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PubMed1988

Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro.

We constructed SP6-human beta-globin derivative plasmids that included possible donor site (5' splice site) sequences at a specified position within the first intron. The runoff transcripts from these templates truncated in the second exon were examined for splicing in a nuclear extract from HeLa cells. In addition to the products from the authentic donor site, a corresponding set of novel products from the inserted, alternative donor site was generated. Thus, a short sequence inserted within an intron can be an active donor site signal in the presence of an authentic donor site. The active donor site sequences included a 9-nucleotide consensus sequence, 14- or 16-nucleotide sequences at the human beta-globin first or second donor, and those at simian virus 40 large T antigen or small t antigen donor. These included 3 to 8 nucleotides of an exon and 6 to 8 nucleotides of an intron. The activity of the inserted donor site relative to that of the authentic donor site depended on the donor sequence inserted. The relative activity also strongly depended on the concentrations of both KCl (40 to 100 mM) and MgCl2 (1.6 to 6.4 mM). At the higher KCl concentrations tested, all the inserted, or proximate, donor sites were more efficiently used. Under several conditions, some inserted donor sites were more active than was the authentic donor site. Our system provides an in vitro assay for donor site activity of a sequence to be tested.

Amino Acid Sequence↗
PubMed2004

Quantitative analysis of total mitochondrial DNA: competitive polymerase chain reaction versus real-time polymerase chain reaction.

An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 x 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders.

DNA, Mitochondrial↗
PubMed1987

The dual effector system for exocytosis in mast cells: obligatory requirement for both Ca2+ and GTP.

The secretory process is a coordinated cellular response, initiated by occupation of surface receptors and comprising an ordered sequence of biochemical steps subject to multiple controls. Conceptually we can divide the sequence into two main sections comprising early, receptor-mediated events leading to generation of intracellular second messengers, and later events leading to membrane fusion and exocytosis. With the discovery that occupation of Ca2+ mobilising receptors leads to activation of polyphosphoinositide phosphodiesterase (PPI-pde) through the mediation of a G-protein (Gp), all the early events can be ascribed to the plasma membrane. Investigation of the exocytotic stage of secretion has been simplified by the use of permeabilised cells in which the composition of the cytosol can be precisely controlled. We have used streptolysin-O, a bacterial cytolysin which generates protein-sized pores in the plasma membrane, to investigate the exocytotic mechanism of rat mast cells. We find that in addition to the activation of PPI-dpe, GTP also acts in concert with Ca2+ at, or close to, the exocytotic site. Exocytosis can occur after substantial depletion of cytosol lactate dehydrogenase and 3-phosphoglycerate kinase indicating that soluble cytosol proteins are unlikely to play any role. There is no absolute requirement for ATP or phosphorylating nucleotide in exocytosis though when present the effective affinities of the two obligatory effectors (i.e. Ca2+ and GTP) are substantially enhanced.

Animals↗
PubMed1995

Frequent patient-to-patient transmission of hepatitis C virus in a haematology ward.

Blood transfusion is a well-documented route of transmission of hepatitis C virus (HCV). However, a persisting high frequency of HCV infections was recorded in our haematology ward even after screening of blood donors had been introduced. We investigated the viral strains in 37 patients with haematological malignant diseases who had developed hepatitis C when treated in the ward during 1990-93. 17 of the patients acquired hepatitis C despite being transfused only with blood components screened by second-generation anti-HCV tests. The viral strains were characterised by PCR genotyping and nucleotide sequencing of the hypervariable region of the E2 gene. Five clusters of closely related or identical viruses were found involving 2, 3, 4, 6, and 15 patients, respectively. Blood components could be ruled out as the common source of infection because no donor had given blood to all patients sharing a specific strain, and even donors whose blood had been given to several patients were negative for HCV RNA. All patients in each cluster had been treated in the ward during overlapping periods. These findings suggest that despite strict hygienic control, HCV transmission occurred between patients treated in the same hospital setting, as has previously been reported in a smaller group of haemodialysis patients.

Adult↗
PubMed2006

Identification and characterization of O-acetylpeptidoglycan esterase: a novel enzyme discovered in Neisseria gonorrhoeae.

Modification of the bacterial cell wall heteropolymer peptidoglycan by addition of an acetyl group to the C-6 hydroxyl group of N-acetylmuramoyl residues is known to inhibit the activity of muramidases (lysozymes) of innate immune systems. The O-acetylation of peptidoglycan also precludes the action of intrinsic lytic transglycosylases, enzymes that require a free C-6 hydroxyl group to generate their 1,6-anhydromuropeptide products. This class of autolysins is ubiquitous in peptidoglycan-synthesizing bacteria as they are responsible for insertion of pores and flagella, spore formation, and the general metabolism of peptidoglycan. We recently discovered a cluster of genes in the Neisseria gonorrhoeae chromosome that are proposed to participate in peptidoglycan O-acetylation (Weadge, J. T., Pfeffer, J. M., and Clarke, A. J. (2005) BMC Microb. 5, 49). In the current study, we demonstrate that one of these genes, ape1 functions as an O-acetylpeptidoglycan esterase. The ape1 gene was cloned and overexpressed in Escherichia coli as a fusion protein with a hexa-histidine tag. The expressed protein was purified to apparent homogeneity and assayed for activity as an esterase using three different assays involving high-performance liquid chromatography and chromogenic detection methods which measured the release of ester-linked acetate from a variety of polymer and soluble substrates. These assays demonstrated that Ape1 has a higher specific activity on O-acetylated peptidoglycan compared to O-acetylated xylan. Consequently, Ape1 represents the first enzyme characterized as an O-acetylpeptidoglycan esterase. The physicochemical and kinetic parameters of Ape1 were determined using soluble chromogenic substrates for convenience. Thus, its pH optima for stability and activity were observed to be 6.0 and 6.2, respectively, while its optimum temperature for activity was 55 degrees C. Two forms of truncated Ape1 are generated in E. coli, one lacked the complete predicted N-terminal signal sequence, while the second involved a proteolytic cleavage within this signal sequence. The smaller truncated form was localized predominantly to the periplasm, whereas the larger form was mainly associated with the outer membrane, and to a lesser extent, the cytoplasmic membrane, sites expected for the maintenance of peptidoglycan.

Acetylation↗
PubMed2004

Dicer is required for embryonic angiogenesis during mouse development.

Dicer is a multi-domain protein responsible for the generation of short interfering RNAs (siRNAs) from long double-stranded RNAs during RNA interference. It is also involved in the maturation of microRNAs, some of which are transcriptional regulators of developmental timing in nematodes. To assess the role of Dicer in mammals, we generated Dicerex1/2 mice with a deletion of the amino acid sequences corresponding to the first and second exons of the dicer gene via homologous recombination. We found that Dicerex1/2 homozygous embryos displayed a retarded phenotype and died between days 12.5 and 14.5 of gestation. Thus, these results show that dicerex1/2 is severely hypomorphic and that Dicer is essential for normal mouse development. Interestingly, we also found that blood vessel formation/maintenance in dicerex1/2 embryos and yolk sacs were severely compromised, suggesting a possible role for Dicer in angiogenesis. This finding is consistent with the altered expression of vegf, flt1, kdr, and tie1 in the mutant embryos. Taken together, the results of this study indicate that Dicer exerts its function on mouse embryonic angiogenesis probably through its role in the processing of microRNAs that regulate the expression levels of some critical angiogenic regulators in the cell.

Animals↗
PubMed2004

Specific Cre/Lox recombination in the mouse proximal tubule.

The present work reports for the first time the construction of a transgenic mouse strain with specific expression of Cre recombinase in the kidney proximal tubule. A Cre/loxP strategy was developed using sglt2 promoter to drive Cre recombinase expression in transgenic mice. The mouse sglt2 5' region consisting of the first exon, the first intron, and part of the second exon was cloned upstream of a nucleotide sequence encoding the Cre recombinase. Transgenic mice were generated by pronuclear injection, and tissue specificity of Cre expression was analyzed using reverse transcription-PCR. The iL1-sglt2-Cre mouse line scored positive for kidney transcription of Cre but not for the other tissues analyzed. Within the kidney, Cre transcripts were demonstrated to be restricted to the proximal tubule only. iL1-sglt2-Cre mice were bred with ROSA26-LacZ reporter mice that contained a loxP-flanked stop sequence upstream of the LacZ gene. X-gal staining and immunohistochemistry using specific antibodies (anti-megalin, anti-Tamm-Horsfall, anti-NaCl co-transporter, and anti-aquaporin 2) revealed that sglt2 drives Cre functional expression specifically in proximal tubules. The iL1-sglt2-Cre mouse therefore represents a powerful tool for Cre-LoxP-mediated conditional expression in the renal proximal tubule.

Animals↗
PubMed1996

An epitope chimeric antigen for the hepatitis C virus serological screening test.

The epitope chimeric antigen, CepCM, composed of the 9 selected major epitope regions (two in NS3, two each in the NS4 of two genotypes, two each in the core of two genotype, and one in the core in hepatitis C virus (HCV) polypeptide), was expressed as a fusion protein of the trpE peptide in E. coli. An ELISA test using this antigen produced the same judgements with most of the panel sera as a second generation HCV screening kit. Though discrepancies were found in twelve samples (5% of the samples), further analysis revealed that eleven samples were indeterminate sera as judged by an immunoblot test. The reactivity found in several seroconversion series sera suggested that CepCM has superior reactivity to HCV infected sera than some second generation kits. These data indicated that an epitope chimeric antigen with a man-made sequence will be a excellent tool for a diagnostic test kit.

Antigens, Viral↗
PubMed2005

Molecular engineering approaches for DNA sequencing and analysis.

High-throughput DNA sequencing development for mutation screening and identification is essential to realize the goal of pharmacogenomics and personalized medicine, which will lead to a new era in clinical medicine and healthcare. Molecular engineering approaches to modify the building blocks of DNA by introducing functional groups for purification and detection has led to the development of high-throughput genetic analysis technologies. This review is focused on the following two DNA sequencing approaches. The first approach is based on the use of molecular affinity and mass spectrometry to perform quick and highly accurate mutation screening, heterozygote identification and insertion/deletion detection. The second approach is based on a sequencing-by-synthesis platform that has the potential for generating DNA sequencing data in a massive, parallel manner. The basic principles, fundamental challenges and methods of implementation of these exciting new technologies will be discussed.

Animals↗
PubMed1994

Response of monkey glabrous skin mechanoreceptors to random-noise sequences: I. Temporal response characteristics.

The response of slowly adapting (SA, Merkel cell), rapidly adapting (RA, Meissner), and Pacinian corpuscle (PC) mechanoreceptors in monkey glabrous skin were recorded while being activated with a vibrotactile indenting stimulus. For one set of experiments, the stimulator was driven by an 800-point (400-msec) pseudorandom-noise (PRN) sequence, repeated 40 times. For a second set of experiments, a non-repeating-noise (NRN) sequence was used. SA impulse patterns generated with the PRN sequence were highly regular and similar for all afferents tested. RA and PC response patterns were more varied, but exhibited basic intraclass similarities. SA and RA PRN stimulus impulse patterns were often nearly indistinguishable at low to medium discharge rates. At higher discharge rates, RA and SA discharge patterns differed, primarily because of the unambiguous suprathreshold velocity sensitivity and shorter recovery periods of RA afferents. PC impulse patterns were substantially different from SA and RA patterns, being clearly dependent on the stimulus velocity at all stimulus intensities. Input-output correlation analysis, coupled with PRN stimulus-response analysis, provided a basis for hypothesizing the basic dynamic encoder function of each type of mechanoreceptor.

Afferent Pathways↗
PubMed1992

Second generation anti-MUC1 peptide monoclonal antibodies.

Second generation antibodies to mammary mucins were produced by immunizing mice with a peptide with a sequence deduced from that of the MUC1 complementary DNA sequence (PAHGVTSAPDTRPAPGSTAP). Four monoclonal antibodies (BCP7-10) were produced which gave different reactions. BCP8 was similar in tissue reactivity (by immunoperoxidase staining) to anti-breast cancer or anti-human milk fat globule membranes (HMFG) antibodies and reacted strongly with most breast cancers and a proportion of other adenocarcinomas, whether formalin fixed or fresh, and reacted less strongly with some normal tissues. The three other antibodies (BCP7, BCP9, BCP10) reacted only with fresh tissues or a single cell line (LS174T of colon cancer origin) and gave variable weak reactions. Like many anti-mucin antibodies BCP8 reacted with HMFG, but more strongly with deglycosylated HMFG; analysis with peptides by enzyme-linked immunosorbent assay indicated reactivity with an epitope contained in the amino acid motif PDTR and using the pepscan method, the minimum epitope was DTR. MAbs BCP7, BCP9, and BCP10 did not react with HMFG; substantial reactions were obtained with deglycosylated HMFG for BCP7 and weaker reactions with BCP9 and BCP10. The finding that BCP7 reacted with breast cancer tissues and deglycosylated HMFG suggested that the epitope recognized by BCP7 was masked in native form and exposed in cancer, indicating that BCP7 could be a useful agent for analyzing differences between normal and cancer mucins. The amino acid epitopes for these antibodies were VTSA (BCP7), GSTAP (BCP9), and RPAP (BCP10). For BCP8, amino acid substitution analysis of SAPDTR indicated that substitutions were poorly tolerated (except Q for T and L/Y for R), contrasting with the substitution analysis of anti-mucin antibody reactions where virtually any amino acid can be substituted for T, indicating that in the native state T (threonine) may be O-glycosylated. The use of synthetic peptides to produce antibodies similar to those produced using crude mucins or tumor extracts represents a major advance in the production of antitumor reagents.

Amino Acid Sequence↗
PubMed1984

Immune response to ferredoxin; nonresponder status is not due to suppression.

Ferredoxin (Fd), a small protein from Clostridium pasteurianum, has been selected for immunologic studies because of its limited number (two) of antigenic determinants. Functionally (as determined by antibody binding), monodeterminant fragments of Fd can be generated enzymatically, leaving molecules only a few amino acids smaller than the native protein, with unaltered solid phase binding properties. These fragments were used to assess the immune response to each of the two determinants. Clear differences in immunologic properties can be assigned to sequences within Fd: the amino terminal tripeptide is responsible for inducing a proliferative response and limited antibody production, whereas the carboxy terminal dipeptide accounts for most of the antibody activity, yet little, if any, T-proliferative activity. Studies with the enzyme-generated fragments of Fd have unmasked a sequence proximal to the amino terminal that represents a second determinant for T cell proliferation but does not have any demonstrable antibody-inducing activity. This third determinant is shown to induce responsiveness to Fd in nonresponder animals after the removal of the amino terminal tripeptide. The results indicate that nonresponsiveness to this molecule in H-2d mice is not a direct effect of suppression.

Animals↗
PubMed1983

Insulin release : the fuel concept.

Insulin release evoked by nutrient secretagogues invariably coincides with an increase in the catabolism of exogenous and/or endogenous nutrients in pancreatic islet cells, resulting in an increased generation rate of reducing equivalents and ATP, and an increase in O2 consumption. This situation was documented in response to a number of carbohydrates, including the anomers of D-glucose and D-mannose, various 2-keto acids, including pyruvate and its poorly oxidized phenylated analog 3-phenylpyruvate, and selected amino acids, including the nonmetabolized analog of L-leucine 2-aminobicyclo[2, 2, 1]heptane-2-carboxylic acid. These convergent observations indicate that the B-cell should be considered as a fuel-sensor organ. Further progress in this field requires both the identification of those circulating nutrients used by the B-cell whether in the resting or stimulated state, and a better understanding of the coupling between the generation of second messengers (H+, NAD(P)H, ATP) and more distal events in the secretory sequence, such as the remodelling of ionic fluxes across the plasma membrane and within the pancreatic B-cell.

Amino Acids↗
PubMed1995

The dopamine transporter: immunochemical characterization and localization in brain.

Antibodies specific for the dopamine transporter (DAT) was developed and characterized by immunoblot analysis, immunoprecipitation, and immunocytochemistry, and used for immunolocalization of transporter protein in rat brain at the light microscopic level. Antibodies targeting the N-terminus, the second extracellular loop, and the C-terminus were generated from fusion proteins containing amino acid sequences from these respective regions. Immunoblot analysis demonstrated that N-terminus and loop antibodies were specific for expressed cloned DAT, recognized transporter protein in rat and human striatal membranes, and were sensitive to preabsorption with excess homologous fusion protein. Immunoprecipitation studies demonstrated that anti-DAT antisera recognized solubilized, radiolabeled DAT protein in a concentration-dependent manner. DAT immunocytochemistry with these antibodies were also sensitive to preabsorption with fusion protein and to lesions of dopaminergic mesostriatal and mesocorticolimbic pathways. Regional distribution of DAT coincided with established dopaminergic innervation of several regions, including ventral mesencephalon, medial forebrain bundle, and dorsal and ventral striatum. However, certain mismatches between immunocytochemical distributions of DAT and tyrosine hydroxylase were apparent, indicating that dopaminergic systems are heterogeneous and may use independent mechanisms for the regulation of dopamine levels in brain. The generation of specific DAT antibodies will permit further characterization of the cellular and subcellular localization of DAT protein, and of dopaminergic circuits in neurological and psychiatric disorders.

Animals↗
PubMed2001

Statistics of the integrated backscatter estimate from a blood-mimicking fluid.

This work evaluates the variance of the integrated backscatter (IBS) from moving blood [or blood-mimicking fluid (bmf)] as a way of determining the quality of the mean IBS estimate. The main motivation for this work comes from the fact that absolute IBS values from tissues adjacent to arterial blood can be found by normalizing the measured backscatter energy with the IBS of moving, deaggregated blood. The paper describes the parameters that control the statistics of the IBS estimate, which is calculated for the stochastic ultrasound backscatter signals from flowing blood. It further formulates how the measurement parameters should be specified so that an appropriately low blood IBS variance is ensured or, alternatively, a specified accuracy of the tissue IBS estimate is obtained. First, the paper provides an analytic formulation of the statistics of the IBS, based on a sequence of sampled echoes from a nonstationary Gaussian scattering medium. The analysis incorporates the correlation between the sample values as well as the correlation between the IBS of the individual echoes. The estimate of the mean IBS has been shown to be chi-squared distributed with a determinable order. With the degree of correlation between the samples and between the IBS of individual echoes specified, the number of measurements required to obtain an IBS estimate with a specified variance is readily calculated. Next, a sequence of synthetic echoes is produced and arranged as columns in a data matrix. The echoes are generated such that the second-order statistics along the rows and columns of the matrix match that of actually observed echoes. The actual variance of the mean IBS estimate for the synthetic echoes is calculated and compared with the variance determined from the analytic model, and a good agreement has been found. Finally, sequences of actual backscattered echoes from circulating blood-mimicking fluid are acquired and analyzed to determine the variance of their mean IBS estimate. Based on the measured second-order statistics of the rows and columns of the data matrix for the actual echoes, the observed variance of the mean IBS estimate was compared with the analytically determined variance and with good agreement. Thus, the paper has shown through modeling, simulations, and experiments how the variance of the IBS estimate of the blood backscatter signal can be quantified and reduced to a specified tolerable level.

Blood↗
PubMed1988

A directed nucleotide-sequencing approach for single-stranded vectors based on recloning intermediates of a progressive DNA synthesis reaction.

A simple method for site-directed nucleotide sequencing is presented that uses a novel procedure for generating nested 'deletions' within inserts of single-stranded clones. In this method, single-stranded template, sequencing primer, and the Klenow fragment of Escherichia coli DNA polymerase I are used to initiate progressive DNA synthesis of the entire insert of the clone. By time-dependent sampling and pooling of intermediates from the synthesis reaction a series of nested double-stranded DNA subfragments of the insert can be created. Nested subclones are then produced by S1-endonuclease treatment and oriented subcloning methods. First, smaller quantities of template DNA can be used, equivalent to a fraction of a small DNA sequencing prep. Second, it works with single-stranded M13 phage DNA rather than requiring the preparation of double-stranded replicative form DNA as in ExoIII-based methods. Third, the 'deletions' it generates can span areas of simple nucleotide sequence or secondary structure that often halt digestion in the single-stranded exonuclease-based method. Last, the method is adaptable to a larger variety of insert cloning sites than the ExoIII-based method. The main disadvantage of the method is that, due to the lower efficiency of subcloning larger DNA fragments, subclone inserts larger than 3 kb are generated only infrequently.

Bacteriophages↗
PubMed1995

Differentiation alters the expression of the two splice variants of the serotonin 5-HT3 receptor-A mRNA in NG108-15 cells.

The serotonin 5-HT3-A receptor (5-HT3R-A) mRNA has been shown recently to be expressed as two forms (5-HT3R-AL and 5-HT3R-AS) varying by the presence or the absence of a sequence of 18 bases in the region corresponding to the second cytoplasmic domain of the receptor, and generated by alternative splicing at the level of the 3' acceptor site of exon 9. As the long form of the receptor exhibits a potential phosphorylation site that is disrupted by the alternative splicing, the hypothesis of functional identity and stochastic expression of these two variants was questioned. In the present study, we used quantitative reverse transcriptase-polymerase chain reaction to examine the possible influence of culture conditions on the expression and the alternative splicing of 5-HT3R-A mRNA in NG108-15 clonal cells. Cell differentiation induced by dibutyryl cyclic AMP or theophyllin plus prostaglandin E1 in the presence of 10% serum reduced by threefold the expression of total 5-HT3R-A mRNA, and favored the short form of the message as the ratio S/L (5-HT3R-AS mRNA/5-HT3R-AL mRNA) shifted from 2.23 to 7.33 after 9 days of treatment. Culture with 0.3% serum (instead of 10%) lowered by 10-fold the level of expression of total 5-HT3R-A mRNA, but only slightly reduced the S/L ratio. However, this ratio fell to 0.06 in the presence of 0.3% serum plus 10 ng/ml basic fibroblast growth factor. These results demonstrate that external factors can influence the differential expression of the two variants of the 5-HT3R-A in NG108-15 cells. Appropriate culture conditions for the almost exclusive expression of 5-HT3R-AS mRNA or 5-HT3R-AL mRNA in NG108-15 cells should allow the identification of possible differences in the respective functional properties of each of these two forms of the native 5-HT3 receptor.

Alternative Splicing↗