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Biomedical subjects

R Lin

Publications and source records attributed to R Lin.

At least 73 records · Page 4Linked to original sources
PubMed1999

Taxol selectively blocks microtubule dependent NF-kappaB activation by phorbol ester via inhibition of IkappaBalpha phosphorylation and degradation.

Activation of the NF-kappa-B transcription factors has been shown to be directly influenced by changes in the microtubule cytoskeleton network. To better understand cytoskeletal regulation of NF-kappaB, experiments were performed to determine whether the microtubule (MT) stabilizing agent taxol could modulate NF-kappaB activation in the presence of different NF-kappa-B inducers. Pretreatment of murine NIH3T3 and human 293 cells with 5 microM taxol resulted in complete inhibition of phorbol, 12-myristate, 13-acetate (PMA) mediated NF-kappaB activation, detected as the loss of DNA binding and reduced NF-kappaB dependent reporter gene activity. Furthermore, in COS-7 and NIH3T3 cells, PMA-induced Ikappa-Balpha turnover was dramatically reduced in taxol treated cells, mediated via the inhibition of IkappaBalpha phosphorylation. However, taxol did not prevent TNF-alpha induced Ikappa-Balpha phosphorylation, degradation, or NF-kappaB activation, indicating that TNF-alpha acts through a microtubule-independent pathway. In vitro kinase assays with PMA stimulated cell extracts demonstrated that taxol reduced protein kinase C activity by 30%, thus implicating the loss of PKC activity as a possible regulatory target of taxol-mediated suppression of NF-kappa-B. Since PMA causes modulation of cytoarchitecture through PKC activation, microtubule integrity and cell morphology was analysed by indirect immunofluorescence. Both PMA and nocodazole, a MT depolymerizing agent, caused microtubule depolymerization, whereas TNF-alpha did not alter MT integrity; concomitant taxol treatment blocked both nocodazole and PMA induced depolymerization of MTs, as well as NF-kappaB induction, thus demonstrating a link between microtubule depolymerization and NF-kappaB activation. These observations illustrate a novel biological activity of taxol as a selective inhibitor of NF-kappa-B activity, suggesting a link between the state of microtubule integrity and gene regulation.

3T3 Cells↗
PubMed1999

'Yes' ifs, ands or buts: examining performance and correlates of the repetition task in the mini-mental state examination.

The aim of this study was to examine whether the type of sentence used in the repetition task included in the Mini-Mental State Examination (MMSE) affected performance in a group of 79 demented and 19 non-demented Hebrew-speaking elderly persons. The cognitive functioning of the participants was assessed using the MMSE and CAMCOG examinations. The performance of the repetition task was evaluated by using three sentences: the literal translation of the English language expression used in the original MMSE; a well-known Hebrew proverb consisting of monosyllabic words and rhythmic effects; and another well-known Hebrew proverb without such attributes. Only a third of the participants successfully repeated the literally translated expression. It showed low predictive value and was highly affected by education. The well-known Hebrew monosyllabic proverb showed moderate predictive value but no discriminatory ability. The other well-known Hebrew proverb performed the best. The translation of the repetition task in the MMSE to other languages is problematic. Strict adherence to the original language proved to be the least desirable choice.

Aged↗
PubMed1999

Biomechanical properties of muscle-tendon unit under high-speed passive stretch.

OBJECTIVE: The purpose of this study was to investigate the strain injury mechanisms of the Achilles muscle-tendon unit during high-speed passive stretch. DESIGN: The high-speed traction device consisted of an impactor which dropped freely to hit one end of a lever, transferring the impact energy to traction energy at the other end. A muscle-tendon unit was attached to the other end of the lever via a force link, and the elongation was recorded with a high-speed camera. BACKGROUND: The muscle-tendon unit is thought to act viscoelastically. It is generally strain rate dependent, exhibiting higher tensile stress at faster strain rates. However, previous studies of passive stretch in muscle-tendon units usually employed low strain rates. METHODS: 16 fresh Achilles muscle-tendon units were subjected to passive stretch at a test speed of 310 cm s(-1). The history of elongation and the traction force of the muscle-tendon unit during the elongation process were analyzed. RESULTS: The muscle-tendon units exhibited highly nonlinear mechanical behavior. Most of the elongation occurred in muscle and resulted in structural failure. Failure was not found in the tendon or muscle-tendon junction. Muscle fibers during stretching reached their maximum mechanical strength and then progressively ruptured. CONCLUSION: The strain rate is an important factor in strain injuries of the muscle-tendon unit due to passive stretch. The muscle is a good energy absorber; the rupture process can absorb a great deal of external energy and prevent complete failure of the muscle, while also protecting bone and joints. RELEVANCE: The study of muscle-tendon unit under high-speed stretch could help us to understand the mechanism of strain injuries over passive stretch in real-life situations.

Achilles Tendon↗
PubMed1999

MOM-4, a MAP kinase kinase kinase-related protein, activates WRM-1/LIT-1 kinase to transduce anterior/posterior polarity signals in C. elegans.

In C. elegans, a Wnt/WG-like signaling pathway down-regulates the TCF/LEF-related protein, POP-1, to specify posterior cell fates. Effectors of this signaling pathway include a beta-catenin homolog, WRM-1, and a conserved protein kinase, LIT-1. WRM-1 and LIT-1 form a kinase complex that can directly phosphorylate POP-1, but how signaling activates WRM-1/LIT-1 kinase is not yet known. Here we show that mom-4, a genetically defined effector of polarity signaling, encodes a MAP kinase kinase kinase-related protein that stimulates the WRM-1/LIT-1-dependent phosphorylation of POP-1. LIT-1 kinase activity requires a conserved residue analogous to an activating phosphorylation site in other kinases, including MAP kinases. These findings suggest that anterior/posterior polarity signaling in C. elegans may involve a MAP kinase-like signaling mechanism.

Amino Acid Sequence↗
PubMed1999

Triggering the interferon response: the role of IRF-3 transcription factor.

The interferon (IFN) regulatory factors (IRF) consist of a growing family of related transcription proteins first identified as regulators of the IFN-alpha/beta gene promoters, as well as the IFN-stimulated response element (ISRE) of some IFN-stimulated genes. IRF-3 was originally identified as a member of the IRF family based on homology with other IRF family members and on binding to the ISRE of the IFN-stimulated gene 15 (ISG15) promoter. Several recent studies have focused attention on the unique molecular properties of IRF-3 and its role in the regulation of IFN gene expression. IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. Following virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues, located in the carboxy-terminus of IRF-3. Phosphorylation causes the cytoplasmic to nuclear translocation of IRF-3, stimulation of DNA binding, and increased transcriptional activation, mediated through the association of IRF-3 with the CBP/p300 coactivator. The purpose of this review is to summarize recent investigations demonstrating the important role of IRF-3 in cytokine gene transcription. These studies provide the framework for a model in which virus-dependent phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN and IFN-responsive genes.

Amino Acid Sequence↗
PubMed1999

Risk factors for development of hepatocellular carcinoma among Australians with hepatitis C: a case-control study.

BACKGROUND: Older patients with cirrhosis due to hepatitis C are at risk of developing hepatocellular carcinoma (HCC), but additional risk factors may vary between countries. AIM: In the present study, we sought to identify additional risk factors for HCC among a cohort of Australian patients with chronic hepatitis C. METHODS: Case-control study of patients with advanced fibrosis stage hepatitis C who developed HCC during five-year follow up at a referral liver clinic. Cases were compared to twice the number of age-matched patients with chronic hepatitis C of similar fibrotic severity who did not develop HCC over a similar interval, using conditional logistic regression analysis (CLRA) and multivariate analysis. The main outcome measures were demographic and disease-related variables at first presentation in relation to the development of HCC. RESULTS: HCC developed in 17 cases, an annual incidence among those considered to be at risk of 2%. The duration of follow up since first assessment was comparable among the cases and 34 selected age-matched controls (4.1 and 5.2 years respectively, p=0.5). Cases were more often male (p=0.03), born in Asia (p=0.05), and had poorer liver function as indicated by serum albumin concentration (p=0.02). Anti-hepatitis B core-antibody (anti-HBc) was detected in 59% (ten/17) of cases, compared to 21% (seven/34) of the controls (p=0.01). No patient with a sustained response to interferon developed HCC during follow up. There were no significant differences in the mode of HCV transmission, HCV genotype, alcohol exposure, serum bilirubin level or prothrombin time between the cases and the controls. Although the data set was small, multivariate CLR analysis identified serum albumin < or = 35 g/L and anti-HBc positivity to be independent risk factors for development of HCC. CONCLUSIONS: Among older Australian patients (over the age of 40 years) with advanced fibrosis stage hepatitis C, the annual incidence of HCC is about 2%. Those who have low serum albumin and evidence of previous exposure to hepatitis B virus (anti-HBc positivity) appear to have the highest risk of developing HCC during follow up, but males and those born in Asia could also be at increased risk.

Aged↗
PubMed1999

IkappaB-mediated inhibition of virus-induced beta interferon transcription.

We have examined the consequences of overexpression of the IkappaBalpha and IkappaBbeta inhibitory proteins on the regulation of NF-kappaB-dependent beta interferon (IFN-beta) gene transcription in human cells after Sendai virus infection. In transient coexpression studies or in cell lines engineered to express different forms of IkappaB under tetracycline-inducible control, the IFN-beta promoter (-281 to +19) linked to the chloramphenicol acetyltransferase reporter gene was differentially inhibited in response to virus infection. IkappaBalpha exhibited a strong inhibitory effect on virus-induced IFN-beta expression, whereas IkappaBbeta exerted an inhibitory effect only at a high concentration. Despite activation of the IkappaB kinase complex by Sendai virus infection, overexpression of the double-point-mutated (S32A/S36A) dominant repressors of IkappaBalpha (TD-IkappaBalpha) completely blocked IFN-beta gene activation by Sendai virus. Endogenous IFN-beta RNA production was also inhibited in Tet-inducible TD-IkappaBalpha-expressing cells. Inhibition of IFN-beta expression directly correlated with a reduction in the binding of NF-kappaB (p50-RelA) complex to PRDII after Sendai virus infection in IkappaBalpha-expressing cells, whereas IFN-beta expression and NF-kappaB binding were only slightly reduced in IkappaBbeta-expressing cells. These experiments demonstrate a major role for IkappaBalpha in the regulation of NF-kappaB-induced IFN-beta gene activation and a minor role for IkappaBbeta in the activation process.

Amino Acid Sequence↗
PubMed1999

Essential role of interferon regulatory factor 3 in direct activation of RANTES chemokine transcription.

Localized and systemic cytokine production in virus-infected cells play an important role in the outcome of viral infection and pathogenicity. Activation of the interferon regulatory factors (IRF) in turn is a critical mediator of cytokine gene transcription. Recent studies have focused on the 55-kDa IRF-3 gene product as a direct transcriptional regulator of type 1 interferon (IFN-alpha and IFN-beta) activation in response to virus infection. Virus infection induces phosphorylation of IRF-3 on specific C-terminal serine residues and permits cytoplasmic-to-nuclear translocation of IRF-3, activation of DNA binding and transactivation potential, and association with the CBP/p300 coactivator. We previously generated constitutively active [IRF-3(5D)] and dominant-negative forms of IRF-3 that control IFN-beta and IFN-alpha gene expression. In an effort to characterize the range of immunoregulatory genes controlled by IRF-3, we now demonstrate that endogenous human RANTES gene transcription is directly induced in tetracycline-inducible IRF-3(5D)-expressing cells or paramyxovirus-infected cells. We also show that a dominant-negative IRF-3 mutant inhibits virus-induced expression of the RANTES promoter. Specific mutagenesis of overlapping ISRE-like sites located between nucleotides -123 and -96 in the RANTES promoter reduces virus-induced and IRF-3-dependent activation. These studies broaden the range of IRF-3 immunoregulatory target genes to include at least one member of the chemokine superfamily.

Base Sequence↗
PubMed1999

Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains.

The interferon regulatory factor 3 (IRF-3) gene encodes a 55-kDa protein which is expressed constitutively in all tissues. In unstimulated cells, IRF-3 is present in an inactive cytoplasmic form; following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues located in the carboxy terminus. Virus-induced phosphorylation of IRF-3 leads to cytoplasmic to nuclear translocation of phosphorylated IRF-3, association with the transcriptional coactivator CBP/p300, and stimulation of DNA binding and transcriptional activities of virus-inducible genes. Using yeast and mammalian one-hybrid analysis, we now demonstrate that an extended, atypical transactivation domain is located in the C terminus of IRF-3 between amino acids (aa) 134 and 394. We also show that the C-terminal domain of IRF-3 located between aa 380 and 427 participates in the autoinhibition of IRF-3 activity via an intramolecular association with the N-terminal region between aa 98 and 240. After Sendai virus infection, an intermolecular association between IRF-3 proteins is detected, demonstrating a virus-dependent formation of IRF-3 homodimers; this interaction is also observed in the absence of virus infection with a constitutively activated form of IRF-3. Substitution of the C-terminal Ser-Thr phosphorylation sites with the phosphomimetic Asp in the region ISNSHPLSLTSDQ between amino acids 395 and 407 [IRF-3(5D)], but not the adjacent S385 and S386 residues, generates a constitutively activated DNA binding form of IRF-3. In contrast, substitution of S385 and S386 with either Ala or Asp inhibits both DNA binding and transactivation activities of the IRF-3(5D) protein. These studies thus define the transactivation domain of IRF-3, two domains that participate in the autoinhibition of IRF-3 activity, and the regulatory phosphorylation sites controlling IRF-3 dimer formation, DNA binding activity, and association with the CBP/p300 coactivator.

Binding Sites↗
PubMed1999

Nasopharyngeal carcinoma: repeat treatment with conformal proton therapy--dose-volume histogram analysis.

PURPOSE: To analyze control, survival, and complication rates of conformal proton radiation for recurrent nasopharyngeal carcinoma. MATERIALS AND METHODS: Sixteen patients with nasopharyngeal carcinoma initially treated with 50.0-88.2 Gy photons were re-treated with protons to additional doses of 59.4-70.2 CGE. Local-regional control and survival were correlated with extent of relapse, recurrence versus persistence, and prescribed dose and were subjected to dose-volume histogram analysis. Mean follow-up was 23.7 months (range, 4-47 months). RESULTS: Twenty-four-month actuarial overall and local-regional progression-free survival rates were both 50%. The 24-month actuarial overall survival rates for patients with "optimal" dose-volume histogram coverage versus "suboptimal" coverage were 83% and 17%, respectively (P = .006). Doses to critical structures were low (0-22.0 Gy); no central nervous system side effects supervened. CONCLUSION: Adequate tumor coverage, as evaluated by using dose-volume histogram analysis, was found to be the most important variable influencing local-regional control and survival. No central nervous system complications were observed; increases in the dose to adjacent critical structures are being evaluated.

Adult↗
PubMed1999

A role for casein kinase II phosphorylation in the regulation of IRF-1 transcriptional activity.

The Interferon Regulatory Factors (IRFS) play an important role in the transcriptional control of growth regulatory and immunoregulatory genes. The inducibility and availability of IRF-1 and IRF-2 are influenced by external stimuli, such as virus infection or interferon treatment. In the present study, we sought to examine the potential modulatory role of phosphorylation on IRF-1 transcriptional activity. During the purification of IRF recombinant proteins, a kinase activity copurified with IRF-1 (and IRF-2) from baculovirus infected Sf9 insect cell extracts, but not from E. coli extracts. The kinase activity was also identified in Jurkat T cells, specifically interacted with IRF proteins in GST affinity chromatography, and phosphorylated IRF-1 with high specificity in vitro. Using an in gel kinase assay with recombinant IRF-1 as substrate, two molecular weight forms of the kinase (43 and 38 kDa) were identified. Biochemical criteria identified the kinase activity as the alpha catalytic subunit of casein kinase II (CKII). Furthermore, far western analysis of protein-protein interactions demonstrated that casein kinase II directly interacted with IRF-1 protein. Deletion mutation analysis of IRF-1 revealed that IRF-1 was phosphorylated at two clustered sites, one located between amino acids 138-150, the other in the C-terminal acidic activation domain between amino acids 219-231. Cotransfection studies comparing wild type and point mutated forms of IRF-1 demonstrated that mutations of the four phosphoaceptor residues in the C-terminal transactivation domain, significantly decreased transactivation by IRF-1, indicating that casein kinase II may be involved in the regulation of IRF-1 function. Strikingly, the casein kinase II clusters in IRF-1 resemble the sites identified in the C-terminal PEST domain of IkappaBalpha. The present experiments, together with previously published studies with IkappaBalpha, c-Jun and other proteins, indicate a broad role for casein kinase II phosphorylation in the regulation of transcription factor activity.

Blotting, Western↗
PubMed1999

alpha1-adrenergic receptor activation of c-fos expression in transfected rat-1 fibroblasts: role of Ca2+.

alpha1-Adrenergic receptors mediate mitogenic responses and increase intracellular free Ca2+ ([Ca2+]i) in vascular smooth muscle cells. Induction of c-fos is a critical early event in cell growth; expression of this gene is regulated by a number of signaling pathways including Ca2+. We wondered whether Ca2+ signaling plays a critical role in the induction of c-fos gene by alpha1-adrenergic receptors. Using stably transfected rat-1 fibroblasts, we confirmed that PE induced c-fos mRNA expression in a time- and dose-dependent manner, and also increased [Ca2+]i (measured with Fura-2 AM). These responses were blocked by the alpha1-adrenergic receptor antagonist doxazosin. Both intracellular Ca2+ chelation (using BAPTA/AM) and extracellular Ca2+ depletion (using EGTA) significantly inhibited PE-induced c-fos expression by alpha1A and alpha1B receptors. Brief (1-min) stimulation of alpha1A and alpha1B receptors with PE did not maximally induce c-fos expression, suggesting that a sustained increase in [Ca2+]i due to Ca2+ influx is required. The calmodulin (CaM) antagonists, R24571, W7, and trifluoperazine, but not the CaM-dependent protein kinases inhibitor KN-62, significantly inhibited c-fos induction by alpha1A and alpha1B receptors. Neither inhibition of protein kinase C nor inhibition of adenylyl cyclase modified c-fos induction by PE. These results suggest that alpha1-adrenergic receptor-induced c-fos expression in rat-1 cells is dependent on a Ca2+/CaM-associated pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

How do cognitively impaired elderly patients define "testament": reliability and validity of the testament definition scale.

The testament definition scale (TDS) is a specifically designed six-item scale aimed at measuring the respondent's capacity to define "testament." We assessed the reliability and validity of this new short scale in 31 community-dwelling cognitively impaired elderly patients. Interrater reliability for the six items ranged from .87 to .97. The interrater reliability for the total score was .77. Significant correlations were found between the TDS score and the Mini-Mental State Examination (MMSE) and the Cambridge Cognitive Examination scores (r = .71 and .72 respectively, p = .001). Criterion validity yielded significantly different means for subjects with MMSE scores of 24-30 and 0-23: mean 3.9 and 1.6 respectively (t(20) = 4.7, p = .001). Using a cutoff point of 0-2 vs. 3+, 79% of the subjects were correctly classified as severely cognitively impaired, with only 8.3% false positives, and a positive predictive value of 94%. Thus, TDS was found both reliable and valid. This scale, however, is not synonymous with testamentary capacity. The discussion deals with the methodological limitations of this study, and highlights the practical as well as the theoretical relevance of TDS. Future studies are warranted to elucidate the relationships between TDS and existing legal requirements of testamentary capacity.

Aged↗
PubMed1999

[Function research of esophageal lower sphincter in infant with hiatal hernia].

OBJECTIVE: To investigate the relationship between gastroesophageal dynamics of infantile hiatal hernia (HH) and gastroesophageal reflux (GER) and to assess the curative effects of antireflux surgery. METHODS: Ambulatory 24-hr esophageal pH-metry and esophageal manometry were studied in 26 patients with HH before and after surgery. RESULTS: By radiology, patients were divided into group of sliding HH (n = 16, group I) and group of para-esophageal or combined HH (n = 10, group II). All patients showed GER. The duration of the longest episode, and the percentage of the time at pH < 4 were significantly greater in lying position than those in upright position (P < 0.05). All parameters were significantly decreased after operation (P < 0.01). Lower esophageal sphincter length (LESL) increased significantly postoperatively. In group I, lower esophageal sphincter pressure (LESP) and barrier pressure (BP) was increased after surgery, but no significant difference was seen in LESP and BP in group II. Intragastric pressure (GP) significantly reduced after operation. GP in group II before operation was higher than that in group I (P < 0.05). A second high pressure zone in the esophagus was found in 67% of patients in group I and 14% patients in group II. CONCLUSIONS: Most patients with infantile HH and GER decreased LESL, LESP and increased GP. Differential gastroesophageal dynamics is associated with types of hiatal hernia. It is very important to remain enough LESL in mechanism of Dor antireflux surgery. To prevent respiratory infection is an important measure to prevent patients from relapse after surgery.

Child, Preschool↗
PubMed1999

[Application of X-ray diffraction to the analysis of traditional Chinese medicine].

OBJECTIVE: To approach the application of powder Xray diffraction in analysis of traditional Chinese medicine(TCM). METHOD: Studying the references published in recent years and making a summary on the subject of application of Xray in the pharmaceutical analysis of TCM. RESULT: This method is effective in identifying and analyzing TCM. CONCLUSION: This technique may have broad prospects for pharmaceutical analysis.

Drug Contamination↗
PubMed1998

Homologue scanning mutagenesis of heregulin reveals receptor specific binding epitopes.

The EGF domain of heregulin has all the receptor binding characteristics of full-length heregulin and has strong homology to the ligands for erbB-1. Despite this, it does not bind erbB-1 but instead binds erbB-3 and erbB-4. The sequence similarity between HRG and the erbB-1 ligands suggest that a few residues are responsible for receptor binding specificity. To determine the sequences involved in receptor binding, we performed homologue scanning mutagenesis on the EGF domain of HRGalpha using sequences of TGFalpha or EGF. We found three sets of mutations in the N-terminal subdomain that were responsible for receptor binding specificity. Mutations in the C-terminal subdomain affected the binding affinity, but did appear to confer any specificity.

Adenocarcinoma↗
PubMed1998

Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3.

The family of interferon (IFN) regulatory factors (IRFs) encodes DNA-binding transcription factors, some of which function as modulators of virus-induced signaling. The IRF-3 gene is constitutively expressed in many tissues and cell types, and neither virus infection nor IFN treatment enhances its transcription. In infected cells, however, IRF-3 protein is phosphorylated at the carboxyl terminus, which facilitates its binding to the CBP/p300 coactivator. In the present study, we demonstrate that overexpression of IRF-3 significantly enhances virus-mediated transcription of the IFNA and IFNB genes in infected cells as well as IFN synthesis. IRF-3-mediated activation of IFN genes depends in part on carboxyl-terminal phosphorylation of a cluster of Ser/Thr residues, because a mutant with Ser/Thr to Ala substitutions activates the IFN promoter less efficiently. However, overexpression of IRF-3 in human 2FTGH cells alone results in the induction of an antiviral state, which depends on functional IFN signaling, because IRF-3 does not induce an antiviral state in mutant 2FTGH cells defective in either JAK-1 or p48 functions; also no antiviral effect of IRF-3 could be demonstrated in Vero cells that lack the IFNA and IFNB genes. This finding indicates that the observed antiviral activity of IRF-3 in 2FTGH cells results mainly from the induction of IFNs. Furthermore, E1A protein inhibited IRF-3-mediated stimulation of the IFNA4 promoter in transient expression assays; this inhibition could be reversed partially by overexpression of CBP/p300 and was not demonstrated with the mutant of E1A that does not bind p300. These results identify IRF-3 and CBP/p300 as integral components of the virus-induced complex that stimulates type 1 IFN gene transcription. The observation that adenovirus E1A antagonizes IRF-3 mediated activation suggests that E1A and IRF-3 may compete for binding to CBP/p300 and implicates a novel mechanism by which adenovirus may overcome the antiviral effects of the IFN pathway.

Adenoviridae↗