Biomedical subjects

T Lu

Publications and source records attributed to T Lu.

At least 73 records · Page 4Linked to original sources

PubMed1999

Loss of USF transcriptional activity in breast cancer cell lines.

USF is a family of transcription factors that are structurally related to the Myc oncoproteins and also share with Myc a common DNA-binding specificity. USF overexpression can prevent c-Myc-dependent cellular transformation and also inhibit the proliferation of certain transformed cells. These antiproliferative activities suggest that USF inactivation could be implicated in carcinogenesis. To explore this possibility, we compared the activities of the ubiquitous USF1 and USF2 proteins in several cell lines derived from either normal breast epithelium or breast tumors. The DNA-binding activities of USF1 and USF2 were present at similar levels in all cell lines. In the non-tumorigenic MCF-10A cells, USF in general, and USF2 in particular, exhibited strong transcriptional activities. In contrast, USF1 and USF2 were completely inactive in three out of six transformed breast cell lines investigated, while the other three transformed cell lines exhibited loss of USF2 activity. Analyses in cells cultured from healthy tissue confirmed the transcriptional activity of USF in normal human mammary epithelial cells. These results demonstrate that a partial or complete loss of USF function is a common event in breast cancer cell lines, perhaps because, like Myc overexpression, it favors rapid proliferation.

Adenocarcinoma↗

PubMed1999

Cytoplasmic amino and carboxyl domains form a wide intracellular vestibule in an inwardly rectifying potassium channel.

We have studied the structural components and architecture of the intracellular vestibule of a strongly rectifying channel (Kir2.1) expressed in Xenopus oocytes. Putative vestibule-lining residues were identified by systematically examining covalent modification by sulfhydryl-specific reagents of cysteine residues engineered into two cytoplasmic regions. In a stretch of 33 amino acids in the amino terminus (from C54 to V86) and 22 amino acids in the carboxyl terminus (from R213 to S234), 15 and 11 residues, respectively, were found to be accessible to methanethiosulfonate ethylammonium (MTSEA) or methanethiosulfonate ethyltrimethylammonium (MTSET) and presumably project into the aqueous intracellular vestibule. The pattern of accessibility suggests that both stretches may adopt an extended loop structure. To explore the physical dimension of the intracellular vestibule, we covalently linked a constrained number (one to four) of positively charged moieties of different sizes to the E224 position and found that this vestibule region is sufficiently wide to accommodate four modifying groups with dimensions of 12 A x 10 A x 6 A. These results suggest that regions in both the amino and carboxyl domains of Kir2.1 channel form a long and wide intracellular vestibule that protrudes beyond the membrane into the cytoplasm.

Amino Acid Sequence↗

PubMed1999

Effects of epoxyeicosatrienoic acids on the cardiac sodium channels in isolated rat ventricular myocytes.

1. Whole-cell Na+ currents (holding potential, -80 mV; test potential, -30 mV) in rat myocytes were inhibited by 8, 9-epoxyeicosatrienoic acid (8,9-EET) in a dose-dependent manner with 22+/-4% inhibition at 0.5 microM, 48+/-5% at 1 microM, and 73+/-5% at 5 microM (mean +/- S.E.M., n = 10, P<0.05 for each dose vs. control). Similar results were obtained with 5,6-, 11,12-, and 14,15-EETs, while 8,9-dihydroxyeicosatrienoic acid (DHET) was 3-fold less potent and arachidonic acid was 10- to 20-fold less potent. 2. 8,9-EET produced a dose-dependent, hyperpolarized shift in the steady-state membrane potential at half-maximum inactivation (V ), without changing the slope factor. 8,9-EET had no effect on the steady-state activation of Na+ currents. 3. Inhibition of Na+ currents by 8,9-EET was use dependent, and channel recovery was slowed. The effects of 8,9-EET were greater at depolarized potentials. 4. Single channel recordings showed 8,9-EET did not change the conductance or the number of active Na+ channels, but markedly decreased the probability of Na+ channel opening. These results were associated with a decrease in the channel open time and an increase in the channel closed times. 5. Incubation of cultured cardiac myocytes with 1 microM [3H]8,9-EET showed that 25% of the radioactivity was taken up by the cells over a 2 h period, and most of the uptake was incorporated into phospholipids, principally phosphatidylcholine. Analysis of the medium after a 2 h incubation indicated that 86% of the radioactivity remained as [3H]8,9-EET while 13% was converted into [3H]8,9-DHET. After a 30 min incubation, 1-2% of the [3H]8,9-EET uptake by cells remained as unesterified EET. 6. These results demonstrate that cardiac cells have a high capacity to take up and metabolize 8,9-EET. 8,9-EET is a potent use- and voltage-dependent inhibitor of the cardiac Na+ channels through modulation of the channel gating behaviour.

8,11,14-Eicosatrienoic Acid↗

PubMed1999

Modulation of rat cardiac sodium channel by the stimulatory G protein alpha subunit.

1. Modulation of cardiac sodium currents (INa) by the G protein stimulatory alpha subunit (Gsalpha) was studied using patch-clamp techniques on freshly dissociated rat ventricular myocytes. 2. Whole-cell recordings showed that stimulation of beta-adrenergic receptors with 10 microM isoprenaline (isoproterenol, ISO) enhanced INa by 68.4 +/- 9.6 % (mean +/- s.e.m.; n = 7, P < 0.05 vs. baseline). With the addition of 22 microgram ml-1 protein kinase A inhibitor (PKI) to the pipette solution, 10 microM ISO enhanced INa by 30.5 +/- 7.0 % (n = 7, P < 0.05 vs. baseline). With the pipette solution containing both PKI and 20 microgram ml-1 anti-Gsalpha IgG or 20 microgram ml-1 anti-Gsalpha IgG alone, 10 microM ISO produced no change in INa. 3. The effect of Gsalpha on INa was not due to changes in the steady-state activation or inactivation curves, the time course of current decay, the development of inactivation, or the recovery from inactivation. 4. Whole-cell INa was increased by 45.2 +/- 5.3% (n = 13, P < 0.05 vs. control) with pipette solution containing 1 microM Gsalpha27-42 peptide (amino acids 27-42 of rat brain Gsalpha) without altering the properties of Na+ channel kinetics. Furthermore, application of 1 nM Gsalpha27-42 to Na+ channels in inside-out macropatches increased the ensemble-averaged INa by 32.5 +/- 6.8 % (n = 8, P < 0.05 vs. baseline). The increase in INa was reversible upon Gsalpha27-42 peptide washout. Single channel experiments showed that the Gsalpha27-42 peptide did not alter the Na+ single channel current amplitude, the mean open time or the mean closed time, but increased the number of functional channels (N) in the patch. 5. Application of selected short amino acid segments (Gsalpha27-36, Gsalpha33-42 and Gsalpha30-39) of the 16 amino acid Gsalpha peptide (Gsalpha27-42 peptide) showed that only the C-terminal segment of this peptide (Gsalpha33-42) significantly increased INa in a dose-dependent fashion. These results show that cardiac INa is regulated by Gsalpha via a mechanism independent of PKA that results in an increase in the number of functional Na+ channels. In addition, a 10 residue domain (amino acids 33-42) near the N-terminus of Gsalpha is important in modulating cardiac Na+ channels.

Adrenergic beta-Agonists↗

PubMed1999

Modulation of desensitization at glutamate receptors in isolated crucian carp horizontal cells by concanavalin A, cyclothiazide, aniracetam and PEPA.

In horizontal cells freshly dissociated from crucian carp (Carassius auratus) retina, we examined the effects of modulators of glutamate receptor desensitization, concanavalin A, cyclothiazide, aniracetam and 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetam ide (PEPA), on responses to rapid application of glutamate and kainate, using whole-cell voltage-clamp techniques. Incubation of concanavalin A suppressed the peak response but weakly potentiated the equilibrium response of horizontal cells to glutamate. Cyclothiazide blocked glutamate-induced desensitization in a dose-dependent manner, which resulted in a steady increase of the equilibrium current. The concentration of cyclothiazide causing a half-maximal potentiation for the equilibrium response was 85 microM. Furthermore, cyclothiazide shifted the dose-response relationship of the equilibrium current to the right, but slightly suppressed the kainate-induced sustained current. These effects of concanavalin A and cyclothiazide are consistent with the supposition that glutamate receptors of carp horizontal cells may be an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-preferring subtype. In order to further characterize the AMPA receptors of horizontal cells, modulation by aniracetam and PEPA of glutamate- and kainate-induced currents was studied. Aniracetam, a preferential modulator of flop variants of AMPA receptors, considerably blocked desensitization of glutamate-induced currents, but only slightly potentiated kainate-induced currents. It was further found that PEPA, a flop-preferring allosteric modulator of AMPA receptor desensitization, slightly suppressed the peak current, while it dramatically potentiated the equilibrium current induced by glutamate in a dose-dependent manner. PEPA was much potent than aniracetam at these receptors and showed the effect on glutamate-induced desensitization even at a concentration as low as 3 microM. PEPA also potentiated non-desensitizing currents induced by kainate, but with much less extent. These modulatory effects of concanavalin A, cyclothiazide, aniracetam and PEPA on AMPA receptors in carp horizontal cells were rather similar to those obtained at AMPA receptors assembled from flop variants expressed in Xenopus oocyte and HEK cell. Consequently, we speculate that the AMPA receptor on carp horizontal cells may predominantly carry the flop splice variants.

Animals↗

PubMed1999

Architecture of a K+ channel inner pore revealed by stoichiometric covalent modification.

Inwardly rectifying K+ channels bind intracellular magnesium and polyamines to generate inward rectification. We have examined the architecture of the inner pore of Kir2.1 channels by covalently attaching a constrained number (from one to four) of positively charged moieties of different sizes to the channel. Our results indicate that the inner pore is formed solely by the second transmembrane segment and is unprecedentedly wide. At a position critical for inward rectification (D172), the pore is sufficiently wide to bind three Mg2+ ions or polyamine molecules simultaneously. Single-channel recordings directly demonstrate that partially modified channels exhibit distinct subconductance levels. Such a wide inner pore may greatly facilitate ion permeation and high-affinity binding of multiple pore blockers to generate strong inward rectification.

Amino Acid Sequence↗

PubMed1999

Investigation of the orientation of purple membrane sheets in Langmuir-Blodgett films by a quartz crystal microbalance.

By means of the quartz crystal microbalance (QCM), a convenient method was developed to determine the degree of orientation of purple membrane (PM) sheets on the air/water interface. Langmuir-Blodgett films from both wild-type and SH-mutant PM (bR D36C) were vertically deposited on the surface of gold-sputtered AT-cut quartz crystals. The shift of resonance frequency of the QCM during a special washing protocol allowed us to differentiate between physically adsorbed PM fragments and any PM attached to the gold surface via chemical bonds. By washing with organic solvents, complete desorption of the wild-type PM was achieved, whereas for the SH-mutant, approximately 60% of the PM fragments could not be detached from the surface. These PM sheets should be oriented with the cytoplasmic side facing the water subphase to that their SH-groups can chemically bind to the gold surface of the quartz plate.

Adsorption↗

PubMed1999

Cytotoxic hammerhead ribozymes.

Small catalytic RNA molecules of the hammerhead ribozyme type were found to have cytotoxic effects unrelated to their intended activity. An expression library of ribozyme sequence variants was constructed in a recA-deficient strain of Escherichia coli such that individual library members differed in regions designed to form base pairs with human immunodeficiency virus-1 (HIV-1) tat mRNA. The parental ribozyme and many variants exhibited a bacteriostatic effect. One variant studied in detail was also bactericidal. When its expression was induced, ribozyme-dependent inhibition of bacterial growth was not observed in recA+ or recA+ lexA3 (Ind-) cells, suggesting that the recombination function of the RecA protein, not the absence of the SOS response, is sufficient to alleviate the cytotoxic effect. These data document the need for careful testing for toxic effects during intracellular studies of ribozyme action.

Base Sequence↗

PubMed1999

Gatifloxacin activity against quinolone-resistant gyrase: allele-specific enhancement of bacteriostatic and bactericidal activities by the C-8-methoxy group.

Antibacterial activities of gatifloxacin (AM1155), a new C-8-methoxy fluoroquinolone, and two structurally related compounds, AM1121 and ciprofloxacin, were studied with an isogenic set of ten quinolone-resistant, gyrA (gyrase) mutants of Escherichia coli. To compare the effect of each mutation on resistance, the mutant responses were normalized to those of wild-type cells. Alleles exhibiting the most resistance to growth inhibition mapped in alpha-helix 4, which is thought to lie on a GyrA dimer surface that interacts with DNA. The C-8-methoxy group lowered the resistance due to these mutations more than it lowered resistance arising from several gyrA alleles located outside alpha-helix 4. These data are consistent with alpha-helix 4 being a distinct portion of the quinolone-binding site of GyrA. A helix change to proline behaved more like nonhelix alleles, indicating that helix perturbation differs from the other changes at helix residues. Addition of a parC (topoisomerase IV) resistance allele revealed that the C-8-methoxy group also facilitated attack of topoisomerase IV. When lethal effects were measured at a constant multiple of the minimum inhibitory concentration for each fluoroquinolone to normalize for differences in bacteriostatic action, gatifloxacin was more potent than the C-8-H compounds, both in the presence and absence of protein synthesis (an exception was observed when alanine was substituted for aspartic acid at position 82). Collectively, these data show that the C-8-methoxy group contributes to the enhanced activity of gatifloxacin against resistant gyrase and wild-type topoisomerase IV.

Alleles↗

PubMed1999

Cell-type-dependent activity of the ubiquitous transcription factor USF in cellular proliferation and transcriptional activation.

USF1 and USF2 are basic helix-loop-helix transcription factors implicated in the control of cellular proliferation. In HeLa cells, the USF proteins are transcriptionally active and their overexpression causes marked growth inhibition. In contrast, USF overexpression had essentially no effect on the proliferation of the Saos-2 osteosarcoma cell line. USF1 and USF2 also lacked transcriptional activity in Saos-2 cells when assayed by transient cotransfection with USF-dependent reporter genes. Yet, there was no difference in the expression, subcellular localization, or DNA-binding activity of the USF proteins in HeLa and Saos-2 cells. Furthermore, Gal4-USF1 and Gal4-USF2 fusion proteins activated transcription similarly in both cell lines. Mutational analysis and domain swapping experiments revealed that the small, highly conserved USF-specific region (USR) was responsible for the inactivity of USF in Saos-2 cells. In HeLa, the USR serves a dual function. It acts as an autonomous transcriptional activation domain at promoters containing an initiator element and also induces a conformational change that is required for USF activity at promoters lacking an initiator. Taken together, these results suggest a model in which the transcriptional activity of the USF proteins, and consequently their antiproliferative activity, is tightly controlled by interaction with a specialized coactivator that recognizes the conserved USR domain and, in contrast to USF, is not ubiquitous. The activity of USF is therefore context dependent, and evidence for USF DNA-binding activity in particular cells is insufficient to indicate USF function in transcriptional activation and growth control.

Amino Acid Sequence↗

PubMed1999

Ethambutol-induced psychosis: a case report.

Clinically, ethambutol (EMB)-induced psychosis is rare. In our review of the literature, most cases of antituberculosis agent-associated psychoses were caused by isoniazid (INH). We report the case of a 51-year-old man with suspected tuberculosis (TB) pleurisy. An anti-TB trial with INH, rifampicin and EMB was given initially. Dizziness, disorientation, and auditory and visual hallucinations developed after seven days of therapy. Laboratory examinations, including routine biochemistry tests, serum titer of antinuclear antibodies, cerebrospinal fluid analysis and computerized tomography of the head showed no abnormal findings. Following discontinuation of anti-TB agents, the psychiatric symptoms subsided. When the patient was challenged with EMB, the same psychiatric symptoms recurred, but resolved again after discontinuation of EMB. It is important to be aware that EMB can induce psychosis when anti-TB medications are prescribed.

Antitubercular Agents↗

PubMed1999

Hepatic radioembolization with Yttrium-90 glass microspheres for treatment of primary liver cancer.

OBJECTIVE: To study the clinical results of hepatic radioembolization with Yttrium-90 (90Y) glass microspheres in the treatment of primary liver cancer. METHODS: Seventeen patients with liver cancer were treated with glass microspheres from August 1996 to May 1998. Hepatic radioembolization with 90Y and lipiodol-ultrafluid was used. Percutaneous port-catheter system (PCS) implantations via femoral artery were performed in 12 patients. RESULTS: In the 17 patients, their mean ratio of absorbed doses between tumor and normal liver was 2.4:1. CT showed a significant reduction in tumor size in 11 of the 17 patients. Average survival was 19.5 months. The indwelling catheters of all the 12 patients were patent and no catheter tip locations were found. CONCLUSIONS: 90Y glass microsphere is one of the best radioisotopes. Not only good responses to the therapy of 90Y glass microspheres can be achieved in patients with metastatic liver cancer, but also in those with primary liver cancer, specially the localized or hypervascular mass. The patients with massive arterioportal shunt should not be limited to this form of radiation therapy. The percutaneous PCS implantation via the femoral artery is a new passageway for the treatment of primary liver cancer with 90Y glass microspheres and other interventional therapy.

Adult↗

PubMed1999

[One stage surgical repair for complex cranio-orbital injury].

OBJECTIVE: To approach clinical effect of early stage surgery on complex cranio-orbital injury. METHODS: Surgical procedures included removal of intracranial hematoma, contused and necrotic brain tissues, correction of cranio-orbital deformity, and repair of dure mater. RESULTS: Early evaluation was completed in 6 weeks, and late evaluation was made in 6 months after surgery according to the scale of Jennett and Bond's plastic surgical outcome and Glasgow outcome scale (GOS). Six weeks after surgery, GOS grade I was observed in 6 patients and GOS grade II in 3 patients. Excellent scale of Jennett and Bond was seen in 4 patients and good scale in 3 patients. Cerebrospinal leakage was effectively obstructed in 5 patients. CONCLUSIONS: Early stage repair for complex cranio-orbital injury may be conducive to improving neural function and decreasing intracranial infection.

Adult↗

PubMed1998

In vitro evaluation and crystallographic analysis of a new class of selective, non-amide-based thrombin inhibitors.

We describe the in vitro evaluation and crystallographic analysis of a new class of potent and selective, non-amino acid-based, small-molecule thrombin inhibitors, exemplified by 14. This class of achiral inhibitors lacks an amide-based backbone, exhibits nM inhibition of thrombin, and is selective for thrombin. Compound 14 does not interact with the active-site catalytic apparatus and is anchored to the enzyme via a single network of hydrogen bonds to Asp189 of the S1 pocket.

Amides↗

PubMed1998

Targeted disruption of the interferon-gamma receptor 2 gene results in severe immune defects in mice.

To study the role of the interferon- (IFN) gammaR2 chain in IFN-gamma signaling and immune function, IFN-gammaR2-deficient mice have been generated and characterized. Cells derived from IFN-gammaR2 -/- mice are unable to activate either JAK/STAT signaling proteins or gene transcription in response to IFN-gamma. The lack of IFN-gamma responsiveness alters IFN-gamma-induced Ig class switching by B cells from these mice. In vitro cultures of T cells demonstrate that the T cells from the IFN-gammaR2 -/- mice have a defect in Th1 cell differentiation. The IFN-gammaR2 (-/-) mice also produce lower amounts of IFN-gamma in response to antigenic challenge. In addition, IFN-gammaR2 -/- mice are defective in contact hypersensitivity and are highly susceptible to infection by Listeria monocytogenes. These results demonstrate that the IFN-gammaR2 is essential for IFN-gamma-mediated immune responses in vivo.

Animals↗

PubMed1998

Structural analysis of thrombin complexed with potent inhibitors incorporating a phenyl group as a peptide mimetic and aminopyridines as guanidine substitutes.

The structure of the noncovalent complex of human alpha-thrombin with a nonpeptide inhibitor containing a central phenyl scaffold, N-[2-[5-methyl-3-(2-chlorophenylsulfonyloxy)phenoxy]ethyl]-N- methyl-4 -aminopyridine (1), has been determined to 2.20 A resolution. In addition, the thrombin-bound structures of two distinct amino acid-based inhibitors (3 and 4) containing different aminopyridine-derived guanidine mimetics have been determined. Each compound occupies the same region of the active site and projects an aminopyridine, a central hydrophobic group, and an aryl group, into the S1, S2, and aryl subsites on thrombin. Nonpeptide 1 forms only one direct intermolecular hydrogen bond to the thrombin active site and forms no hydrogen bonds to ordered molecules of solvent. Close contacts are observed between main-chain carbonyl groups on thrombin and the edges of the central phenyl and aminopyridine rings and the sulfonyl group of 1 such that atoms carrying opposite partial charges are juxtaposed. Aminopyridine groups in 3 and 4 also form close contacts with the edges of carbonyl groups on thrombin and are flexibly accommodated in the S1 subsite. Superposition of the bound conformations of 1 and D-Phe-Pro-amidobutylguanidine (2) revealed that the central phenyl scaffold of 1 substitutes for the peptide main chain of 2.

Aminopyridines↗

PubMed1998

Is GRO J1744-28 a strange star?

The unusual hard x-ray burster GRO J1744-28 recently discovered by the Compton Gamma-Ray Observatory can be modeled as a strange star with a dipolar magnetic field of </=10(11) gauss. According to this model, when the accreted mass of the star exceeds some critical mass, its crust breaks, resulting in the conversion of the accreted matter into strange matter and a release of energy. Subsequently, a fireball forms and expands relativistically outward. The expanding fireball interacts with the surrounding interstellar medium, causing its kinetic energy to be radiated in shock waves and producing a burst of x-ray radiation. The burst energy, duration, interval, and spectrum derived from such a model are consistent with the observations of GRO J1744-28.

Journal Article↗

PubMed1998

Antimycobacterial evaluation of germacranolides.

The minimum inhibitory concentrations (MIC) against Mycobacterium tuberculosis and M. avium of parthenolide, costunolide, 1 (10)-epoxycostunolide and other germacranolide-type sesquiterpene lactones and derivatives were determined by use of a radiorespirometric bioassay. Structure-activity relationship studies with natural and semisynthetic sesquiterpene lactones suggested that the alpha-methylene-gamma-lactone moiety is an essential, but not sufficient, structural requirement for significant in vitro activity against M. tuberculosis and M. avium.

Anti-Bacterial Agents↗