Fabrication of plastic microfluid channels by imprinting methods

Microfluidic devices have been fabricated on poly(methyl methacrylate) substrates by two independent imprinting techniques. First-generation devices were fabricated using a small-diameter wire to create an impression in plastics softened by low-temperature heating. The resulting devices are limited to only simple linear channel designs but are readily produced at low cost. Second-generation devices with more complex microchannel arrangements were fabricated by imprinting the plastic substrates using an inverse three-dimensional image of the device micromachined on a silicon wafer. This micromachined template may be used repeatedly to generate devices reproducibly. Fluorescent analtyes were used to demonstrate reproducible electrophoretic injections. An immunoassay was also performed in an imprinted device as a demonstration of future applications.     

The satellite RNA of barley yellow dwarf virus-RPV is supported by beet western yellows virus in dicotyledonous protoplasts and plants

The subgroup II luteovirus barley yellow dwarf virus-RPV (BYDV-RPV) acts as a helper virus for a satellite RNA (satRPV RNA). The subgroup II luteovirus beet western yellows virus (BWYV) and the ST9-associated RNA (ST9a RNA), a BWYV-associated RNA that encodes a polymerase similar to those of subgroup I luteoviruses, were assayed for their ability to support replication of satRPV RNA. SatRPV RNA was replicated in tobacco protoplasts in the presence of BWYV RNA or a mixture of BWYV plus the ST9a RNA, but not in the presence of ST9a RNA alone. ST9a RNA stimulated BWYV RNA accumulation which, in turn, increased the accumulation of satRPV RNA. SatRPV RNA was encapsidated in BWYV capsids primarily as circular monomers, which differs from the linear monomers found in BYDV (RPV + PAV) particles. SatRPV RNA was transmitted to Capsella bursa-pastoris plants by aphids only in the presence of BWYV and ST9a RNA. SatRPV RNA reduced accumulation of both BWYV helper and ST9a nonhelper RNAs in plants but did not affect symptoms. The replication of satRPV RNA only in the presence of subgroup II luteoviral RNAs but not in the presence of RNAs with subgroup I-like polymerase genes, in both monocotyledonous and dicotyledonous hosts, suggests that the specificity determinants of satRPV RNA replication are contained within the polymerase genes of supporting viruses rather than in structural genes or host plants.     

Effect of CYP2D1 inhibition on the behavioural effects of d-amphetamine

In rats, amphetamine (AMP) conversion to 4-OH-AMP is metabolized by CYP2D1, the rat equivalent of the human enzyme CYP2D6. To determine the impact of impaired AMP metabolism on its behavioural effects, AMP-induced hyperactivity, AMP discrimination and AMP self-administration were examined in male Wistar rats with or without pretreatment with the CYP2D1 inhibitors quinine and budipine. In vivo, quinine (20 mg/kg) and budipine (10 mg/kg) increased the plasma area under the curve of AMP 4-fold and 3.6-fold respectively, and decreased the plasma levels of 4-OH-AMP, 3-fold and 8.6-fold, confirming that the doses used suppressed CYP2D1 activity. Both inhibitors prolonged AMP-induced hyperactivity (0.3 mg/kg) and prolonged the duration of AMP-appropriate responding for periods of up to 90 min post-AMP administration in a drug discrimination procedure. In rats given a preload dose of AMP (0.8 mg/kg) 3 h prior to the self-administration test session, CYP2D1 inhibition resulted in fewer AMP infusions being taken compared with rats receiving the AMP preload dose alone. These studies indicate that AMP is responsible for the behavioural effects seen in rats and that a rat phenocopy model of the human CYP2D6 deficiency state can be produced by CYP2D1 inhibitors.     

Widely dispersed p53 mutation in respiratory epithelium. A novel mechanism for field carcinogenesis

Individuals with one aerodigestive tract malignancy have a high incidence of second primary aerodigestive tumors. The mechanism for this field effect has not been determined. We studied an individual with widespread dysplastic changes in the respiratory epithelium but no overt carcinoma. The entire tracheobronchial tree obtained at autopsy was embedded in paraffin, and bronchial epithelial cells were isolated by microdissection. DNA extracted from the microdissected cells was analyzed for point mutations in the p53 tumor suppressor gene. A single, identical point mutation consisting of a G:C to T:A transversion in codon 245 was identified in bronchial epithelium from 7 of 10 sites in both lungs. Epithelium at sites containing the p53 mutation was morphologically abnormal, exhibiting squamous metaplasia and mild to moderate atypia. No invasive tumor was found in the tracheobronchial tree or any other location. Cells from peripheral blood, kidney, liver, and lymph node exhibited no abnormality in the p53 gene. The widespread presence of a single somatic p53 point mutation in the bronchi of a smoker suggests that a single progenitor bronchial epithelial clone may expand to populate broad areas of the bronchial mucosa-a novel mechanism for field carcinogenesis in the respiratory epithelium that may be of importance in assessing individuals for risk of a second primary tumor as well as in devising effective strategies for chemoprevention of lung cancer.     

Structural and thermodynamic characteristics of the binding of the lambda N protein to a RNA hairpin

The specific binding of the lambda N protein to a 15 nucleotide RNA oligomer that forms a hairpin structure has been investigated by biophysical methods. Using fluorescence spectroscopy and equilibrium ultra-centrifugation, it was found that the N protein binds specifically to this RNA hairpin as a monomer. Circular dichroism experiments show that both the N protein and the RNA hairpin undergo structural change upon association of the complex.     

Immunochemical identification and differential phosphorylation of alternatively spliced forms of the alpha 1A subunit of brain calcium channels

Biochemical properties of the alpha 1 subunits of class A brain calcium channels (alpha 1A) were examined in adult rat brain membrane fractions using a site-directed anti-peptide antibody (anti-CNA3) specific for alpha 1A. Anti-CNA3 specifically immunoprecipitated high affinity receptor sites for omega-conotoxin MVIIC (Kd approximately 100 pM), but not receptor sites for the dihydropyridine isradipine or for omega-conotoxin GVIA. In immunoblotting and immunoprecipitation experiments, anti-CNA3 recognized at least two distinct immunoreactive alpha 1A polypeptides, a major form with an apparent molecular mass of 190 kDa and a minor, full-length form with an apparent molecular mass of 220 kDa. The 220- and 190-kDa alpha 1A polypeptides were also specifically recognized by both anti-BI-Nt and anti-BI-1-Ct antibodies, which are directed against the NH2- and COOH-terminal ends of alpha 1A predicted from cDNA sequence, respectively. These data indicate that the predicted NH2 and COOH termini are present in both size forms and therefore that these isoforms of alpha 1A are created by alternative RNA splicing rather than post-translational proteolytic processing of the NH2 or COOH termini. The 220-kDa form was phosphorylated preferentially by cAMP-dependent protein kinase, whereas protein kinase C and cGMP-dependent protein kinase preferentially phosphorylated the 190-kDa form. Our results identify at least two distinct alpha 1A subunits with different molecular mass, demonstrate that they may result from alternative mRNA splicing, and suggest that they may be differentially regulated by protein phosphorylation.     

Genomic imprinting and Igf2 influence liver tumorigenesis and loss of heterozygosity in SV40 T antigen transgenic mice

Maternal-specific loss of heterozygosity (LOH) and allelic imbalances [i.e., partial LOH (pLOH)] observed in SV40 T/t antigen-induced liver tumors suggests that an imprinted gene on chromosome 7 is involved in liver tumorigenesis. Maternal-specific LOH/pLOH may reflect the loss of a maternally expressed tumor suppressor gene or the acquisition of paternally active alleles of a growth promoter. In addition, two oppositely imprinted genes on distal chromosome 7, Igf2 and H19, are re-expressed in most liver tumors from an SV40 T/t antigen transgenic line (M11T-G). Igf2 is a paternally expressed growth promoter, and H19 is a maternally expressed gene that can suppress growth in some tumor cell lines. We studied the role of Igf2 during liver tumorigenesis by creating Igf2 (+/-) M11T-G mice. These mice are essentially null for Igf2 expression because imprinting normally precludes maternal Igf2 expression. M11T-G, Igf2 (+/-) males exhibit a 15-fold reduction in the frequency of large tumors. Igf2 (+/-) tumors do not express maternal Igf2, indicating rigid imprinting control in the liver. LOH/pLOH analysis was performed on the tumors and indicates that acquisition of paternally active Igf2 alleles is a major selective event for M11T-G liver tumorigenesis. This also implies the existence of an imprinted, maternally expressed tumor suppressor gene on chromosome 7 that is unlikely to be H19.     

The role of low-molecular-weight heparin in unstable angina, acute myocardial infarction and post-elective percutaneous transluminal coronary angioplasty

A young woman with a large astigmatic refractive error obtained no visual improvement with glasses. Repeated manifest refractions revealed persistent variations in the apparent cylinder axis. A suitable choice of spectacle frames facilitated a satisfactory outcome. The pitfalls inherent in the clinical specification of cylinder axis, and the potential visual consequences, are discussed.