Biomedical subjects

Z Li

Publications and source records attributed to Z Li.

At least 19 recordsLinked to original sources

PubMed2025

HLA-E and NKG2A Mediate Resistance to BCG Immunotherapy in Non-Muscle-Invasive Bladder Cancer.

Bacillus Calmette-Guérin (BCG) is the first-line therapy for high-grade non-muscle-invasive bladder cancer (NMIBC), yet many patients experience recurrence due to immune evasion. We identify HLA-E and NKG2A as mediators of adaptive resistance involving chronic activation of NK and T cells in BCG-unresponsive tumors. Prolonged IFN-γ exposure enhances HLA-E and PD-L1 expression on recurrent tumors, accompanied by the accumulation of NKG2A+ NK and CD8 T cells. HLA-Ehigh tumor cells preferentially cluster near CXCL12-rich stromal regions with dense effector cell presence, underscoring a spatially segregated tumor architecture. Although cytotoxic lymphocytes retain effector potential, their activity is restrained by HLA-E/NKG2A and PD-L1/PD-1 pathways located in their immediate neighborhood within the bladder tumor microenvironment. These data reveal a spatially organized immune escape program that limits anti-tumor immunity. Our findings support dually targeting NKG2A and PD-L1 checkpoint blockade as a rational, bladder-sparing strategy for patients with BCG-unresponsive NMIBC.

BCG-unresponsive↗

PubMed1995

Tryptophan 250 on the alpha subunit plays an important role in flavin and aldehyde binding to bacterial luciferase. Effects of W-->Y mutations on catalytic function.

Bacterial luciferase is a heterodimer (alpha beta) that catalyzes the oxidation of FMNH2 and a fatty aldehyde, resulting in light emission. To explore the nature of the flavin binding site with respect to the role of tryptophan residues, the catalytic and binding properties of single-point mutants of Xenorhabdus luminescens luciferase with one of the eight tryptophans converted to a tyrosine residue were investigated by luminescence and fluorescence measurements. Conversion of tryptophans 194 and 250 on the alpha subunit to tyrosine had relatively large effects on the properties of luciferase with only minor changes in the properties on mutation of the other four tryptophans on alpha and the two on the beta subunit. Mutation of alpha W250 decreased the binding to FMNH2, FMN, aldehyde, and fatty acid, causing major changes in luminescence emission and decay. The results are consistent with alpha W250 interacting with flavin which in turn affects aldehyde binding. Mutation of alpha W194 did not affect the interaction with flavin or aldehyde but did change the relative rate of decay of light emission with aldehydes of different chain lengths as well as the activation energy for this process. Moreover, these results provide evidence for alpha W250, and to a lesser extent alpha W194, being in contact with the isoalloxazine ring of flavin, a proposal that has been recently made based on a model with flavin bound to the alpha subunit and anchored at a binding site for the phosphate moiety of FMN(H2) identified in the crystal structure of Vibrio harveyi luciferase [Fisher, A. J. Raushel, F. M., Baldwin, T. O., & Rayment, I. (1995) Biochemistry 34, 6581-6586].

Aldehydes↗

PubMed1995

1 alpha,25-dihydroxyvitamin D3 analogs featuring aromatic and heteroaromatic rings: design, synthesis, and preliminary biological testing.

Aromatic compounds 2a-c, analogs of 1 alpha, 25-dihydroxyvitamin (calcitriol, 1), and heteroaromatic compounds 4a-c and 5a-c, analogs of 19-nor-1 alpha, 25-dihydroxyvitamin D3 (3), were designed to simulate the topology of their biologically potent parent compounds while avoiding previtamin D equilibrium. Convergent and facile total syntheses of the analogs (+)-2b, (+)-2c, (-)-4b, and (-)-5b were achieved via carbonyl addition of regiospecifically formed organolithium nucleophiles to the enantiomerically pure C,D-ring ketone (+)-17, characteristic of natural calcitriol (1). Likewise, hybrid analogs 20a-c were prepared to determine whether incorporation of a known potentiating side chain would lead to increased biological activity. Preliminary in vitro biological testing showed that aromatic analogs (+)-2b, (+)-2c, and 20a-c as well as heteroaromatic analogs (-)-4b and (-)-5b have very low affinities for the calf thymus vitamin D receptor but considerable antiproliferative activities in murine keratinocytes at micromolar concentration. No biological advantage was observed in this keratinocyte assay for the doubly modified hybrid analogs 20a-c over the singly modified parent (+)-2b. Analog (+)-2b, but surprisingly not the corresponding analog 20b differing from (+)-2b only in the side chain, showed considerable activity in nongenomic opening of calcium channels in rat osteosarcoma cells.

Animals↗

PubMed1995

Characterization of the tryptophan binding site of Escherichia coli tryptophan holorepressor by phosphorescence and optical detection of magnetic resonance of a tryptophan-free mutant.

The L-tryptophan binding site of the Escherichia coli tryptophan holorepressor (trpR) is characterized by low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Measurements are made on a tryptophan-free mutant of trpR, W19/99F, in which both intrinsic tryptophan residues of apo-trpR have been replaced with phenylalanine. Thus, essentially all of the phosphorescence that is observed from trpR originates from the bound L-tryptophan corepressor. The phosphorescence and ODMR results for the bound corepressor agree quite well with those obtained previously for the corepressor site in both single tryptophan-containing mutants, W19F and W99F [Burns, L.E., & Maki, A.H. (1994) J. Fluorescence 4, 217-226]. A red shift of the L-tryptophan phosphorescence origin as well as a decrease in the D-E ODMR frequency result from an increase in the local polarizability upon binding at the corepressor binding site. A large decrease in the ODMR line widths signals a reduction of local heterogeneity upon binding. Subsequent binding of trpR to a self-complementary DNA sequence that mimics the trp operator, 5'-CGTACTAGTTAACTAGTACG-3', produces a further decrease in line widths and additional changes in the ODMR frequencies, attributable to an increase in both the D and E parameters. This result demonstrates that binding of holo-trpR to the operator affects the local environment of the bound corepressor.

Bacterial Proteins↗

PubMed1995

The tRNA processing enzyme RNase T is essential for maturation of 5S RNA.

The maturation of 5S RNA in Escherichia coli is poorly understood. Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated. We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis. Our results indicate that no mature 5S RNA is made in RNase T-deficient strains. Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate. Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type. Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction. In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation. Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action. These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival.

Base Sequence↗

PubMed1995

Sequence analysis of a 5.6 kb fragment of chromosome II from Saccharomyces cerevisiae reveals two new open reading frames next to CDC28.

The sequence of a 5653 bp DNA fragment of the right arm of chromosome II of Saccharomyces cerevisiae contains two unknown open reading frames (YBR1212 and YBR1213) next to gene CDC28. Gene disruption reveals both putative genes as non-essential. ORF YBR1212 encodes a predicted protein with 71% similarity and 65% identity (total polypeptide of 376 aa) with the 378 aa Surl protein of S. cerevisiae, while the putative product of ORF YBR1213, which is strongly expressed, has 28% identity with a Lactococcus lactis-secreted 45 kDa protein and 24% identity with the Saccharomyces cerevisiae AGA1 gene product.

Amino Acid Sequence↗

PubMed1995

Desmosome assembly and disassembly are regulated by reversible protein phosphorylation in cultured epithelial cells.

Desmosomes are one component of the intercellular junctional complex in epithelia. In cultures of epithelial cells, desmosome assembly can be regulated by modulating the calcium concentrations of the growth media. At present, very little is known about the intracellular signal transduction mechanisms that regulate desmosome assembly and disassembly in response to changing extracellular calcium concentrations. We have used inhibitors of protein kinases and phosphatases in a combined biochemical and morphological approach to analyze the role of protein phosphorylation in the assembly and disassembly of desmosomes in Madin-Darby canine kidney epithelial cells. Our results suggest that desmosomal proteins (desmoplakins I/II and desmoglein 1) are primarily phosphorylated on serine residues. Electron microscopic analyses of desmosome assembly upon induction of cell-cell contact, in the presence of protein kinase inhibitor, H-7, revealed an apparently normal assembly of desmosomes. However, complete disassembly of desmosomes was inhibited by H-7 upon removal of extracellular calcium. Under these conditions, although desmosomes split, desmosomal plaques and their associated cytokeratin filaments can not be internalized. In contrast, treatment of the cultures with okadaic acid (OA), an inhibitor of protein phosphatases, inhibited desmosome assembly but had no effect on disassembly. In addition, the inhibitory effect of okadaic acid on desmosome assembly was specific to this junction since we observed apparently normal tight junction and adherens junction in okadaic acid-treated cultures. These results suggest that assembly and disassembly of desmosomes may be regulated by extracellular Ca2+ via reversible protein phosphorylation involving both protein kinase and protein phosphatases.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Solid-phase fluorescent labeling reaction of picomole amounts of insulin in very dilute solutions and their analysis by capillary electrophoresis.

The fluorescent labeling of peptides at concentrations as low as 10(-8) M can be achieved by using a solid-phase reactor. Using oxidized insulin chain B as a test peptide, we demonstrate the use of an Immobilon CD membrane to capture and preconcentrate peptides. Insulin chain B can then be labeled with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde, while it is still attached to the membrane. Unwanted fluorescent products (attributed to secondary reactions) can be washed away with methanol without significant removal of the labeled insulin chain B, which then can be extracted with a low pH buffer. The analysis by micellar electrokinetic capillary chromatography with post-column laser-induced fluorescence detection (mass limit of detection of 2.4 x 10(-21) moles insulin chain B) results in electropherograms that show great improvement in terms of unwanted peaks and high number of theoretical plates (up to 20 million). The use of the solid-phase reactor allows easy handling of as little as 5 picomoles of insulin chain B.

Animals↗

PubMed1995

Correlation of DNA fragment sizes within loci in the presence of non-detectable alleles.

At present most forensic databases of DNA profiling of individuals consist of DNA fragment sizes measured from Southern blot restriction fragment length polymorphism (RFLP) analysis. Statistical studies of these databases have revealed that, when fragment sizes are measured from RFLP analysis, some of the single-band patterns of individuals may actually be due to heterozygosity of alleles in which fragment size resulting from one allele remains undetected. In this work, we evaluate the effect of such allelic non-detectability on correlation of fragment sizes within individuals at a locus, and its impact on the inference of independence of fragment sizes within loci. We show that when non-detectable alleles are present in a population at a locus, positive correlations of fragment sizes are expected, which increase with the proportion of non-detectable alleles at the locus. Therefore, a non-zero positive correlation is not a proof of allelic dependence within individuals. Applications of this theory to the current forensic RFLP databases within the US show that there is virtually no evidence of significant allelic dependence within any of the loci. Therefore, the assumption that DNA fragment sizes within loci are independent is valid, and hence, the population genetic principles of computing DNA profile frequencies by multiplying binned frequencies of fragment sizes are most likely to be appropriate for forensic applications of DNA typing data.

Alleles↗

PubMed1995

Guanylin, an endogenous ligand for C-type guanylate cyclase, is produced by goblet cells in the rat intestine.

BACKGROUND & AIMS: Guanylin activates an intestinal guanylate cyclase (GCC) and stimulates electrolyte movement across the gut epithelium. Cells expressing guanylin messenger RNA have been localized to the epithelial cell layer of the intestine; however, the identity of the guanylin-producing cells has not been determined. The aim of this study was to identify cells that express guanylin in the rat intestine. METHODS: Antibodies were raised against defined proguanylin epitopes, evaluated by Western blotting, and used for immunoperoxidase histochemistry. RESULTS: Guanylin-like immunoreactivity was localized to a subset of goblet cells. In the small intestine, most, perhaps all, goblet cells in the villi were immunopositive, as were some goblet cells in upper crypts; however, goblet cells deep within crypts were unlabeled. In the colon, goblet cells clustered in the necks and around the openings of crypts were immunopositive, whereas (as in the small intestine) goblet cells in deeper crypt regions were unlabeled. In some animals, immunoreactive columnar epithelial cells were also observed in the colon (although such cells were not apparent in the small intestine). Relative labeling of columnar cells varied from animal to animal. CONCLUSIONS: Guanylin is expressed in mature goblet cells. If secreted in conjunction with mucin, it could play a role in the hydration of mucus.

Animals↗

PubMed1995

Calpain as a novel target for treating acute neurodegenerative disorders.

Calpains are cytosolic, neutral proteases that normally exist in an inactive or quiescent state. They require higher than normal levels of calcium for activation which, once accomplished, lead to irreversible proteolysis of numerous cytoskeletal, membrane-associated and regulatory proteins. Because of these characteristics, calpain is gaining attention as a potentially important pathogenic variable in ischemic neuronal death. This manuscript explores this hypothesis by briefly reviewing current support for the role played by calpain in ischemic neurodegeneration, and then discussing a series of recently published studies which: 1. offer further evidence for the hypothesis, and 2. provide direct support for the idea that selective inhibition of calpain can greatly limit the neuronal damage that would normally occur following both global as well as focal brain ischemia. Thus, the data reviewed in this manuscript support the ideas that unregulated activation and proteolysis of intraneuronal calpain plays a significant role in the brain damage that occurs following an ischemic event and that delivering selective and membrane permeant calpain inhibitors to ischemic tissue may provide a powerfully effective therapeutic means of limiting neuronal damage.

Animals↗

PubMed1995

Inhibitory action of metabolites of Pseudomonas aeruginosa against gram-negative bacteria.

Fifty clinical isolates of Pseudomonas aeruginosa were tested for inhibition of growth of clinical isolates of Escherichia coli, Salmonella infantis, Klebsiella pneumoniae and other Gram-negative bacteria in the authors' laboratory. Pseudomonas aeruginosa was strongly active against both E. coli and Enterobacter cloacae, with 89.4% and 94.7% inhibition respectively, but weakly active against S. infantis, K. pneumoniae and Proteus mirabilis with 56.3%, 48.8% and 23.8% inhibition, respectively. The pigmented strains were found to have stronger antimicrobial activity than the unpigmented strains. Pyocyanin, the major metabolite of Pseudomonas aeruginosa, has been shown to inhibit Escherichia coli, Proteus spp. and other Gram-negative bacteria, by research with a few strains of P. aeruginosa and a single inhibited strain. However, little attempt has been made to determine the inhibitory action of many strains of P. aeruginosa against a large number of clinical isolates such as Escherichia spp., Klebsiella spp., and Salmonella spp., up to now. For this reason, in this study we examined 50 randomly selected clinical isolates of P. aeruginosa for inhibition of growth of a wide range of Gram-negative bacteria, including 30 strains of E. coli, 30 of K. pneumoniae, 30 of S. infantis, 6 of Enterobacter cloacae and 9 of Proteus mirabilis.

Antibiosis↗

PubMed1995

Minimization of target positioning error in accelerator-based radiosurgery.

The stereotactic radiosurgery system used at the Mallinckrodt Institute of Radiology is patterned after that developed at the Joint Center for Radiation Therapy (Brigham & Women's Hospital, Boston, MA) and uses the Brown-Roberts-Wells computed tomography (CT) stereotactic system. The patient's head is attached to a stand that rotates with the treatment couch. The irradiation is conducted using a set of converging arcs of irradiation. Because of mechanical limitations, no accelerator or treatment couch is capable of placing the center of the radiation beam at precisely the same point for all gantry and couch angles and a compromise must be made when locating the nominal isocenter. The stand settings are checked by placing a radiopaque QA sphere at the desired target location. The QA sphere is imaged using a series of eight films exposed at a set of couch and gantry angles that encompass the treatment angles. The distances between the QA sphere image and the center of the radiation field indicate if the correct coordinates were set on the stand and if the radiation beam converges to a sufficiently small region (< 0.1-cm diameter) for treatment. A mathematical procedure has been developed to use the film-measured position errors to determine a stand offset that will minimize the distance between the accelerator isocenter and the target. The technique is capable of reducing the average placement error, as measured by imaging the QA sphere, to 0.035 cm with a maximum deviation of 0.07 cm.

Humans↗

PubMed1995

Monte Carlo aided dosimetry of the microselectron pulsed and high dose-rate 192Ir sources.

Despite the large number of single-stepping source pulsed and high dose-rate (HDR) remote after-loading devices in clinical use, the published literature contain little data characterizing dose-rate distributions around the high-intensity (4 x 10(3)-4 x 10(4) microGy m2h-1) 192Ir sources currently used in these devices. We have used the Monte Carlo method to calculate complete two-dimensional dose-rate distributions about the most widely used high dose-rate source design, as well as the Nucletron pulsed dose-rate (PDR) 192Ir source. A Monte Carlo photon transport code, incorporating the detailed internal geometry of the source, was used to calculate the dose rate per unit air-kerma strength in water medium on the transverse bisecting axis over the 0.15-12 cm distance range. In addition, polar dose profiles were calculated at distances ranging from 0.25 to 5 cm. The PDR and HDR dose-rate distributions are tabulated using the formalism endorsed by the Interstitial Collaborative Working Group and the AAPM Task Group 43, and includes dose-rate constant, radial dose function, anisotropy function, geometry function, and anisotropy factors. The dose-rate constants, lambda, of the MicroSelectron/HDR and PDR sources were found to be 1.115 and 1.128 cGy h-1 per unit air-kerma strength, respectively, in good agreement with previously published data for low dose-rate interstitial 192Ir sources. Oblique filtration by the high-density iridium metal core resulted in deviations from anisotropy as large as 35%-55% near the longitudinal axis of the source. Dose-rate distributions are also presented in Cartesian ("away" and "along") coordinates.

Brachytherapy↗

PubMed1995

Adenovirus type 12-induced fragility of the human RNU2 locus requires U2 small nuclear RNA transcriptional regulatory elements.

Infection of human cells with oncogenic adenovirus type 12 (Ad12) induces four specific chromosome fragile sites. Remarkably, three of these sites appear to colocalize with tandem arrays of genes encoding small, abundant, ubiquitously expressed structural RNAs--the RNU1 locus encoding U1 small nuclear RNA (snRNA), the RNU2 locus encoding U2 snRNA, and the RN5S locus encoding 5S rRNA. Recently, an artificial tandem array of the natural 5.8-kb U2 repeat unit has been shown to generate a new Ad12-inducible fragile site (Y.-P. Li, R. Tomanin, J. R. Smiley, and S. Bacchetti, Mol. Cell. Biol. 13:6064-6070, 1993), demonstrating that the U2 repeat unit alone is sufficient for virally induced fragility. To identify elements within the U2 repeat unit that are required for virally induced fragility, we generated cell lines containing artificial tandem arrays of the entire 5.8-kb repeat unit, an 834-bp fragment spanning the U2 gene alone, or the same 834-bp fragment from which key U2 transcriptional regulatory elements had been deleted. The U2 snRNA coding regions within each artificial array were marked by an innocuous single base change (U to C at position 87) so that the relative expression of supernumerary and endogenous U2 genes could be monitored by a primer extension assay. We find that artificial arrays of both the 5.8- and the 0.8-kb U2 repeat units are fragile but that arrays lacking either the distal sequence element or both the distal and the proximal sequence elements of the promoter are not. Surprisingly, variations in repeat copy number and/or transcriptional activity of the artificial arrays do not appear to correlate with the degree of Ad12-inducible fragility. We conclude that U2 transcriptional regulatory elements are required for virally induced fragility but not necessarily U2 snRNA transcription per se.

Adenovirus Infections, Human↗

PubMed1995

Feedback inhibition of the yeast ribosomal protein gene CRY2 is mediated by the nucleotide sequence and secondary structure of CRY2 pre-mRNA.

The Saccharomyces cerevisiae CRY1 and CRY2 genes, which encode ribosomal protein rp59, are expressed at a 10:1 ratio in wild-type cells. Deletion or inactivation of CRY1 leads to 5- to 10-fold-increased levels of CRY2 mRNA. Ribosomal protein 59, expressed from either CRY1 or CRY2, represses expression of CRY2 but not CRY1. cis-Acting elements involved in repression of CRY2 were identified by assaying the expression of CRY2-lacZ gene fusions and promoter fusions in CRY1 CRY2 and cry1-delta CRY2 strains. Sequences necessary and sufficient for regulation lie within the transcribed region of CRY2, including the 5' exon and the first 62 nucleotides of the intron. Analysis of CRY2 point mutations corroborates these results and indicates that both the secondary structure and sequence of the regulatory region of CRY2 pre-mRNA are necessary for repression. The regulatory sequence of CRY2 is phylogenetically conserved; a very similar sequence is present in the 5' end of the RP59 gene of the yeast Kluyveromyces lactis. Wild-type cells contain very low levels of both CRY2 pre-mRNA and CRY2 mRNA. Increased levels of CRY2 pre-mRNA are present in mtr mutants, defective in mRNA transport, and in upf1 mutants, defective in degradation of cytoplasmic RNA, suggesting that in wild-type repressed cells, unspliced CRY2 pre-mRNA is degraded in the cytoplasm. Taken together, these results suggest that feedback regulation of CRY2 occurs posttranscriptionally. A model for coupling ribosome assembly and regulation of ribosomal protein gene expression is proposed.

Base Sequence↗

PubMed1995

Mouse chick chimera: a new model to study the in ovo developmental potentialities of mammalian somites.

Chimeras were prepared by transplanting somites from 9-day post-coïtum mouse embryos or somitic dermomyotomes from 10-day post-coïtum mouse embryos into 2-day-old chick embryos at different axial levels. Mouse somitic cells then differentiated in ovo in dermis, cartilage and skeletal muscle as they normally do in the course of development and were able to migrate into chick host limb. To trace the behavior of somitic myogenic stem cells more closely, somites arising from mice bearing a transgene of the desmin gene linked to a reporter gene coding for Escherichia coli beta-galactosidase (lacZ) were grafted in ovo. Interestingly, the transgene was rapidly expressed in myotomal muscles derived from implants. In the limb muscle mass, positive cells were found several days after implantation. Activation of desmin nls lacZ also occurred in in vitro cultures of somite-derived cells. Our experimental method facilitates investigation of the mechanisms of mammalian development, allowing the normal fate of implanted mouse cells to be studied and providing suitable conditions for identification of descendants of genetically modified cells.

Animals↗

PubMed1995

[The diagnosis and treatment of the epithelial salivary gland tumors in children and adolescents].

Sixty-six cases with epithelial salivary gland tumors in children and adolescents, who were less than 16 years old, were analized. Those cases accounted for 3.6% of all epithelial salivary gland tumors in our school. Twenty-five cases (37.9%) were benign tumors, among which pleomorphic adenoma was the most common. Forty-one cases (62.1%) were malignant tumors, in which mucoepidermoid carcinoma was the most common one. Three quarters of cases were in the group over 10 years old. Six cases were in the group less than 5 years old. Among them 5 cases were malignant tumors. The younger the children is, the more possible the malignant tumor is. Two cases with malignant tumors had the history of receiving radiotherapy because of the hemangiomas at the same locations. It indicates that the occurrence of the malignant tumors was related to radiotherapy before. The diagnosis, differential diagnosis and treatment of the tumors were discussed. The initial radical resection of the malignant tumors was emphasized.

Adolescent↗