51 and HLA-B*52 are two close human leukocyte antigen (HLA) allele groups with minor amino acid differences. However, they are associated with two different vasculitides (HLA-B*51 in Behçet's disease and HLA-B*52 in Takayasu's arteritis (TAK)) and with major clinical and immunological differences. In this study, we aimed to screen a large cohort of TAK patients from Turkey for the presence of HLA-B*51 and HLA-B*52 as susceptibility and severity factors.

Methods

TAK patients (n = 330) followed at a total of 15 centers were included in the study. The mean age of the patients was 37.8 years, and 86% were women. DNA samples from the patients and healthy controls (HC; n = 210) were isolated, and the presence of HLA-B*51 or HLA-B*52 was screened for by using PCR with sequence-specific primers.

Results

We found a significant association of HLA-B*52 with TAK (20.9% vs HC = 6.7%, P = 0.000, OR = 3.7, 95% CI = 2.02 to 6.77). The distribution of HLA-B*51 did not differ between TAK patients and HCs (22.7% vs 24.8%, OR = 0.9, 95% CI = 0.60 to 1.34). The presence of HLA-B*52 decreased in late-onset patients (> 40 years of age; 12.0%, P = 0.024, OR = 0.43, 95% CI = 0.20 to 0.91). Patients with angiographic type I disease with limited aortic involvement also had a lower presence of HLA-B*52 compared to those with all other disease subtypes (13.1% vs 26%, P = 0.005, OR = 0.43, 95% CI = 0.23 to 0.78).

Conclusions

In this study, the previously reported association of TAK with HLA-B*52 in other populations was confirmed in patients from Turkey. The functional relevance of HLA-B*52 in TAK pathogenesis needs to be explored further.     

Monitoring signal transduction in cancer: tyrosine kinase gene expression profiling.

Abnormal expression of tyrosine kinase (TK) genes is common in tumors, in which it is believed to alter cell growth and response to external stimuli such as growth factors and hormones. Although the etiology and pathogenesis of carcinomas of the thyroid or breast remain unclear, there is evidence that the expression of TK genes, such as receptor tyrosine kinases, or mitogen-activated protein kinases, is dysregulated in these tumors, and that overexpression of particular TK genes due to gene amplification, changes in gene regulation, or structural alterations leads to oncogenic transformation of epithelial cells. We developed a rapid scheme to measure semiquantitatively the expression levels of 50-100 TK genes. Our assay is based on RT-PCR with mixed based primers that anneal to conserved regions in the catalytic domain of TK genes to generate gene-specific fragments. PCR products are then labeled by random priming and hybridized to DNA microarrays carrying known TK gene targets. Inclusion of differently labeled fragments from reference or normal cells allows identification of TK genes that show altered expression levels during malignant transformation or tumor progression. Examples demonstrate how this innovative assay might help to define new markers for tumor progression and potential targets for disease intervention. (J Histochem Cytochem 49:673-674, 2001)     

ROLE OF DICTYOSOMES IN WALL FORMATION DURING CELL DIVISION OF CHLORELLA VULGARIS

The behavior of dictyosomes in wall formation during cell division of Chlorella vulgaris follows a definite pattern. During formation of the partition membrane they migrate into the equatorial plane and pair. There is a close spatial relationship between the dictyosomes and the partition membrane which, itself, may be derived from the fusion of dictyosomal vesicles. Dictyosomes also may participate significantly in the deposition of new wall material.