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Biomedical subjects

T Lin

Publications and source records attributed to T Lin.

At least 37 records · Page 2Linked to original sources
PubMed2002

Mechanisms involved in development of resistance to adenovirus-mediated proapoptotic gene therapy in DLD1 human colon cancer cell line.

To evaluate resistance that develops in cancer cells during treatment with adenoviral vectors expressing proapoptotic genes, we repeatedly treated the human colon cancer cell line DLD1 with adenoviral vectors expressing the human Bax gene and the human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene. DLD1 cells resistant to the Bax- or TRAIL-expressing adenoviral vectors were then selected and designated as DLD1/Bax-R or DLD1/TRAIL-R cells, respectively. Further study showed that resistance in DLD1/Bax-R cells was caused by resistance to adenoviral infection, which can be overcome by dose escalation of the adenoviral vectors. However, resistance in DLD1/TRAIL-R cells was caused by resistance to the TRAIL gene. Therefore, different mechanisms are involved in the development of resistance during adenovirus-mediated proapoptotic gene therapy. A survey of molecules involved in TRAIL- or Bax-mediated apoptotic pathways showed no significant change in expression of death receptors, death decoy receptors; FLIP; Bcl-2; Bcl-xS; Bax; Bak; XIAP or caspase-2, -7, -8, or -9 in either DLD1/Bax-R or DLD1/TRAIL-R cells. Bcl-xL expression detected in both mRNA and protein level assays was three times higher in DLD1/TRAIL-R cells than in parental or DLD1/Bax-R cells. However, transfection of DLD1 cells with the Bcl-xL gene showed that overexpression of Bcl-xL is not sufficient for the resistance. Moreover, DLD1/Bax-R cells were sensitive to adenoviral vectors that expressed the TRAIL gene, but resistant to adenoviral vectors that expressed the Bak gene. In contrast, DLD1/TRAIL-R cells were sensitive to adenoviral vectors that expressed either Bax or Bak gene. Thus, alternative application of adenoviral vectors that expressed proapoptotic genes in different pathways or different cell killing models may delay or prevent development of resistance in adenovirus-mediated proapoptotic gene therapy.

Adenoviridae↗
PubMed2002

Combined TRAIL and Bax gene therapy prolonged survival in mice with ovarian cancer xenograft.

We evaluated the antitumor activity of the Bax gene and green fluorescent protein/tumor necrosis factor-related apoptosis-inducing ligand (GFP/TRAIL) fusion gene driven by the human telomerase reverse transcriptase promoter both separately and combined in the human ovarian cancer lines SKOV3ip and DOV13 and human lung cancer line H1299. In vitro study showed that both TRAIL- and Bax-expressing vectors elicited significant cell killing in H1299 and SKOV3ip cells, but only the GFP/TRAIL gene elicited significant cell killing in DOV13 cells. Combined TRAIL and Bax therapy also produced more profound cell killing in SKOV3ip and H1299 cells, but not DOV13 cells without escalation of the vector doses. To further evaluate the combined effects of Bax and TRAIL, abdominally spread tumors were established in nude mice via intraperitoneal inoculation of SKOV3ip cells followed by that of adenoviral vectors. Tumor growth, ascites formation, survival duration and toxicity were evaluated after treatment. We found that treatment using the Bax- or TRAIL-expressing vector alone significantly suppressed tumor growth and ascites formation, and prolonged animal survival when compared with that of using PBS or a control vector. Combined TRAIL and Bax therapy further prolonged survival significantly when compared with therapy using the TRAIL or Bax gene alone. Transgene expression and apoptosis induction were not detected in normal human ovarian epithelial cells in vitro or normal mouse tissues in vivo after intraperitoneal vector administration. Also, liver toxicity was not detected after either treatment. Thus, combined TRAIL and Bax gene therapy may be useful for treatment of abdominally spread tumors.

Animals↗
PubMed2002

Cowpea mosaic virus: from the presentation of antigenic peptides to the display of active biomaterials.

The potential of cowpea mosaic virus (CPMV), a plant icosahedral virus, for the presentation of foreign peptides and proteins is reported. The most prominent feature at the virus surface is a region of the smaller of the two coat proteins (S) which has been extensively used for the insertion of foreign peptides. Given the availability of the three-dimensional structure of the native virus and the amenability of foreign peptide-expressing CPMV chimeras to crystallisation, immunological data can be correlated with the conformational state of the foreign insert. The latter is influenced by proteolysis which occurs within the foreign inserts. In an effort to offer an alternative context for peptide expression, extensive exploration of a second region of the S protein is reported with respect to tolerance to small insertions. Moreover, to make CPMV suitable for a wider spectrum of presentation, a technique was developed to allow surface coupling of a peptide which can serve as the anchoring point for a range of proteins. This new approach is also widely applicable for the direct chemical cross-linking of peptides and full-length protein domains to the viral capsid.

Amino Acid Sequence↗
PubMed2001

Accumulation and activation-induced release of preformed Fas (CD95) ligand during the pathogenesis of experimental graft-versus-host disease.

Fas (CD95/APO-1) ligand (FasL)-mediated cytotoxicity has been implicated in tissue destruction in a variety of diseases, including acute graft-vs-host disease (GVHD). In this study, we have analyzed FasL expression and regulation during the course of experimental murine acute GVHD. Although activation-induced FasL-mediated cytotoxicity in control T cells was sensitive to the immunosuppressant cyclosporin A, we observed that functional FasL expression of GVHD T cells became increasingly cyclosporin A unresponsive. This was found to be the result of a massive in vivo accumulation and intracellular storage of FasL protein and its release in a transcription- and protein synthesis-independent manner. Immunohistochemistry analysis of FasL expression in situ revealed accumulation of FasL-expressing cells in the spleen, the liver, and small intestine, with a typical cytoplasmic and granular expression pattern. Thus, we conclude that the release of preformed FasL by infiltrating donor T cells may contribute to recipient tissue damage during the pathogenesis of acute GVHD.

Acute Disease↗
PubMed2001

IL-7 prevents both caspase-dependent and -independent pathways that lead to the spontaneous apoptosis of i-IEL.

Intestinal intraepithelial lymphocytes (i-IEL) readily undergo spontaneous apoptosis in vitro through an unclear mechanism. Here we examined the relationship between caspases, which plays a major role in apoptosis, and IL-7 in the spontaneous apoptosis of i-IEL in vitro. We demonstrated that IL-7 and zVAD prevented the spontaneous apoptosis of i-IEL by approximately 50% and 25% respectively with no additive protection seen when both are used. IL-7 preferentially prevented the apoptosis of gammadelta i-IEL, while zVAD equally prevented the apoptosis of gammadelta and alphabeta i-IEL. Lastly, we demonstrated that the spontaneous apoptosis of i-IEL is associated with a marked increase in caspase activity. Caspase activity was completely inhibited by zVAD, but only slightly by IL-7. Overall these results suggest that two pathways lead to the spontaneous apoptosis of i-IEL, one which is caspase dependent and the other which is caspase independent. IL-7 appears to exert its effect on i-IEL undergoing spontaneous by partially inhibiting both apoptotic pathways.

Amino Acid Chloromethyl Ketones↗
PubMed2001

p53-dependent radiation-induced crypt intestinal epithelial cells apoptosis is mediated in part through TNF-TNFR1 system.

Radiation induces apoptosis of crypt intestinal epithelial cells (IEC) through a pathway that is largely dependent on p53. However, exactly how p53 mediates IEC apoptosis is unclear. Studies in vitro suggest that one mechanism by which p53 mediates apoptosis is through its ability to transactivate members of the TNF receptor family of 'Death Receptors'. Here, we examined the role of one of its member, TNF receptor type 1 (TNFR1), in an in vivo model of p53-dependent radiation-induced IEC apoptosis. We demonstrate that mice genetically engineered to be deficient in TNF receptor type 1 (TNFR1(-/-)) and mice injected with TNFR1-fusion chimeric protein (TNFR1-Fc; a competitive inhibitor of TNFR1) were partially protected (30-40%) from p53-dependent radiation-induced IEC apoptosis. However, we found no evidence to support the possibility p53 transcriptionally regulates the expression of TNFR1 nor increases the susceptibility of IEC to TNF-mediated apoptosis. Interestingly, we found that injection of TNF readily induced IEC apoptosis and that radiation induced a p53-dependent increase in the intestinal level of TNF. Furthermore, injection of a neutralizing anti-TNF mAb reduced p53-dependent radiation-induced IEC apoptosis by approximately 60%. Overall, these results suggest that p53-dependent radiation-induced IEC apoptosis is mediated in part through ability of p53 to regulate TNF, which subsequently induces IEC apoptosis through TNFR1.

Animals↗
PubMed2001

Wavenumber Measurements of CO(2) Transitions in 1.5-µm Atmospheric Window Using an External-Cavity Diode Laser.

Using an external-cavity diode laser (ECDL) as the light source, we have observed the spectrum of CO(2) in the 1.5-µm atmospheric window including the (30(0)1)(I) <-- (00(0)0) and (31(1)1)(I) <-- (01(1)0)(I) bands. Good signal-to-noise ratio allowed us to lock the frequency of our ECDL to the absorption line centers. Wavenumber measurements of the transitions with accuracy about 6.6 x 10(-4) cm(-1) are made with the help of a precision wavemeter calibrated to the accurate C(2)H(2) frequency references in the 1.5-µm wavelength region. Molecular constants are obtained by making the least-squares fits of the measured transition wavenumbers. The rotational and centrifugal distortion constants are consistent with the previous results using high-resolution Fourier transform spectroscopy. However, the band centers are different with previous results by several thousandth reciprocal centimeters. Copyright 2001 Academic Press.

Journal Article↗
PubMed2001

Purification, crystallization, and preliminary X-ray analysis of L-A: a dsRNA yeast virus.

TheL-A virus (LAV) particle is a specialized compartment for the transcription and replication of double-stranded RNA. It is 390 A in diameter and infects yeast. The particle is formed by a capsid containing 120 copies of a 680-residue gene product arranged with T = 1 icosahedral symmetry, approximately two copies of an RNA-directed RNA polymerase, and a 4.6-kb linear, duplex RNA. LAV crystals diffracting to at least 4.5-A resolution were grown in a combination of polyethylene glycol 8000, ethylene glycol, and lithium chloride. Following crystallization the reservoir solution was replaced by a 2x concentrated reservoir solution in order for ethylene glycol to function as a cryoprotectant even though initial crystals would not grow at sufficiently high concentrations of ethylene glycol for cryoprotection. A complete data set was collected to 6-A resolution from a frozen crystal obtained with this procedure. The crystals belong to space group P2(1). The unit cell dimensions are a = 406.7 A, b = 403.3 A, c = 572.5 A, beta = 90.3 degrees with two virus particles in the unit cell. The particle orientation was determined with the rotation function and the particle center was estimated on the basis of packing considerations.

Capsid↗
PubMed2001

Direct costs-of-illness of patients with diabetes mellitus in Taiwan.

The purpose of the present study was to examine the characteristics of healthcare costs for diabetic patients in Taiwan. The study analyzed claim data from the Bureau of National Health Insurance for the period from July 1997 to June 1998. There were 536159 documented diabetic patients who were treated within the universal healthcare system in Taiwan during this study period. The annual number of visits of these diabetic patients was 6.2% of the total outpatient visits of all patients due to all causes during the one-year study period. Diabetes-related problems were the causes of 25.2% of outpatient visits among diabetic patients, while 74.8% of visits were for causes unrelated to diabetes. The distribution of treatment for the diabetic patients was by oral hypoglycemic agents 88.3%, insulin only 6.9%, and a combination of insulin and oral agents 4.8%. Diabetic patients accounted for 4,724,711 hospital inpatient days during the study period, which was 22.1% of the total inpatient days in Taiwan. Of the inpatient admissions, 13.9% were for diabetes as the principal cause, 23.4% were for diabetes-related disease, and 62.7% were for causes unrelated to diabetes. The direct costs of healthcare for the documented diabetic patients was 11.5% of the total costs of healthcare in Taiwan, and was 4.3 times higher than the average costs of care for non-diabetic individuals.

Ambulatory Care↗
PubMed2001

Dissociation pathways of alkali-cationized peptides: opportunities for C-terminal peptide sequencing.

Dissociation pathways of alkali-cationized peptides have been studied using multiple stages of mass spectrometry (MSx) with a quadrupole ion trap mass spectrometer. Over 100 peptide ions ranging from 2 to 10 residues in length and containing each of the 20 common amino acids have been examined. The formation of the [b(n-1) + Na + OH]+ product ion is the predominant dissociation pathway for the majority of the common amino acids. This product corresponds to a sodium-cationized peptide one residue shorter in length than the original peptide. In a few cases, product ions such as [b(n-1) + Na - H]+ and those formed by loss, or partial loss, of a sidechain are observed. Both [b(n-1) + Na + OH]+ and [b(n-1) + Na - H]+ product ions can be selected as parent ions for a subsequent stage of tandem mass spectrometry (MS/MS). It is shown that these dissociation patterns provide opportunities for peptide sequencing by successive dissociation from the C-terminus of alkali-cationized peptides. Up to seven stages of MS/MS have been performed on a series of [b + Na + OH]+ ions to provide sequence information from the C-terminus. This method is analogous to Edman degradation except that the cleavage occurs from the C-terminus instead of the N-terminus, making it more attractive for N-terminal blocked peptides. The isomers leucine and isoleucine cannot be differentiated by this method but the isobars lysine and glutamine can.

Alkalies↗
PubMed2001

The structure of pariacoto virus reveals a dodecahedral cage of duplex RNA.

The 3.0 A resolution crystal structure of Pariacoto virus (PaV) reveals extensive interactions between portions of the viral RNA genome and the icosahedral capsid. Under the protein shell of the T = 3 quasi equivalent capsid lies a dodecahedral cage composed of RNA duplex that accounts for approximately 35% of the single-stranded RNA genome. The highly basic N-terminal regions (residues 7-54) of the subunits, forming pentamers (A subunits) are clearly visible in the density map and make numerous interactions with the RNA cage. The C-terminal segments (residues 394-401) of the A subunits lie in channels near the quasi three-fold axes. Electron cryo-microscopy and image reconstruction of PaV particles clearly show the dodecahedral RNA cage.

Amino Acid Sequence↗
PubMed2001

Dominant effector genetics in mammalian cells.

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.

Amino Acid Motifs↗
PubMed2001

Corneal surface area: an index of anterior segment growth.

Corneal surface area and perimeter were assessed as novel indices to monitor anterior segment growth, using chicks reared under different photoperiods. We obtained central and mid-peripheral corneal curvatures using photokeratometry. Anatomical tracings of the anterior corneal surface also were made from freeze-dried non-fixed preparations of the anterior segments of the same eyes. Using either photokeratometry or anatomical data, the profile of the anterior corneal surface was fit to a general equation for conical sections; corneal surface area was estimated from surfaces of revolution. Optical techniques modeled the chick cornea as a circle or as an ellipse closely resembling a circle. The anatomical technique, in contrast, modeled the chick corneal profile as a hyperbola. Potential explanations of this discrepancy are discussed. Regardless of which model is evaluated, the corneal surface area and perimeter of two-week-old chicks are affected by the photoperiod of rearing. Corneal surface area in particular proved more sensitive than conventional measurements in identifying anterior segment effects of rearing under different photoperiods. Analysis of corneal area may prove useful in evaluating the mechanisms governing anterior segment growth.

Animals↗
PubMed2001

Influence of smoking, hypertension, and sex on the phenotypic expression of familial intracranial aneurysms in siblings.

OBJECTIVE: To investigate the effects of smoking, hypertension, and sex on the phenotypic expression of familial intracranial aneurysms (FIAs). METHODS: We retrospectively reviewed the case records of 806 consecutive patients undergoing aneurysm surgery at our institution (1986-1995) and discovered 24 families with at least two affected siblings. Prevalence rates for the smoking, hypertension, and sex risk factors in these nuclear families were compared with those of patients with sporadic intracranial aneurysms (SIAs) and population-based control patients. RESULTS: Affected family members with FIAs exhibited prevalence rates of smoking and hypertension (74% and 43%, respectively) that tended to be higher than those of population-based control patients (52% [P < 0.005] and 36% [P = not significant (NS)], respectively) and comparable to those of patients with SIAs (64% [P = NS] and 40% [P = NS], respectively). A positive association existed between FIA formation and female sex but was somewhat less strong than that observed in the SIAs (59% FIAs, 71% SIAs, 50% control patients). In addition, the prevalence rates of smoking, hypertension, and female sex were higher in affected family members with FIAs than in their unaffected siblings (58% [P < 0.05], 28% [P = 0.06], and 39% [P < 0.05], respectively). Individuals in families with expressed FIAs who had high aneurysmal penetrance had a greater tendency to be smokers, hypertensive, and female (74%, 59%, and 55%, respectively) than did their low-penetrance counterparts (61% [P = 0.1], 27% [P < 0.05], and 45% [P = NS], respectively). CONCLUSION: Together these data suggest that hypertension, smoking, and female sex increase the likelihood that a member of a family with an expressed FIA will have an aneurysm. These observations may prove helpful in guiding the use of screening studies and encouraging education about the potential risks of continued tobacco use and untreated hypertension in this patient cohort.

Cohort Studies↗
PubMed2001

Comparison of the anti-influenza virus activity of RWJ-270201 with those of oseltamivir and zanamivir.

We have recently reported an influenza virus neuraminidase inhibitor, RWJ-270201 (BCX-1812), a novel cyclopentane derivative discovered through structure-based drug design. In this paper, we compare the potency of three compounds, RWJ-270201, oseltamivir, and zanamivir, against neuraminidase enzymes from various subtypes of influenza. RWJ-270201 effectively inhibited all tested influenza A and influenza B neuraminidases in vitro, with 50% inhibitory concentrations of 0.09 to 1.4 nM for influenza A neuraminidases and 0.6 to 11 nM for influenza B neuraminidases. These values were comparable to or lower than those for oseltamivir carboxylate (GS4071) and zanamivir (GG167). RWJ-270201 demonstrated excellent selectivity (>10,000-fold) for influenza virus neuraminidase over mammalian, bacterial, or other viral neuraminidases. Oral administration of a dosage of 1 mg/kg of body weight/day of RWJ-270201 for 5 days (beginning 4 h preinfection) showed efficacy in the murine model of influenza virus infection as determined by lethality and weight loss protection. RWJ-270201 administered intranasally at 0.01 mg/kg/day in the murine influenza model demonstrated complete protection against lethality, whereas oseltamivir carboxylate and zanamivir at the same dose demonstrated only partial protection. In the delayed-treatment murine influenza model, oral administration of a 10-mg/kg/day dose of RWJ-270201 or oseltamivir (GS4104, a prodrug of GS4071) at 24 h postinfection showed significant protection against lethality (P < 0.001 versus control). However, when the treatment was delayed for 48 h, no significant protection was observed in either drug group. No drug-related toxicity was observed in mice receiving 100 mg/kg/day of RWJ-270201 for 5 days. These efficacy and safety profiles justify further consideration of RWJ-270201 for the treatment and prevention of human influenza.

Acetamides↗
PubMed2001

Genetic heterogeneity of Borrelia burgdorferi sensu lato in the southern United States based on restriction fragment length polymorphism and sequence analysis.

Fifty-six strains of Borrelia burgdorferi sensu lato, isolated from ticks and vertebrate animals in Missouri, South Carolina, Georgia, Florida, and Texas, were identified and characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. A total of 241 to 258 bp of intergenic spacers between tandemly duplicated rrf (5S) and rrl (23S) was amplified by PCR. MseI and DraI restriction fragment polymorphisms were used to analyze these strains. PCR-RFLP analysis results indicated that the strains represented at least three genospecies and 10 different restriction patterns. Most of the strains isolated from the tick Ixodes dentatus in Missouri and Georgia belonged to the genospecies Borrelia andersonii. Excluding the I. dentatus strains, most southern strains, isolated from the ticks Ixodes scapularis and Ixodes affinis, the cotton rat (Sigmodon hispidus), and cotton mouse (Peromyscus gossypinus) in Georgia and Florida, belonged to Borrelia burgdorferi sensu stricto. Seven strains, isolated from Ixodes minor, the wood rat (Neotoma floridana), the cotton rat, and the cotton mouse in South Carolina and Florida, belonged to Borrelia bissettii. Two strains, MI-8 from Florida and TXW-1 from Texas, exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Eight Missouri tick strains (MOK-3a group) had MseI patterns similar to that of B. andersonii reference strain 21038 but had a DraI restriction site in the spacer. Strain SCGT-8a had DraI restriction patterns identical to that of strain 25015 (B. bissettii) but differed from strain 25015 in its MseI restriction pattern. Strain AI-1 had the same DraI pattern as other southern strains in the B. bissettii genospecies but had a distinct MseI profile. The taxonomic status of these atypical strains needs to be further evaluated. To clarify the taxonomic positions of these atypical Borrelia strains, the complete sequences of rrf-rrl intergenic spacers from 20 southeastern and Missouri strains were determined. The evolutionary and phylogenetic relationships of these strains were compared with those of the described genospecies in the B. burgdorferi sensu lato species complex. The 20 strains clustered into five separate lineages on the basis of sequence analysis. MI-8 and TXW-1 appeared to belong to two different undescribed genospecies, although TXW-1 was closely related to Borrelia garinii. The MOK-3a group separated into a distinct deep branch in the B. andersonii lineage. PCR-RFLP analysis results and the results of sequence analyses of the rrf-rrl intergenic spacer confirm that greater genetic heterogeneity exists among B. burgdorferi sensu lato strains isolated from the southern United States than among strains isolated from the northern United States. The B. andersonii genospecies and its MOK-3a subgroup are associated with the I. dentatus-cottontail rabbit enzootic cycle, but I. scapularis was also found to harbor a strain of this genospecies. Strains that appear to be B. bissettii in our study were isolated from I. minor and the cotton mouse, cotton rat, and wood rat. The B. burgdorferi sensu stricto strains from the south are genetically and phenotypically similar to the B31 reference strain.

Animals↗
PubMed2001

Epidermal growth factor receptor and tyrosine phosphorylation of estrogen receptor.

Activation of estrogen receptor-alpha (ERalpha) by growth factors in the absence of estrogen is a well-documented phenomenon. To study further this process of ligand-independent receptor activation, COS-7 cells without ER were transfected with both ER and epidermal growth factor receptor (EGFR). In the absence of estrogen, epidermal growth factor (EGF) stimulated rapid tyrosine phosphorylation of ER in transfected COS-7 cells. Similarly, in MCF-7 breast cancer cells that have natural expression of ER and EGFR, EGF promoted acute phosphorylation of serine and tyrosine residues in ER, and a direct interaction between ER and EGFR after treatment with EGF was found. In confirmation of a direct interaction between ER and EGFR, activation of affinity-purified EGFR tyrosine kinase in vitro stimulated the phosphorylation of recombinant ER. The cross-communication between EGFR and ER appears to promote significant stimulation of cell proliferation and a reduction in the apoptotic loss of those cells that express both receptor signaling pathways. However, COS-7 cells transfected with both ER and EGFR show minimal stimulation of classical estrogen response element (ERE)-dependent transcriptional activity after stimulation by EGF ligand. This suggests that the proliferative and antiapoptotic activity of EGF-induced ER activation may be dissociated from ERE-dependent transcriptional activity of the ER.

Animals↗