Monitoring signal transduction in cancer: cDNA microarray for semiquantitative analysis.

This study targeted the development of a novel microarray tool to allow rapid determination of the expression levels of 58 different tyrosine kinase (tk) genes in small tumor samples. The goals were to define a reference probe for multi-sample comparison and to investigate the variability and reproducibility of the image acquisition and RT-PCR procedures. The small number of tk genes on our arrays enabled us to define a reference probe by artificially mixing all genes on the arrays. Such a probe provided contrast reference for comparative hybridization of control and sample DNA and enabled cross-comparison of more than two samples against one another. Comparison of signals generated from multiple scanning eliminated the concern of photo bleaching and scanner intrinsic noise. Tests performed with breast, thyroid, and prostate cancer samples yielded distinctive patterns and suggest the feasibility of our approach. Repeated experiments indicated reproducibility of such arrays. Up- or downregulated genes identified by this rapid screening are now being investigated with techniques such as in situ hybridization.     

Plant Yield and Nitrogen Content of a Digitgrass in Response to Azospirillum Inoculation

Two Australian soils, a vertisol (pH 6.8, 0.299% N) and a sandy yellow podzol (pH 6.2, 0.042% N), were used with digitgrass, Digitaria sp. X46-2 (PI 421785), in a growth room experiment. Comparisons were made between plants inoculated with live and autoclaved bacterial suspensions of Australian and Brazilian isolates of Azospirillum brasilense. Seedlings were inoculated on days 10 and 35. Acetylene-reducing activity was measured five times during the experiment. Dry matter yields of the digitgrass on the podzol (low N) inoculated with live bacteria were 23% higher than those of the controls. On the vertisol (high N), yield increases from inoculation with live bacteria were 8.5%. The higher-yielding plants had significantly lower percent nitrogen, but when total nitrogen of the tops was calculated, the inoculated plants had a higher total N than did the controls (P=0.04). Acetylene-reducing activity was variable in the experiment, ranging from 0.5 to 11.9 μmol of C₂H₄ core⁻¹ day⁻¹. Live bacterial treatment induced a proliferation of roots, possible earlier maturity, higher percent dry matter, and a higher total N in the tops.     

Neuroprotection against CCl4 induced brain damage with crocin in Wistar rats

Owing to its lipophilic property, carbon tetrachloride (CCl₄) is rapidly absorbed by both the liver and brain. We investigated the protective effects of crocin against brain damage caused by CCl₄. Fifty rats were divided into five groups of ten: control, corn oil, crocin, CCl₄ and CCl₄ + crocin. CCl₄ administration decreased glutathione (GSH) and total antioxidant status (TAS) levels, and catalase (CAT) activity, while significant increases were observed in malondialdehyde (MDA) and total oxidant status (TOS) levels and superoxide dismutase (SOD) activity. The cerebral cortex nuclear lamina developed a spongy appearance, neuronal degeneration was observed in the hippocampus, and heterochromatic and pyknotic neurons with increased cytoplasmic eosinophilia were observed in the hippocampus after CCl₄ treatment. Because crocin exhibits strong antioxidant properties, crocin treatment increased GSH and TAS levels and CAT activities, and decreased MDA and TOS levels and SOD activity; significant improvements also were observed in histologic architecture. We found that crocin administration nearly eliminated CCl₄ induced brain damage by preventing oxidative stress.     

Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries

Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few megabasepairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of overlapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wavelength intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific microdissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region-specific paints, but do not readily allow positioning of breakpoints on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses.     

Rational design of landmark probes for quantitative DNA fiber mapping (QDFM)

Rapid construction of high-resolution physical maps requires accurate information about overlap between DNA clones and the size of gaps between clones or clone contigs. We recently developed a procedure termed ‘quantitative DNA fiber mapping’ (QDFM) to help construct physical maps by measuring the overlap between clones or the physical distance between non-overlapping contigs. QDFM is based on hybridization of non-isotopically labeled probes onto DNA molecules that were bound to a solid support and stretched homogeneously to ~2.3 kb/µm. In this paper, we describe the design of probes that bind specifically to the cloning vector of DNA recombinants to facilitate physical mapping. Probes described here delineate the most frequently used cloning vectors such as BACs, P1s, PACs and YACs. As demonstrated in representative hybridizations, vector-specific probes provide valuable information about molecule integrity, insert size and orientation as well as localization of hybridization domains relative to specifically-marked vector sequences.     

Multilocus genetic analysis of single interphase cells by spectral imaging

Numerical chromosome aberrations are detrimental to early embryonic, fetal and perinatal development of mammals. When fetuses carrying a chromosomal imbalance survive to term, an aberrant gene dosage typically leads to stillbirth or causes a severely altered phenotype. Aneuploidy of any of the 24 chromosomes will negatively impact on human development, and a preimplantation and prenatal genetic diagnosis test should thus score as many chromosomes as possible. Since cells available for analysis are likely to be in interphase, we set out to develop a rapid enumeration procedure based on hybridization of chromosome-specific probes and spectral imaging detection. The probe set was chosen to allow the simultaneous enumeration of ten chromosome types and was expected to detect more than 70% of all numerical chromosome aberrations responsible for spontaneous abortions, i.e., human chromosomes 9, 13, 14, 15, 16, 18, 21, 22, X, and Y. Cell fixation protocols were optimized to achieve the desired detection sensitivity and reproducibility. We were able to resolve and identify ten separate chromosomal signals in interphase nuclei from different types of cells, including lymphocytes, uncultured amniocytes, and blastomeres. In summary, this study demonstrates the strength of spectral imaging, allowing us to construct partial spectral imaging karyotypes for individual interphase cells by assessing the number of each of the target chromosome types.     

sn-1-Acylglycerol-3-phosphate acyltransferase of Escherichia coli causes insertion of cis-11 eicosenoic acid into the sn-2 position of transgenic rapeseed oil

The plsC gene of Escherichia coli encoding sn-1-acylglycerol-3-phosphate acyltransferase was modified by inserting an endoplasmic reticulum retrieval signal to its 3 end and introduced into rapeseed (Brassica napus L.) plants under the control of a napin promotor. In developing seeds from transgenic plants an sn-1-acylglycerol-3-phosphate acyltransferase activity was detectable which showed substrate specificities typical of the E. coli enzyme. Moreover, seed oil from the transformants unlike that from untransformed plants contained substantial amounts of triacylglycerol species esterified with very-long-chain fatty acids at each glycerol position. Analysis of fatty acids at the sn-2 position of triacylglycerol showed hardly any very-long-chain fatty acids in untransformed plants, but in certain transformants these fatty acids were present, namely about 4% erucic acid and 9% eicosenoic acid. These data demonstrate that the bacterial acyltransferase can function in developing rapeseed and alters the stereochemical composition of transgenic rape seed oil by directing very-long-chain fatty acids, especially cis-11 eicosenoic acid, to its sn-2 position.     

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.     

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.