New methods for determining the enantiomeric purity of erythro -sphingosine.
(17/5815)
The enantiomeric purity of erythro -sphingosine samples can be determined simply, reliably, and accurately from 1H or 19F nuclear magnetic resonance spectra of the alpha-methoxy-alpha-(trifluoromethyl)phenylacetate (MTPA) derivative. As little as 0.1% of the minor enantiomer could be observed in a 1-mg sample, and detection limits of 1% and 5% were estimated for samples of 100 microg and 10 microg. The two threo -sphingosine enantiomers and four dihydrosphingosine stereoisomers were also differentiated by this technique, which served as an effective method for assessing the purity of sphingosine and dihydrosphingosine samples. Enantiomeric and diastereomeric purities could also be determined by normal-phase high performance liquid chromatographic analysis of the MTPA derivatives. (+info)
Human African trypanosomiasis: a latex agglutination field test for quantifying IgM in cerebrospinal fluid.
(18/5815)
LATEX/IgM, a rapid agglutination test for the semi-quantitative detection of IgM in cerebrospinal fluid of patients with African trypanosomiasis, is described in this article. The lyophilized reagent has been designed for field use and remains stable at 45 degrees C for one year. The test has been evaluated on cerebrospinal fluid samples from trypanosome-infected and non-infected patients, by comparison with commercial latex agglutination, radial immunodiffusion, and nephelometry. All test systems yielded similar results. (+info)
Molecular cloning and characterization of a cDNA for an iron-superoxide dismutase in rice (Oryza sativa L.).
(19/5815)
We have isolated a cDNA encoding Fe-SOD from rice (Oryza sativa L.). The deduced amino acid sequence consists of a polypeptide with 255 amino acids, including a putative transit peptide (40 a.a.) in amino-terminal residues. This sequence is similar to the known plant Fe-SODs but not classified in the group of known Fe-SODs. The metal analysis and SOD assays of the partial purified recombinant protein expressed in E. coli showed that this cDNA encodes an iron-containing SOD. However this SOD activity was not inhibited by the treatment with hydrogen peroxide, which was expected to inhibit known Fe-SOD activity. mRNA of rice Fe-SOD was detected in all vegetative tissues examined, being especially abundant in calli, and strongly increased by light induction. These results suggested that this cDNA encodes rice Fe-SOD, which is apparently distinct from known plant Fe-SODs. (+info)
NMDAR channel segments forming the extracellular vestibule inferred from the accessibility of substituted cysteines.
(20/5815)
In NMDA receptor channels, the M2 loop forms the narrow constriction and the cytoplasmic vestibule. The identity of an extracellular vestibule leading toward the constriction remained unresolved. Using the substituted cysteine accessibility method (SCAM), we identified channel-lining residues of the NR1 subunit in the region preceding M1 (preM1), the C-terminal part of M3 (M3C), and the N-terminal part of M4 (M4N). These residues are located on the extracellular side of the constriction and, with one exception, are exposed to the pore independently of channel activation, suggesting that the gate is at the constriction or further cytoplasmic to it. Permeation of Ca2+ ions was decreased by mutations in M3C and M4N, but not by mutations in preM1, suggesting a functionally distinct contribution of the segments to the extracellular vestibule of the NMDA receptor channel. (+info)
Functional expression of exogenous proteins in mammalian sensory hair cells infected with adenoviral vectors.
(21/5815)
To understand the function of specific proteins in sensory hair cells, it is necessary to add or inactivate those proteins in a system where their physiological effects can be studied. Unfortunately, the usefulness of heterologous expression systems for the study of many hair cell proteins is limited by the inherent difficulty of reconstituting the hair cell's exquisite cytoarchitecture. Expression of exogenous proteins within hair cells themselves may provide an alternative approach. Because recombinant viruses were efficient vectors for gene delivery in other systems, we screened three viral vectors for their ability to express exogenous genes in hair cells of organotypic cultures from mouse auditory and vestibular organs. We observed no expression of the genes for beta-galactosidase or green fluorescent protein (GFP) with either herpes simplex virus or adeno-associated virus. On the other hand, we found robust expression of GFP in hair cells exposed to a recombinant, replication-deficient adenovirus that carried the gene for GFP driven by a cytomegalovirus promoter. Titers of 4 x 10(7) pfu/ml were sufficient for expression in 50% of the approximately 1,000 hair cells in the utricular epithelium; < 1% of the nonhair cells in the epithelium were GFP positive. Expression of GFP was evident as early as 12 h postinfection, was maximal at 4 days, and continued for at least 10 days. Over the first 36 h there was no evidence of toxicity. We recorded normal voltage-dependent and transduction currents from infected cells identified by GFP fluorescence. At longer times hair bundle integrity was compromised despite a cell body that appeared healthy. To assess the ability of adenovirus-mediated gene transfer to alter hair cell function we introduced the gene for the ion channel Kir2.1. We used an adenovirus vector encoding Kir2.1 fused to GFP under the control of an ecdysone promoter. Unlike the diffuse distribution within the cell body we observed with GFP, the ion channel-GFP fusion showed a pattern of fluorescence that was restricted to the cell membrane and a few extranuclear punctate regions. Patch-clamp recordings confirmed the expression of an inward rectifier with a conductance of 43 nS, over an order of magnitude larger than the endogenous inward rectifier. The zero-current potential in infected cells was shifted by -17 mV. These results demonstrate an efficient method for gene transfer into both vestibular and auditory hair cells in culture, which can be used to study the effects of gene products on hair cell function. (+info)
Targeted correction of an episomal gene in mammalian cells by a short DNA fragment tethered to a triplex-forming oligonucleotide.
(22/5815)
Triplex-forming oligonucleotides (TFOs) can bind to polypurine/polypyrimidine regions in DNA in a sequence-specific manner and provoke DNA repair. We have coupled a TFO to a short donor fragment of DNA that shares homology to a selected gene as a strategy to mediate gene targeting and correction. In this bifunctional oligonucleotide, the TFO domain is designed to bind the target gene and stimulate repair and recombination, with the donor domain positioned for recombination and information transfer. A series of these tethered donor-TFO (TD-TFO) molecules with donor domains of 40-44 nucleotides and TFO domains in both the purine and pyrimidine triplex motifs were tested for their ability to mediate either gene correction or mutation of a supF reporter gene contained in a SV40 shuttle vector in mammalian cells. In vitro binding assays revealed that the attachment of the donor domain via a flexible linker did not significantly alter the binding affinity of the TFO domain for the polypurine site in the supF target DNA, with equilibrium dissociation constants in the 10(-8) M range. Experiments in which the target vector and the linked TD-TFOs were pre-incubated in vitro and co-transfected into cells led to conversion frequencies approaching 1%, 4-fold greater than with the two domains unlinked. When cells that had been previously transfected with the SV40 vector were electroporated with the TD-TFOs, frequencies of base pair-specific gene correction were seen in the range of 0.04%, up to 50-fold over background and at least 3-fold over either domain alone or in unlinked combinations. Sequence conversion by the TD-TFOs was achieved using either single- or double-stranded donor domains and either triplex motif. Substitution of either domain in the TD-TFO with control sequences yielded reagents with diminished activity, as did mixtures of unlinked TFO and donor DNA segments. The boost in activity provided by the attached TFO domain was reduced in cells deficient in the nucleotide excision repair factor XPA but was restored in a subclone of these cells expressing XPA cDNA, suggesting a role for nucleotide excision repair in the pathway of triple helix-stimulated gene conversion. The ability to correct or mutate a specific target site in mammalian cells using the TD-TFO strategy may provide a useful tool for research and possibly for therapeutic applications. (+info)
Issues in immunohistochemistry.
(23/5815)
Immunohistochemistry is a powerful technique used to localize specific molecules in tissues. Literally thousands of immunoreagents specific for an incredibly diverse number of molecules are commercially available, and some of these immunoreagents will detect minute quantities of target molecule in tissues. However, like any powerful technique, the staining study design must be carefully conceived and controlled to avoid misinterpretation. (+info)
Multiple kinetic components and the Ca2+ requirements of exocytosis.
(24/5815)
The use of caged-Ca2+ compounds to stimulate Ca(2+)-dependent exocytosis has substantially increased our understanding of this complex process. By this approach, the existence of multiple kinetic components of exocytosis has been established. These components may correspond to a series of sequential steps that lead to a single fusion-ready state (sequential mechanism) or, alternatively, to heterogeneity in secretory vesicles or in fusion-ready states (parallel mechanism). It is suggested that both of these mechanisms can underlie exocytosis of a single type of vesicle (mixed sequential-parallel mechanism). Studies with caged-Ca2+ compounds have also indicated that the Ca2+ requirement for exocytosis is substantially greater than that suggested by conventional methodologies. This discrepancy is mainly attributable to the underestimation, by imaging studies with high-affinity Ca2+ indicators (due to dye saturation), of the local increases in cytosolic Ca2+ concentration that trigger the exocytosis of individual vesicles. The effects of local saturation of such indicators are explored by means of a simple theory. (+info)