Triple fluorescence energy transfer in covalently trichromophore-labeled DNA.
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Coloring Agents↗
Biomedical subjects
J Ju
Publications and source records attributed to J Ju.
DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3'-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.
Base Sequence↗
A tissue sealant based on reactive multifunctional polyethylene glycol.
A rapidly gelling synthetic tissue sealant was developed from tetra-succinimidyl and tetra-thiol-derivatized polyethylene glycol (PEG). The two reagents were dissolved in aqueous buffers at 20% (w/v) solids and sprayed on the tissue site, with the use of a sprayer/mixer device. Good adhesion to collagen membranes, PTFE grafts, and carotid artery was observed in vitro. In a burst test on collagen membranes with a 2-mm orifice defect, the gel sustained fluid pressures of 125 +/- 36 mm Hg (n = 18), fivefold greater than capillary blood pressure and one-half that observed in hypertension. On 0.4-mm-diameter puncture defects in PTFE grafts, pressures of 390-490 mm Hg were sustained, and on 0.6-0.9-mm puncture defects in carotid arteries, pressures of 490 to 840 mm Hg were sustained. In vitro data corresponded to results in vivo, where bleeding in rabbit arteries was stopped immediately in five out of six trials. A significant reduction in time to hemostasis and blood loss, compared to controls, was observed. Carotid artery and subcutaneous implant data in rabbits showed that the formula was compatible with biological tissue. Rapid gelling and effective sealing were dependent on the presence of active succinimidyl ester and thiol groups on PEG. HPLC and chemical substitution methods were useful in predicting whether batches of derivatized PEG would perform satisfactorily.
Adhesives↗
Combinatorial fluorescence energy transfer tags for multiplex biological assays.
We report an approach for developing combinatorial fluorescence energy transfer (CFET) tags by tuning the tags' fluorescence emission signatures. The tags can all be excited at a single wavelength and analyzed by a simple optical system. We constructed eight CFET tags with unique fluorescence signatures, detected by a three-color capillary array electrophoresis (CAE) system with 488 nm excitation, using only three fluorescent dyes. A 1',2'-dideoxyribose phosphate spacer was used to separate the donor and acceptor to tune the energy transfer efficiency, generating unique fluorescence signatures. The spacer also served as an electrophoretic mobility tag to tune the mobility of CFET-labeled DNA for multiplex detection of single-nucleotide polymorphisms (SNPs). Six nucleotide variations were identified simultaneously using six CFET tags on synthetic DNA templates and on a PCR product from the retinoblastoma tumor suppressor gene.
Chemistry↗
Ancestral divergence, genome diversification, and phylogeographic variation in subpopulations of sorbitol-negative, beta-glucuronidase-negative enterohemorrhagic Escherichia coli O157.
The O157:H7 lineage of enterohemorrhagic Escherichia coli is a geographically disseminated complex of highly related genotypes that share common ancestry. The common clone that is found worldwide carries several markers of events in its evolution, including markers for acquisition of virulence genes and loss of physiological characteristics, such as sorbitol fermentation ability and beta-glucuronidase production. Populations of variants that are distinct with respect to motility and the sorbitol and beta-glucuronidase markers appear to have diverged at several points along the inferred evolutionary pathway. In addition to these variants, distinct subpopulations of the contemporary non-sorbitol-fermenting, beta-glucuronidase-negative O157:H7 clone were recently detected among bovine and human clinical isolates in the United States by using high-resolution genome comparison. In order to determine if these recently described subpopulations were derived from a regional or ancestral divergence event, we used octamer-based genome scanning, marker sorting, and DNA sequence analysis to examine their phylogenetic relationship to populations of non-sorbitol-fermenting, beta-glucuronidase negative O157:H7 and O157:H- strains from Australia. The inferred phylogeny is consistent with the hypothesis that subpopulations on each continent resulted from geographic spread of an ancestral divergence event and subsequent expansion of distinct subpopulations. Marker sorting and DNA sequence analyses identified sets of monophyletic markers consistent with the pattern of divergence and demonstrated that phylogeographic variation occurred through emergence of regional subclones and concentration of regional polymorphisms among distinct subpopulations. DNA sequence analysis of representative polyphyletic markers showed that genome diversity accrued through random drift and bacteriophage-mediated events.
Animals↗
Clinical study on treatment of chronic renal failure with shenshuailing.
The therapeutic effects of Shenshuailing Kou Fu Ye (SKFY [symbol: see text], the Oral Liquid for Renal Failure) and Shenshuailing Guan Chang Ye (SGCY [symbol: see text], the Enema for Renal Failure) were evaluated in treatment of chronic renal failure, with coateg aldehyde oxystarch as the controls. The changes in the clinical symptoms, serum creatinine, blood urea nitrogen and creatinine clearance rate were observed. The total effective rate in the former was 90.46%, and the latter 60.43%.
Aged↗
[Degradation of phenanthrene and pyrene in contaminated soil by immobilized Zoogloea sp. and Fusarium sp].
Immobilized with PVA, sodium alginate and activated carbon, both Zoogloea sp. and Fusarium sp. strains could degrade phenanthrene and pyrene efficiently. The optimal carrier was made of 100 rho.g-1 L PVA, 5 sodium alginate rho.g-1 L and 50 activated carbon rho.g-1 L. The degradation rates of phenanthrene and pyrene in 10 days were 87.48% and 75.34% by the immobilized bacterium, 37.04% and 20.85% higher than those by the free bacterium, and the rates in 15 days were 84.36% and 74.87% by the immobilized fungus, 5.35% and 11.23% higher than those by the free fungus.
Biodegradation, Environmental↗
Correlation of estradiol, parathyroid hormone, interleukin-6, and soluble interleukin-6 receptor during the normal menstrual cycle.
Rodent models suggest that estradiol deficiency promotes bone loss through increasing interleukin-6 (IL-6) activity. However, it is controversial as to whether these findings are applicable to humans. To evaluate estradiol-mediated modulation of IL-6 activity in relation to bone metabolism in humans, we measured serum IL-6, soluble interleukin-6 receptor (sIL-6R), estradiol (E2), progesterone, luteinizing hormone, follicle-stimulating hormone, intact parathyroid hormone (PTH), serum and urine Ca, and bone biochemical markers (serum bone-specific alkaline phosphatase, osteocalcin, and serum and urine deoxypyridinoline [Dpd]) across one menstrual cycle for 211 women. Neither IL-6 nor sIL-6R levels differed between the follicular phase (FP) and the luteal phases (LP). However, IL-6 was negatively correlated with E2 during the FP (p =0.003). Furthermore, IL-6 correlated positively with serum Ca over the entire cycle (p = 0.0091. Serum Ca correlated positively with serum (p = 0.040) and urine (p = 0.006) Dpd. PTH was significantly higher during the FP than in the LP (p = 0.004). PTH was negatively related to E2 (p = 0.002), serum Ca (p < 0.001), and urine Ca (p = 0.036), whereas it was positively correlated with IL-6 (p = 0.027). These data demonstrate that IL-6 and PTH fluctuate with E2, and serum II-6 is associated with PTH levels during the menstrual cycle. However, the role of 11-6 in bone remodeling during the normal menstrual cycle remains to be determined.
Adult↗
A model for pulse jet fabric filters.
A new model for pulse jet fabric filtration is proposed. In contrast to the earlier model of Ravin and Humphries, which was formulated on the steady state assumption, the present study is aimed at developing a predictive capability for both transient and steady state operations, taking into account the compression effect of filter cakes. The model's relative simplicity allows frequent updating of the model parameter values, thus improving the accuracy of predictions. As a result, the model is particularly useful in developing control algorithms and designing controllers of pulse jet fabric filtration systems.
Air Pollution↗
The Bacillus subtilis GTP binding protein obg and regulators of the sigma(B) stress response transcription factor cofractionate with ribosomes.
Obg, an essential GTP binding protein of Bacillus subtilis, is necessary for stress activation of the sigma(B) transcription factor. We investigated Obg's cellular associations by differential centrifugation of crude B. subtilis extracts, using an anti-Obg antibody as a probe to monitor Obg during the fractionation, and by fluorescent microscopy of a B. subtilis strain in which Obg was fused to green fluorescent protein. The results indicated that Obg is part of a large cytoplasmic complex. In subsequent analyses, Obg coeluted with ribosomal subunits during gel filtration of B. subtilis lysates on Sephacryl S-400 and specifically bound to ribosomal protein L13 in an affinity blot assay. Probing the gel filtration fractions with antibodies specific for sigma(B) and its coexpressed regulators (Rsb proteins) revealed coincident elution of the upstream components of the sigma(B) stress activation pathway (RsbR, -S, and -T) with Obg and the ribosomal subunits. The data implicate ribosome function as a possible mediator of the activity of Obg and the stress induction of sigma(B).
Bacillus subtilis↗
A clinical investigation on garlicin injectio for treatment of unstable angina pectoris and its actions on plasma endothelin and blood sugar levels.
To investigate the therapeutic effects and mechanisms of garlicin for treatment of unstable angina pectoris (UAP), garlicin injectio was intravenously dripped 60 mg/day in 34 cases for 10 days. Nitroglycerine was used in 21 cases of the control group. The results showed that the total effective rates in improving symptoms and electrocardiogram after garlicin treatment were respectively 82% and 62%, and that the plasma endothelin and blood sugar levels were markedly lowered in cases with hyperglycemia.
Aged↗
[Clinical study on Shenning Mixture in treating IgA nephropathy].
OBJECTIVE: To study the clinical therapeutic effect of Shenning Mixture (SNM) in treating IgA nephropathy. METHODS: Patients were treated separately with SNM or prednisone by randomized grouping. The criteria, including clinical symptoms, signs, hematuria, albuminuria and immune globulin were observed. RESULTS: The total effective rate and the complete remission rate in the SNM group were 97.1% and 45.7% respectively, while in the control group, they were 37.1% and 8.6% respectively. Comparison of the two groups showed that the therapeutic effect in the former was better than that in the latter significantly, P < 0.01. The hematuria and albuminuria extenuated and serum IgA lowered more significantly in the SNM group than those in the control group (P < 0.05, P < 0.01). CONCLUSION: SNM has obvious therapeutic effect on the hematuria and albuminuria in treating IgA nephropathy.
Adolescent↗
Regulation of p53 expression by thymidylate synthase.
Previous studies showed that thymidylate synthase (TS), as an RNA binding protein, regulates its own synthesis by impairing the translation of TS mRNA. In this report, we present evidence that p53 expression is affected in a similar manner by TS. For these studies, we used a TS-depleted human colon cancer HCT-C cell that had been transfected with either the human TS cDNA or the Escherichia coli TS gene. The level of p53 protein in transfected cells overexpressing human TS was significantly reduced when compared with its corresponding parent HCT-C cells. This suppression of p53 expression was the direct result of decreased translational efficiency of p53 mRNA. Similar results were obtained upon transfection of HCT-C cells with pcDNA 3.1 (+) containing the E. coli TS gene. These findings provide evidence that TS, from diverse species, specifically regulates p53 expression at the translational level. In addition, TS-overexpressing cells with suppressed levels of p53 are significantly impaired in their ability to arrest in G1 phase in response to exposure to a DNA-damaging agent such as gamma-irradiation. These studies provide support for the in vivo biological relevance of the interaction between TS and p53 mRNA and identify a molecular pathway for controlling p53 expression.
Cell Cycle↗
Sigma factor displacement from RNA polymerase during Bacillus subtilis sporulation.
As Bacillus subtilis proceeds through sporulation, the principal vegetative cell sigma subunit (sigma(A)) persists in the cell but is replaced in the extractable RNA polymerase (RNAP) by sporulation-specific sigma factors. To explore how this holoenzyme changeover might occur, velocity centrifugation techniques were used in conjunction with Western blot analyses to monitor the associations of RNAP with sigma(A) and two mother cell sigma factors, sigma(E) and sigma(K), which successively replace sigma(A) on RNAP. Although the relative abundance of sigma(A) with respect to RNAP remained virtually unchanged during sporulation, the percentage of the detectable sigma(A) which cosedimented with RNAP fell from approximately 50% at the onset of sporulation (T(0)) to 2 to 8% by 3 h into the process (T(3)). In a strain that failed to synthesize sigma(E), the first of the mother cell-specific sigma factors, approximately 40% of the sigma(A) remained associated with RNAP at T(3). The level of sigma(A)-RNAP cosedimentation dropped to less than 10% in a strain which synthesized a sigma(E) variant (sigma(ECR119)) that could bind to RNAP but was unable to direct sigma(E)-dependent transcription. The E-sigma(E)-to-E-sigma(K) changeover was characterized by both the displacement of sigma(E) from RNAP and the disappearance of sigma(E) from the cell. Analyses of extracts from wild-type and mutant B. subtilis showed that the sigma(K) protein is required for the displacement of sigma(E) from RNAP and also confirmed that sigma(K) is needed for the loss of the sigma(E) protein. The results indicate that the successive appearance of mother cell sigma factors, but not necessarily their activities, is an important element in the displacement of preexisting sigma factors from RNAP. It suggests that competition for RNAP by consecutive sporulation sigma factors may be an important feature of the holoenzyme changeovers that occur during sporulation.
Bacillus subtilis↗
The "pro" sequence of the sporulation-specific sigma transcription factor sigma(E) directs it to the mother cell side of the sporulation septum.
sigma(E), a mother cell-specific transcription factor of sporulating Bacillus subtilis, is derived from an inactive precursor protein (pro-sigma(E)). Activation of sigma(E) occurs when a sporulation-specific protease (SpoIIGA) cleaves 27 amino acids from the pro-sigma(E) amino terminus. This reaction is believed to take place at the mother cell-forespore septum. Using a chimera of pro-sigma(E) and green fluorescent protein (GFP) to visualize the intracellular location of pro-sigma(E) by fluorescence microscopy, and lysozyme treatment to separate the mother cell and forespore compartments, we determined that the pro-sigma(E)::GFP signal, localized to the forespore septum prior to lysozyme treatment, is restricted to the mother cell compartment after treatment. Thus, pro-sigma(E)::GFP had been sequestered to the mother cell side of the septum. This segregation of pro-sigma(E)::GFP, and presumably pro-sigma(E), to the mother cell is likely to be the reason why sigma(E) activity is restricted to that compartment.
Bacillus subtilis↗
Thymidylate synthase protein and p53 mRNA form an in vivo ribonucleoprotein complex.
A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.
Animals↗
Changes in bone resorption during the menstrual cycle.
To determine if the cyclic changes of female sex hormones during the menstrual cycle are related to changes in bone formation and resorption, we measured serum bone-specific alkaline phosphatase (BAP) and osteocalcin (OC) and bone resorption markers, serum and urine deoxypyridinoline (Dpyr), three times per week during one menstrual cycle in 20 healthy premenopausal women. Serum estradiol (E2) and progesterone (P) showed characteristic cyclic fluctuations. Serum Dpyr was higher during the follicular phase (FP) than in the luteal phase (p = 0.027). Serum BAP, OC, and urine Dpyr levels did not change substantially across the cycle. Serum Dpyr correlated negatively with serum E2 values measured 6 (p = 0.011) and 8 (p = 0.001) days earlier and with P measured concurrently (p = 0.033) 2 (p = 0.002), 4 (p = 0.003), and 6 (p = 0.014) days earlier. BAP correlated negatively with E2 measured 6 days earlier (p = 0.006). We found no statistically significant correlations of E2 or P with OC or urine Dpyr within women over their cycles. BAP was positively correlated with concurrent serum Dpyr (p = 0.015) during the menstrual cycle. Serum OC levels correlated inversely with age (rs = -0.48, p = 0.036). Women with higher mean urine Dpyr levels had higher mean serum OC levels (rs = 0.49, p = 0.033) and showed a trend toward lower hip bone mineral density (rs = -0.40, p = 0.078). We conclude that the low level of E2 and/or P observed during the FP of the normal menstrual cycle is associated with increased bone resorption. These relationships suggest that normal women experience monthly episodes of increased bone resorption from menarche to menopause.
Adult↗
An immunoprecipitation-RNA:rPCR method for the in vivo isolation of ribonucleoprotein complexes.
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Cell Extracts↗