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.     

Comparative study of aflatoxins M1 and B1 production in solid-state and shaking liquid cultures

Production of aflatoxins M1 (AFM) and B1 (AFB) by Aspergillus flavus NRRL 3251 in solid-state and shaking liquid cultures using rice as the carbon source was compared. In general, solid-state cultures produced more aflatoxins than shaking liquid cultures on an equal rice weight basis. Solid-state cultures with continuous shaking yielded higher levels of toxins than those with intermittent shaking. However, intermittent shaking is a feasible replacement for the continuous shaking method for AFM production. A typical solid rice culture supplemented with yeast extract produced 30 and 2600 mg per kg rice of AFM and AFB, respectively, in 8 days at 29 degrees C. The optimal culture conditions for toxin production in a shaking liquid culture were also studied. Parameters under consideration included the amount of carbon (rice) and nitrogen source, initial medium pH, and aeration rate. At optimum conditions, a representative shaking liquid culture produced 18 and 1680 mg per kg rice of AFM and AFB, respectively, in 5 days at 29 degrees C. This shaking liquid culture appears feasible for scaling up and routine production of AFM and AFB for toxicological investigations.     

Lactose repressor protein modified with dansyl chloride: activity effects and fluorescence properties

Chemical modification using 5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl chloride) has been used to explore the importance of lysine residues involved in the binding activities of the lactose repressor and to introduce a fluorescent probe into the protein. Dansyl chloride modification of lac repressor resulted in loss of operator DNA binding at low molar ratios of reagent/monomer. Loss of nonspecific DNA binding was observed only at higher molar ratios, while isopropyl beta-D-thiogalactoside binding was not affected at any of the reagent levels studied. Lysine residues were the only modified amino acids detected. Protection of lysines-33 and -37 from modification by the presence of nonspecific DNA correlated with maintenance of operator DNA binding activity, and reaction of lysine-37 paralleled operator binding activity loss. Energy transfer between dansyl incorporated in the core region of the repressor protein and tryptophan-201 was observed, with an approximate distance of 23 A calculated between these two moieties.     

Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides.

A sequence-specific genomic delivery system for the correction of chromosomal mutations was designed by incorporating two different binding domains into a single-stranded oligonucleotide. A repair domain (RD) contained the native sequence of the target region. A third strand-forming domain (TFD) was designed to form a triplex by Hoogsteen interactions. The design was based upon the premise that the RD will rapidly form a heteroduplex that is anchored synergistically by the TFD. Deoxyoligonucleotides were designed to form triplexes in the human adenosine deaminase (ADA) and p53 genes adjacent to known point mutations. Transfection of ADA-deficient human lymphocytes corrected the mutant sequence in 1-2% of cells. Neither the RD or TFD individually corrected the mutation. Transfection of p53 mutant human glioblastoma cells corrected the mutation and induced apoptosis in 7.5% of cells.     

Effects of Suanzaorentang on behavior changes and central monoamines

Previously, it was found that the ancient Chinese remedy of Suanzaorentang could be a promising anxiolytic drug (Chen and Hsieh, 1985a, Chen and Hsieh, 1985b). To understand the mechanism of the action of Suanzaorentang, the effects of Suanzaorentang on behavior changes and central monoamines and their metabolites were studied in rats. It was found that Suanzaorentang significantly (1) prolonged the period from the onset of clonic to tonic convulsions induced by pentylenetetrazol or picrotoxin, (2) prolonged the sleep duration induced by hexobarbital, (3) reduced locomotor activity, (4) enhanced the hypomotility induced by alpha-MT, (5) reduced the locomotor stimulation produced by levodopa plus benserazide, and (6) reduced central HVA, VMA, and 5-HIAA, but had no significant effects on central DA, NA, and 5-HT. These facts implied that Suanzaorentang decreased the turnover rate of central monoamines and central catecholaminergic activity.     

Carrier detection and prenatal diagnosis of alpha-thalassemia of Southeast Asian deletion by polymerase chain reaction

Summary Alpha-thalassemia of Southeast Asian deletion (-- SEA/) is very common in Southeast Asia. Homozygosity of this genotype is the major cause of Hb Bart's hydrops fetalis in Taiwan. With polymerase chain reaction using three oligonucleotide primers bridging the common deletion breakpoint, a DNA fragment of 194 basepairs (bp) was amplified in chromosomes with the-- SEA determinant and a DNA fragment of 287 bp was amplified in chromosomes without this deletion. In our pilot study including 8 normal subjects, 20 obligate carriers, and 11 homozygotes of the deletion, all the genotypes were determined and then confirmed by Southern blotting and DNA hybridization with globin gene probe. For prenatal diagnosis, 55 at-risk pregnancies were collected. Chorionic villus sampling was done in 51 cases and early amniocentesis was done in 4 cases. Fourteen cases (25.5%) were diagnosed as normal, 25 (45.5%) as heterozygotes, and 16 (29%) as homozygotes of -- SEA. All of the diagnoses were also confirmed as aforementioned. With polymerase chain reaction, the determination of the -- SEA deletion is straightforward and is much quicker and easier than with conventional Southern blotting and DNA hybridization. In areas with a high prevalence of -- SEA deletion, this method provides a rapid tool for carrier detection and prenatal diagnosis.     

Electromagnetic mediated pharmacokinetics in a three-layer diffusional system

The effect of an applied electromagnetic field on drug diffusion in a one dimensional, three-layer drug-receptor model has been analyzed and expressed in terms of a normalized turnover rate parameter. The analysis reveals that an imposed harmonic time-varying electromagnetic field may enhance or retard the drug turnover rate depending on the diffusional pattern, the equivalent Michaelis constant, the maximum drug turnover rate of the intrinsic drug-receptor system, as well as the power density and frequency of the applied electromagnetic field. It is estimated that the power density in the order of magnitude of 1μW/cm² at 100 MHz frequency range may be required to induce significant rate effects.     

Microbial cooxidation of naphthalene for the production of 1,2-dihydroxy-1,2-dihydronaphthalene

A microbial cooxidation process for 1,2-dihydroxy-1,2-dihydronaphthalene from naphthalene has been demonstrated. A Pseudomonas putida it119 mutant strain grown with glucose as the sole carbon and energy source was used to oxidize naphthalene. Growth characteristics of the P. putida mutant strain were studied in both batch and continuous fermentation experiments. The rate of product formation was found to depend on naphthalene particle sizes, initial naphthalene and glucose concentrations. Kinetic models were developed to quantify the microbial cooxidation process and a two-stage fermentation process is proposed for further studies.     

Effects of Luteolin on Human Breast Cancer Using Gene Expression Array: Inferring Novel Genes

Taraxacum officinale (dandelion) is often used in traditional Chinese medicine for the treatment of cancer; however, the downstream regulatory genes and signaling pathways mediating its effects on breast cancer remain unclear. The present study aimed to explore the effects of luteolin, the main biologically active compound of T. officinale, on gene expression profiles in MDA-MB-231 and MCF-7 breast cancer cells. The results revealed that luteolin effectively inhibited the proliferation and motility of the MDA-MB-231 and MCF-7 cells. The mRNA expression profiles were determined using gene expression array analysis and analyzed using a bioinformatics approach. A total of 41 differentially expressed genes (DEGs) were found in the luteolin-treated MDA-MB-231 and MCF-7 cells. A Gene Ontology analysis revealed that the DEGs, including AP2B1, APP, GPNMB and DLST, mainly functioned as oncogenes. The human protein atlas database also found that AP2B1, APP, GPNMB and DLST were highly expressed in breast cancer and that AP2B1 (cut-off value, 75%) was significantly associated with survival rate (p = 0.044). In addition, a Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the DEGs were involved in T-cell leukemia virus 1 infection and differentiation. On the whole, the findings of the present study provide a scientific basis that may be used to evaluate the potential benefits of luteolin in human breast cancer. Further studies are required, however, to fully elucidate the role of the related molecular pathways.