Reconstitution of mammalian DNA replication.
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Animals↗
Biomedical subjects
L Huang
Publications and source records attributed to L Huang.
Isolation and structure identification of an active DNA strand-scission agent, (+)-3,4-di-hydroxy-8,9-methylenedioxypterocarpan.
A new pterocarpan, (+)-3,4-dihydroxy-8,9-methylenedioxypterocarpan [1], was isolated from the flowers of Petalostemon purpureus by a DNA strand-scission assay-guided fractionation procedure. Compound 1 demonstrated activity in a standard in vitro DNA strand-scission assay, and cytotoxicity toward a KB tumor cell line. Two other related pterocarpans [2, 3] isolated from same plant were found to be moderately active for KB cells, but were inactive in the DNA strand-scission assay. (+)-4-Hydroxy-3-methoxy-8,9-methylenedioxypterocarpan [2] has not been reported previously as a natural product, while (+)-maackiain [3] has been isolated only as an optically inactive racemate along with its optical antipode, the (-)-isomer.
Antineoplastic Agents↗
The introduction of two silent mutations into a CFTR cDNA construct allows improved detection of exogenous mRNA in gene transfer experiments.
Phase one clinical trials for gene therapy of cystic fibrosis are in progress using either liposomes or adenoviral vectors for CFTR gene transfer to epithelial cells in the airways. In addition to electrophysiological measurements, expression of vector CFTR is usually assessed by RT-PCR. We have developed a CFTR-expression vector, pCFAS, that simplifies the distinction of transgene-derived CFTR mRNA from endogenous mRNA. Two point mutations were introduced into CFTR cDNA which eliminated a SphI restriction site and created a new, unique AgeI restriction site. Neither mutation altered the predicted amino acid sequence of the protein. Restriction digestion of RT-PCR products from cells transfected with pCFAS allowed the differentiation of transgene and endogenous CFTR transcripts. To verify function of the mutated CFTR, the plasmid was transferred into freshly obtained nasal epithelial cells from CF patients ex vivo using cationic liposomes. Fluorescence microscopy using the halide-sensitive fluorophore SPQ demonstrated restoration of cAMP-mediated Cl- secretion. This plasmid will be useful for CFTR gene transfer studies in vitro and in vivo.
Base Sequence↗
Role of macrophages and dendritic cells in primary cytotoxic T lymphocyte responses.
The successful induction of class I restricted cytotoxic T lymphocytes (CTL) responses with soluble non-replicating antigens relies upon vehicles which deliver antigen in vivo appropriately to antigen presenting cells (APC), which for CTL may be dendritic cells (DC). In this study, we have followed the distribution of liposomes and their incorporated antigen and compared the efficacy of splenic macrophages (Mo) and DC at inducing primary CTL responses in vitro. Our results show that whereas liposomes locate predominantly in the splenic red pulp and marginal zone locations, some of their soluble antigen contents redistribute to the central white pulp, comprising mainly DC and T cells. Such antigen redistribution was most apparent following administration of pH-sensitive liposomes. In comparisons of the APC activity of Mo and DC taken at various times post-injection, DC were always superior to Mo. However, if Mo were depleted prior to antigen exposure, DC were ineffective APC for CTL induction. Furthermore, addition of supernatant from OVA-liposome treated Mo to naive DC-T cell cultures in vitro resulted in OVA-specific T cell responses. These studies indicate a role for Mo in enhancing the antigen presenting function of DC.
Animals↗
Molecular cloning of the biotinylated subunit of 3-methylcrotonyl-coenzyme A carboxylase of Arabidopsis thaliana.
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Arabidopsis↗
Uptake kinetics and intracellular localization of hypocrellin photosensitizers for photodynamic therapy: a confocal microscopy study.
Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The intracellular localization and uptake kinetics of hypocrellin B and several selected hypocrellin congeners were determined semiquantitatively by fluorescence confocal microscopy in monolayer cultures of EMT6/Ed murine tumor cells. Each compound had unique uptake kinetics. Although no compound tested to date has demonstrated nuclear labeling, most could be detected in lysosomes, Golgi, endoplasmic reticulum and, to a minor extent, in cellular membranes. No two compounds gave identical labeling distributions. The differences are assumed to originate in physicochemical properties characteristic of each compound, which may ultimately impact upon the primary modality of phototoxicity.
Animals↗
Mechanical modeling of palatal snoring.
The mechanism of human snoring caused by vibration of the soft palate and the characteristics of the noise are investigated. The soft palate becomes unstable and vibrates violently once the inspiratory flow exceeds a critical speed. The physiological phenomenon is modeled by studying flow over a flexible plate. In determining the stability of this flow, the trailing edge conditions are crucial. It is found that the noise generated in the simple experimental configuration has distinct characteristics found in human snores. For example, there is an antiphase relation between unsteady pressures from the oral and nasal channels and this provides a feature that distinguishes snoring by vibration of the soft palate from that caused by other parts of the human airway.
Acoustics↗
Binding of cellular repressor protein or the IE2 protein to a cis-acting negative regulatory element upstream of a human cytomegalovirus early promoter.
We have previously shown that the human cytomegalovirus early UL4 promoter has upstream negative and positive cis-acting regulatory elements. In the absence of the upstream negative regulatory region, the positive element confers strong transcriptional activity. The positive element contains a CCAAT box dyad symmetry and binds the cellular transcription factor NF-Y. The effect of the negative regulatory element is negated by the viral IE2 protein (L. Huang, C.L. Malone, and M.F. Stinski, J. Virol. 68:2108, 1994). We investigated the binding of cellular or viral IE2 protein to the negative regulatory region. The major cis-acting negative regulatory element was located between -168 and -134 bp relative to the transcription start site. This element could be transferred to a heterologous promoter, and it functioned in either orientation. Mutational analysis demonstrated that a core DNA sequence in the cis-acting negative regulatory element, 5'-GTTTGGAATCGTT-3', was required for the binding of either a cellular repressor protein(s) or the viral IE2 protein. The cellular DNA binding activity was present in both nonpermissive HeLa and permissive human fibroblast cells but more abundant in HeLa cells. Binding of the cellular repressor protein to the upstream cis-acting negative regulatory element correlates with repression of transcription from the early UL4 promoter. Binding of the viral IE2 protein correlates with negation of the repressive effect.
Base Sequence↗
AIDS-related Kaposi sarcoma of the lung: radiographic findings and staging system with bronchoscopic correlation.
PURPOSE: To determine the chest radiographic findings in patients with tracheobronchial Kaposi sarcoma (KS) and to develop a radiographic staging system that allows comparison of radiographic to bronchoscopic findings and allows assessment of the typical temporal progression of pulmonary KS. MATERIALS AND METHODS: The chest radiographs and medical records of 76 male patients, aged 23-60 years (mean, 36 years 4 months) with bronchoscopically proved KS were retrospectively reviewed. Tumor extent at bronchoscopy (grade 1-3) was compared with severity of parenchymal disease (radiographic stage 0-3). RESULTS: There was a statistically significant relationship between extent of tracheobronchial disease and radiographic stage (P = .01). However, in some patients, parenchymal KS of advanced stage was present with no visible endobronchial disease. CONCLUSION: The proposed staging system describes features of early, moderate, and advanced KS. Although there is a correlation between tracheobronchial and parenchymal disease, the latter can occur in the absence of endobronchial lesions.
Acquired Immunodeficiency Syndrome↗
Delivery of superoxide dismutase to pulmonary epithelium via pH-sensitive liposomes.
Respiratory insufficiency, when treated with oxygen supplementation, or exposure to diverse pulmonary toxins can cause lung damage as a result of increased oxygen radical production. Enzymes such as superoxide dismutase (SOD) may attenuate this pathological process, but the intracellular delivery and antioxidant action of SOD is impeded by its inability to cross cellular membranes. One approach for facilitating intracellular delivery of macromolecules is to entrap SOD into liposomes. The delivery of SOD to lung cells was accomplished using pH-sensitive liposomes, made with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1-oleoyl-2-oleoyl-sn-glycero-3-succinate (DOSG), added to cultured fetal rat lung distal epithelial (FRLE) cells. FRLE cells, obtained from fetuses at day 19 gestation, expressed a high-affinity receptor for surfactant protein A (SP-A) with an apparent dissociation constant (Kd) = 3.6 +/- 0.2 micrograms/ml (5.5 x 10(-9) M) and a capacity of 130 +/- 3 ng/10(6) cells (125,000 +/- 3,000 binding sites/cell). This receptor was utilized for targeting liposomes to cells, after incorporating SP-A during liposome membrane formation. Liposomes were uniformly small (180 +/- 77 nm; mean +/- SD) and stable at 4 degrees C for 1 wk, entrapping 10 +/- 4% of initially added SOD. After incubation of pH-sensitive liposomes containing entrapped SOD with cultured FRLE cells, cell-associated SOD activity was increased 5.1-fold from 7.8 +/- 2.5 to 40.1 +/- 3.3 U SOD/mg cell protein. Incorporation of SP-A into liposomes increased by 6.2-fold the delivery of liposomal SOD to cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Suspected Pneumocystis carinii pneumonia with a negative induced sputum examination. Is early bronchoscopy useful?
In U.S. patients with the acquired immunodeficiency syndrome (AIDS), Pneumocystis carinii pneumonia is the most frequent AIDS-defining opportunistic infection. Sputum induction and bronchoscopy are effective techniques for obtaining specimens used to identify P. carinii although debate continues over their optimal use, specifically whether to perform bronchoscopy after a negative induced sputum examination for P. carinii. To evaluate the usefulness of bronchoscopy in this situation, we reviewed all cases of suspected P. carinii pneumonia in which sputum induction for P. carinii was performed at San Francisco General Hospital during a 4-yr period. Bronchoscopy, performed after a negative induced sputum examination, yielded a diagnosis in 50.5% of evaluations. The most frequent diagnoses were P. carinii pneumonia (192), tracheobronchial Kaposi's sarcoma (93), tuberculosis (28), and Cryptococcus neoformans pneumonia (9). Bronchoscopy provided either the only or an earlier diagnosis in 64.3% of tuberculosis cases. Bronchoscopy with BAL was free of complications, and, importantly, a negative BAL examination for P. carinii allowed physicians to discontinue empiric P. carinii pneumonia treatment in 95%. In patients with suspected P. carinii pneumonia with a negative induced sputum examination for P. carinii, early bronchoscopy with BAL should be performed.
AIDS-Related Opportunistic Infections↗
Delivery of protein antigen to the major histocompatibility complex class I-restricted antigen presentation pathway.
Major histocompatibility complex (MHC) class I-restricted antigen presentation normally requires a protein antigen to be synthesized in the cytosol of the antigen presenting cell (APC). Exogenous protein antigen could gain access to the class I presentation pathway if the protein is introduced into the cytosolic compartment of the APC. Approaches which release the protein antigen from endocytic vesicles have been employed to deliver protein antigen for the recognition by class I-restricted cytotoxic T lymphocytes (CTL). These include osmotic shock, electroporation, cationic and pH-sensitive liposomes. An alternative approach is to deliver a gene that encodes the protein antigen. In this case, the APC is transfected with a gene which synthesizes the "exogenous protein" in the cytosol. Delivery of protein antigen targeted for CTL induction in vivo follows a different strategy and generally requires an antigen carrier of lipidic/membranous nature, such as liposomes, immunostimulating complexes, and/or lipid conjugates. Macrophages that are responsible for scavenging the antigen play an important role in CTL induction. An optimal CTL inductive vaccine must contain other immuno-modulatory activities in addition to its activity in delivering antigen to the class I pathway. Attempts to attenuate viral infection and to improve anti-tumor immunity have been successful by delivering the exogenous antigen entrapped in liposomes. These animal model studies should be of great value in the development of potential vaccine formulation.
Amino Acid Sequence↗
Design, synthesis, DNA sequence preferential alkylation and biological evaluation of N-mustard derivatives of Hoechst 33258 analogues.
The design, synthesis and biological evaluation of a series of bis-benzimidazole analogues of Hoechst 33258 bearing nitrogen mustard moieties is described. The novel compounds show clear evidence of interstrand cross-linking of linear lambda DNA, in contrast to their distamycin nitrogen mustard counterparts. Interference of the cross-linking reaction by the minor groove-selective distamycin suggests that this process takes place in the minor groove of DNA. Sequence preferential alkylation is revealed by high-resolution polyacrylamide gel electrophoresis and autoradiography. Alkylation occurs predominantly at the 5'-A or 5'-G termini of mixed sequences, determined largely by the sequence-recognizing properties of the bis-benzimidazole carrier moiety. An analysis of the frequency of bases around the alkylation sites reveals marked individual base preferences, especially for A at -3 and +3 positions. All of the compounds tested showed cytotoxic properties against the human tumor cell line KB in culture.
Alkylation↗
DNA alkylation by minor groove-binding N1-alkoxyalkyl-bis-benzimidazoles.
Two groups of isomeric N1-alkoxyalkyl-bis-benzimidazoles differing in the orientation of the N-alkoxyalkyl group (R) with respect to the DNA minor groove have been examined as to their reaction with DNA. Agarose gel mobility shift assay demonstrates that the 'R-inward' isomers alkylate and cause thermally induced strand breakage, in contrast to a much weaker reaction of the 'R-outward' isomers. Complementary studies using high-resolution polyacrylamide gel electrophoresis confirmed relatively weak but definite alkylation-induced thermal cleavage at all available G base sites, but at selected A sites. The alkylation reaction is interpreted in terms of an SN2 displacement of the alkoxy group by nucleophiles within the groove, in contrast to the complete lack of such nucleophilic displacement of these drugs in bulk solution. Reaction with all available Gs is interpreted in terms of nucleophilic strength within the DNA minor groove whereas, in contrast, reaction at the A residues appears to be determined primarily by initial molecular recognition of a DNA site by the drug, followed by an SN2 displacement. The relative cytotoxic potencies of these drugs against KB human tumor cells may be explained on the basis of this mechanism.
Alkylation↗
[An investigation of medicinal Trichosanthes].
This paper reports the distribution, habitats and medical usages of 14 species of medicinal Trichosanthes produced in China. Among them, there is a new species named Trichosanthes mianyangensis. A key for the identification of all these 14 species has been compiled and presented.
China↗
[Cytogenetic analysis of four cases of hermaphroditism].
On the basis of cytogenetics karyotype analysis, southern blot hybridization using sex-determining region of the Y chromosome (SRY) probe and amplified cation of the genomic DNA between the SRY code region by specific primers through polymerase chain reaction (PCR) have been performed in four cases of hermaphroditism. The results showed that, in 1 46, XY female and 1 45, XO/46, XY female, the SRY specific hybridization band and PCR amplified band could not be detected; in 1 46, XX male, the specific hybridization and PCR band showed SRY gene as in normal male; in another true hermaphrodite, the karyotype is 46, XX/47,XXY, PCR band is detectable but not hybridization band. The mechanism of the abnormal sex differentiation of the cases was analyzed and the two detection methods were compared.
Adult↗
[Effect of U-50, 488H administered centrally in rats on urination and activity of TH-IR neurons in PVH].
The effect of a kappa-receptor agonist U-50, 488H on urine flow and the activity of TH-IR (tyrosine hydroxylase-immunoreactivity) neurons in PVH (Paraventricular neucleus of the hypothalamus) was studied in ethanol-anesthetized rats. U-50, 488H (5 micrograms/1 microliter) injected into PVH induced a potent diuretic response with no significant action on mean arterial pressure and heart rate. The diuretic response began within 20 min after injection, reached a peak value 41-60 min later and subsided at about 100 min. This diuretic response could be blocked by pretreatment with kappa-receptor antagonist NBT (Nor-Binaltorphimine Tetrahydrate). Both the number of neurons and staining density of TH-IR in the PVH tended to decrease at 20 min after the injection of U-50, 488H (10 micrograms/10 microliters) into the third cerebroventricle. The effect was most evident at 50 min and recovered at about 100 min after injection.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
[Establishment of three-dimensional finite element models by using a series of plane section images processed with an image analysis system].
The geometric similarity and mesh dividing of finite element models directly create an effect on the results of analysis when a theoretical stress analysis is performed by the use of finite element method. The model is routinely established by means of sectioning the specimen. In this paper, a series of plane section CT images of the teeth and mandible, an image analysis system and a specially designed software for modeling three-dimensional finite element were used to establish a three-dimensional finite element model. It is a new preprocessing method for finite element applications in the field of oral biomechanism. The technique is characterized by the advantages of high similarity of the model to specimens and no damage to specimens; it is simple and easy. The images and figures can be repetitively used.
Biomechanical Phenomena↗