Microchip module for blood sample preparation and nucleic acid amplification reactions.

A computer numerical control-machined plexiglas-based microchip module was designed and constructed for the integration of blood sample preparation and nucleic acid amplification reactions. The microchip module is comprised of a custom-made heater-cooler for thermal cycling, a series of 254 microm x 254 microm microchannels for transporting human whole blood and reagents in and out of an 8--9 microL dual-purpose (cell isolation and PCR) glass-silicon microchip. White blood cells were first isolated from a small volume of human whole blood (<3 microL) in an integrated cell isolation--PCR microchip containing a series of 3.5-microm feature-sized "weir-type" filters, formed by an etched silicon dam spanning the flow chamber. A genomic target, a region in the human coagulation Factor V gene (226-bp), was subsequently directly amplified by microchip-based PCR on DNA released from white blood cells isolated on the filter section of the microchip mounted onto the microchip module. The microchip module provides a convenient means to simplify nucleic acid analyses by integrating two key steps in genetic testing procedures, cell isolation and PCR and promises to be adaptable for additional types of integrated assays.

Agilent 2100 bioanalyzer for restriction fragment length polymorphism analysis of the Campylobacter jejuni flagellin gene.

The Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.) utilizes capillary electrophoresis on a microchip device (LabChip 7500; Caliper Technologies, Mountain View, Calif.) that is capable of rapidly sizing small DNA fragments. To determine whether the system could replace conventional restriction fragment length polymorphism (RFLP) typing by agarose gel electrophoresis, we compared the analyzer with conventional flagellin RFLP for typing Campylobacter jejuni. Ninety-seven isolates representing 46 Fla types were initially analyzed. Correct Fla types were detected in 59% of the isolates. The major problem with the system was in resolving samples containing multiple DNA fragments differing from 8 to 20 bp. Overall, the bioanalyzer has the potential to replace conventional RFLP analysis by gel electrophoresis, but improvements in the chip separation are needed.

Oxidative stress contributes to the anti-proliferative effects of flavone acetic acid on endothelial cells.

The synthetic flavonoid flavone acetic acid (FAA) has anti-tumor activity against a variety of transplanted tumors in mice through mechanisms which likely involve effects on tumor vasculature and the host immune system. The aims of the present in vitro study were to compare the sensitivity of tumor and endothelial cells to FAA treatment and to assess if nitric oxide and superoxide are involved in the FAA-mediated suppression of cell proliferation. FAA at 1 mM concentration was approximately two times more effective in suppressing proliferation of endothelial than tumor cells. The anti-proliferative effect of 1 mM FAA on endothelial cells was partially blocked by inhibitors to various superoxide-producing enzymes (xanthine oxidase, cyclooxygenase, poly-ADP-ribose polymerase, ribonucleotide reductase) and completely inhibited by the direct scavengers of superoxide lucigenin and Tiron. In contrast, inhibitors of nitric oxide were unable to prevent the effects of FAA on proliferation. FAA induced apoptosis of endothelial cells, which was not affected by inhibitors of nitric oxide or superoxide. Our data imply that FAA inhibits proliferation of endothelial cells by a superoxide-dependent mechanism and induces apoptosis by a nitric oxide and superoxide-independent mechanism.

Flavone acetic acid induces a G2/M cell cycle arrest in mammary carcinoma cells.

Flavone acetic acid (FAA) is a synthetic flavonoid that demonstrated extraordinary anti-tumour properties in murine models but was not effective in clinical trials. In an effort to better understand the molecular mechanisms by which FAA asserts its tumouricidal activities, we have examined the effect of FAA on the cell cycle. We observed FAA-mediated G2/M cell cycle arrest in mammary carcinoma cells at a concentration previously demonstrated to have anti-tumour effects in rodent models. The cell cycle arrest was accompanied by an increase in the P34cdc2 (cdc2) cyclin-dependent kinase activity. Morphological cytogenetic analysis demonstrated a colcemid-like effect of FAA on cytokinesis by causing accumulation of condensed C-metaphases of a sustained mitotic block. The cell cycle effect was blocked by the antioxidants ADPC and ascorbate, the superoxide scavenger Tiron, and the sphingosine kinase inhibitor L-cycloserine, but not by inhibitors of nitric oxide synthase. Based on these data, we propose that FAA may induce cell cycle arrest by stimulating the activity of acidic sphingomyelinase leading to the generation of reactive oxygen species.

Tetraparesis following dental extraction: case report and discussion of preventive measures for cervical spinal hyperextension injury.

This concerns a patient with compression myelopathy following passive hyperextension of the cervical spine during a dental procedure. Although he had been asymptomatic prior to the procedure, subsequent cervical spinal imaging revealed advanced spondylosis and spinal stenosis. Spinal stenosis is often asymptomatic for a long time. However, when radiculomyelopathy occurs after minor trauma to the head or neck, the patient is often found to have spinal stenosis. Specifically, hyperextension of a cervical spine with spondylotic changes can lead to compression myelopathy. Acquired spinal stenosis correlates positively with aging. As the size of the elderly population continues to increase the prevalence of cervical spondylotic radiculo-myelopathy will likely increase as well. Since appropriate precautions against potential neurologic damage can be undertaken, we suggest radiographic screening for pre-existing spinal stenosis prior to a procedure requiring hyperextension of the neck. Preventive measures for individuals with asymptomatic spondylotic changes and education of all health-care professionals to avoid abrupt or prolonged hyperextension of the cervical spine is emphasized.

Intrapartum transmission of group A streptococcus.

Intrapartum transmission of group A streptococcus has not been well documented. As the incidence of severe infections due to this organism has recently increased, it is important to assess if such transmission occurs. We observed two cases of severe neonatal infections due to group A streptococcus, one of which was fatal, that appeared to have been transmitted from the mother during birth. Perinatal prophylaxis, which has been recommended for infections due to group B streptococcus, should be evaluated for infections due to group A streptococcus.

Regulation of genetic expression in shear stress-stimulated endothelial cells.

There is increasing evidence that endothelial cells respond to the initiation of mechanical stress by the generation of certain second messengers and the activation of specific metabolic pathways. These rapid alterations in cellular function are accompanied by alterations in protein synthesis that are detectable several hours after initiation of the mechanical stress. The molecular mechanisms by which changes in the cytosol are converted to altered genetic expression in the nucleus are not known. Because agonist-induced modulations in the rate of synthesis of tPA and ET have been associated with the Fos and Jun protein families, it seems reasonable to propose that genetic expression in shear stress- or mechanical strain-stimulated endothelial cells is also regulated by selective induction of fos and jun gene products. Testing of this hypothesis is actively under way in our laboratory.