Capillary electrophoresis with laser-induced fluorescence detection for the analysis of free and immune-complexed green fluorescent protein.

Naturally fluorescing green fluorescent protein (GFP) was separated by capillary electrophoresis (CE) and detected by laser-induced fluorescence (LIF). Exploiting recombinant technology and the natural fluorescence of GFP presents the capability of preempting the need for fluorescent derivatization. Such an approach would circumvent the obstacles typically associated with covalently labeling and purifying analytes that undergo fluorescent labeling. The unique property of GFP to fluoresce naturally was combined with CE-LIF to compare GFP isoforms prepared recombinantly or in vitro with wild-type GFP isoforms isolated from native jellyfish Aequorea victoria. Second, GFP antisera were reacted with wild-type GFP and the formation of the GFP antigen-antibody complex was monitored. A simple borate buffer, pH 8.5, was ample for resolving the two isoforms of the naturally fluorescent GFP in less than 5 min. The separation of GFP from GFP-Ab was complete in less than 7 min with the individual components detectable at the picogram level. A number of factors influence CE separation and/or LIF detection including sample buffer pH and incorporation of the additive 1,4-diamino butane. Remarkably, conditions that severely impair fluorescence detection of free GFP do not diminish fluorescence detection of the GFP antigen-antibody complex in a similar manner. Thus, the antibody appears to preserve the natural fluorophore of GFP. These data lend credence to the utility of coupling naturally fluorescent GFP to the speed, automation, and reduced sample size benefits of CE-LIF analysis for efficient separation and detection of an immunoreaction. In principle, a fusion protein of antibody with GFP as the label in CE-based immunoassays offers an advantageous alternative to the fluor-labeling process usually required in LIF detection.

Long term exposure to manganese in rural well water has no neurological effects.

BACKGROUND: There is debate on the neurological impact of chronic exposure to Manganese (MN). METHODS: MN burden from rural well water was studied cross-sectionally in two proband cohorts from rural dwellings located in northern Germany. Both cohorts had exposure times for up to 40 years and were separated on the basis of well water MN content. Group A (41 subjects; mean age 57.5 years) was exposed to MN water contents of at least 0.300 mg/l (range 0.300 to 2.160), while group B (74 subjects; mean age 56.9 years) was exposed to concentrations of less than 0.050 mg/l. Both proband groups were homogenous with regard to age, sex, nutritional habits, and drug intake. Neurological assessments by clinical investigators blinded for proband's exposure status was done using structured questionnaires, standardized neurological examination with assessment of possible Parkinsonian signs by the Columbia University Rating Scale, and instrumental tests of fine motor coordination. RESULTS: No significant difference in any neurological measure was found between groups. Results were not confounded by demographic and dietary features. CONCLUSION: Exposure to high body burden of MN does not result in detectable neurological impairment. Exposure to MN in drinking water does not seem to be a risk factor for idiopathic Parkinson's disease.

Proposed semi-analytical formulae for the determination of (L/rho)medair and Prepl for electron beams as used in radiotherapy.

To convert an ionization measurement to absorbed dose, the TG-21, the IAEA, and the TG-25 protocols for the calibration of high energy electron therapy machines demand an accurate knowledge of the mean restricted collision mass stopping power ratio, (L/rho)medair, and the electron fluence correction factor, Prepl. This paper presents a semi-analytical expression to calculate (L/rho)medair at depths in the range of 0 to 30 g/cm2 and for any energy in the range of 4-60 MeV, for water, polystyrene, and acrylic relative to air, and another expression to calculate Prepl for any cylindrical ionization chamber with an inner diameter in the range of 3 to 7 mm. The published values for (L/rho)medair were fitted to a single analytical expression with 10 coefficients using a nonlinear least square routine. Another expression with 4 coefficients was used to fit the electron fluence correction factor, Prepl, for cylindrical ionization chambers, as a function of chamber diameter and the mean electron energy at depth of measurement. In the electron energy range of 4-60 MeV, and for all published depths, the calculated (L/rho)medair values agree with the published values within +/- 1% in 99% of the cases. The calculated Prepl values agree with the tabulated values within +/- 1% in all of the cases. The proposed equations now allow one to use a computer to calculate the values of (L/rho)medair and Prepl accurately, doing away with the time-consuming procedure of interpolating between values from tables during the routine calibration of high energy electron therapy machines.

Activation of c-fos gene expression by a kinase-deficient epidermal growth factor receptor.

The intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR) has been shown to be responsible for many of the pleiotropic intracellular effects resulting from ligand stimulation [W.S. Chen, C.S. Lazar, M. Poenie, R.Y. Tsien, G.N. Gill, and M.G. Rosenfeld, Nature (London) 328:820-823, 1987; A.M. Honegger, D. Szapary, A. Schmidt, R. Lyall, E. Van Obberghen, T.J. Dull, A. Ulrich, and J. Schlessinger, Mol. Cell. Biol. 7:4568-4571, 1987]. Recently, however, it has been shown that addition of ligand to cells expressing kinase-defective EGFR mutants can result in the phosphorylation of mitogen-activated protein kinase (R. Campos-González and J.R. Glenney, Jr., J. Biol. Chem. 267:14535-14538, 1992; E. Selva, D.L. Raden, and R.J. Davis, J. Biol. Chem. 268:2250-2254, 1993), as well as stimulation of DNA synthesis (K.J. Coker, J.V. Staros, and C.A. Guyer, Proc. Natl. Acad. Sci. USA 91:6967-6971, 1994). Moreover, mitogen-activated protein kinase has been shown to phosphorylate the transcription factor p62TCF in vitro, leading to enhanced ternary complex formation between p62TCF, p67SRF, and the c-fos serum response element (SRE) [H. Gille, A.D. Sharrocks, and P.E. Shaw, Nature (London) 358:414-417, 1992]. On the basis of these observations, we have investigated the possibility that the intrinsic tyrosine kinase activity of the EGFR may not be necessary for transcriptional activation mediated via p62TCF. Here, we demonstrate that a kinase-defective EGFR mutant can signal ligand-induced expression of c-fos protein and that a significant component of this induction appears to be mediated at the transcriptional level. Investigation of transcriptional activation mediated by the c-fos SRE shows that this response is impaired by mutations in the SRE which eliminate binding of p62(TCF). These data indicate that information inherent in the structure of the EGFR can be accessed by ligand stimulation independent of the receptor's catalytic kinase function.

Activation of a muscle-specific actin gene promoter in serum-stimulated fibroblasts.

Treatment of AKR-2B mouse fibroblasts with serum growth factors or inhibitors of protein synthesis, such as cycloheximide, results in a stimulation of cytoskeletal beta-actin transcription but has no effect on transcription of muscle-specific isotypes, such as the vascular smooth muscle (VSM) alpha-actin gene. Deletion mapping and site-specific mutagenesis studies demonstrated that a single "CArG" element of the general form CC(A/T)6GG was necessary and possibly sufficient to impart serum and cycloheximide-inducibility to the beta-actin promoter. Although the VSM alpha-actin promoter exhibits at least three similar sequence elements, it remained refractory to serum and cycloheximide induction. However, deletion of a 33 base pair sequence between -191 and -224 relative to the transcription start site resulted in the transcriptional activation of this muscle-specific promoter in rapidly growing or serum-stimulated fibroblasts. Although the activity of this truncated promoter was potentiated by cycloheximide in a manner indistinguishable from that of the beta-actin promoter, this was dependent on a more complex array of interacting elements. These included at least one CArG box and a putative upstream activating element closely associated with the -191 to -224 inhibitory sequences. These results demonstrate that the expression of a muscle-specific actin gene in fibroblasts is suppressed by a cis-acting negative control element and that in the absence of this element, the promoter is responsive to growth factor-induced signal transduction pathways.

Developmental and tissue-specific expression of U4 small nuclear RNA genes.

U4 RNA is one of several small nuclear RNAs involved in the splicing of mRNA precursors. The domestic chicken has two genes per haploid genome that are capable of encoding U4 RNA. The U4X RNA gene (which encodes a sequence variant of U4 RNA that was unknown prior to the cloning of the gene) and the U4B RNA gene were both expressed in vivo in each of seven adult and three embryonic chicken tissues examined. However, the ratio of U4B RNA to U4X RNA can vary more than sevenfold in both a tissue- and stage-specific manner.

Structural and functional analysis of chicken U4 small nuclear RNA genes.

Two distinct chicken U4 RNA genes have been cloned and characterized. They are closely linked within 465 base pairs of each other and have the same transcriptional orientation. The downstream U4 homology is a true gene, based on the criteria that it is colinear with chicken U4B RNA and is expressed when injected into Xenopus laevis oocytes. The upstream U4 homology, however, contains seven base substitutions relative to U4B RNA. This sequence may be a nonexpressed pseudogene, but the pattern of base substitutions suggests that it more probably encodes a variant yet functional U4 RNA product not yet characterized at the RNA level. In support of this, the two U4 genes have regions of homology with each other in their 5'-flanking DNA at two positions known to be essential for the efficient expression of vertebrate U1 and U2 small nuclear RNA genes. In the case of U1 and U2 RNA genes, the more distal region (located near position-200 with respect to the RNA cap site) is known to function as a transcriptional enhancer. Although this region is highly conserved in overall structure and sequence among U1 and U2 RNA genes, it is much less conserved in the chicken U4 RNA genes reported here. Interestingly, short sequence elements present in the -200 region of the U4 RNA genes are inverted (i.e., on the complementary strand) relative to their usual orientation upstream of U1 and U2 RNA genes. Thus, the -200 region of the U4 RNA genes may represent a natural evolutionary occurrence of an enhancer sequence inversion.

Chicken U2 and U1 RNA genes are found in very different genomic environments but have similar promoter structures.

We have cloned and analyzed a gene that codes for chicken U2 small nuclear RNA (snRNA). In the haploid chicken genome, there are approximately 35-40 copies of the U2 RNA gene arranged in tandemly repeated units 5.35 kilobase pairs in length. This U2 gene organization contrasts with that of chicken U1 RNA genes, which are found in heterogeneous genomic environments. Although U snRNA genes are transcribed by RNA polymerase II, they lack the usual TATA and CAAT homologies found in the 5' control regions of most RNA polymerase II transcription units. Nevertheless, a comparison of chicken U2 and U1 RNA gene 5'-flanking DNA sequences reveals two upstream blocks of homology which are also evolutionarily conserved in U2 and U1 RNA genes of other vertebrate species. The first block of conserved sequence is centered around position -55 relative to the RNA cap site, and the other is located near position -200. Interestingly, stretches of sequence with the potential to form Z DNA are located either within or immediately adjacent to both of these two conserved upstream sequence elements, suggesting a possible role for Z DNA in U1/U2 gene expression. Moreover, the chicken U2 and U1 gene promoter regions also contain specific short sequences (i.e., the hexamer GGGCGG and the octamer ATGCAAAT) that have been shown to be required for the expression of a number of mRNA-encoding genes. These findings suggest that the transcription of snRNA genes is controlled by a complex set of factors, some shared with other RNA polymerase II transcription units and others which may be unique to the snRNA genes.