Characterizing Class-Specific Exposure-Viral Load Suppression Response of HIV Antiretrovirals Using A Model-Based Meta-Analysis.

We applied model-based meta-analysis of viral suppression as a function of drug exposure and in vitro potency for short-term monotherapy in human immunodeficiency virus type 1 (HIV-1)-infected treatment-naïve patients to set pharmacokinetic targets for development of nonnucleoside reverse transcriptase inhibitors (NNRTIs) and integrase strand transfer inhibitors (InSTIs). We developed class-specific models relating viral load kinetics from monotherapy studies to potency normalized steady-state trough plasma concentrations. These models were integrated with a literature assessment of doses which demonstrated to have long-term efficacy in combination therapy, in order to set steady-state trough concentration targets of 6.17- and 2.15-fold above potency for NNRTIs and InSTIs, respectively. Both the models developed and the pharmacokinetic targets derived can be used to guide compound selection during preclinical development and to predict the dose-response of new antiretrovirals to inform early clinical trial design.

Factors in the prioritization of information needs among Hong Kong Chinese breast cancer patients.

OBJECTIVE: The study aims to examine the prioritization of information needs in breast cancer patients, using the Information Needs Questionnaire (INQ); and to identify the demographic and clinical characteristics associated with that prioritization. METHODS: A cross-sectional exploratory study was conducted, by means of consecutive sampling. The INQ was used to examine participants' preferences on information needs. Their demographic and clinical characteristics were collected by means of a structured questionnaire and review of medical records. Backward multivariable logistic regression analysis was performed to examine the association between prioritization of patients' information needs and their demographic and clinical characteristics. RESULTS: A total of 275 breast cancer patients took part in the analysis. Of the nine INQ items, most participants ranked as their top four needs information about the likelihood of a cure (79%), extent of the disease (76%), treatment options (55%), and family risk of developing breast cancer (51%). Certain demographic and clinical characteristics-religious belief, whether living alone or not, household income, educational level, and time since cancer diagnosis-influenced patients' prioritization of information needs. CONCLUSION: Understanding and meeting the information needs of breast cancer patients are crucial to improving their quality of care. Different patients are likely to have different priorities in information needs according to their demographic and clinical characteristics. An awareness of these associated factors will allow better tailor-made educational interventions to be provided to meet patients' individual needs in a more adequate way.

LRIG1 modulates aggressiveness of head and neck cancers by regulating EGFR-MAPK-SPHK1 signaling and extracellular matrix remodeling.

EGFR overexpression and chromosome 3p deletion are two frequent events in head and neck cancers. We previously mapped the smallest region of recurrent copy-number loss at 3p12.2-p14.1. LRIG1, a negative regulator of EGFR, was found at 3p14, and its copy-number loss correlated with poor clinical outcome. Inducible expression of LRIG1 in head and neck cancer TW01 cells, a line with low LRIG1 levels, suppressed cell proliferation in vitro and tumor growth in vivo. Gene expression profiling, quantitative RT-PCR, chromatin immunoprecipitation, and western blot analysis demonstrated that LRIG1 modulated extracellular matrix (ECM) remodeling and EGFR-MAPK-SPHK1 transduction pathway by suppressing expression of EGFR ligands/activators, MMPs and SPHK1. In addition, LRIG1 induction triggered cell morphology changes and integrin inactivation, which coupled with reduced SNAI2 expression. By contrast, knockdown of endogenous LRIG1 in TW06 cells, a line with normal LRIG1 levels, significantly enhanced cell proliferation, migration and invasiveness. Such tumor-promoting effects could be abolished by specific MAPK or SPHK1 inhibitors. Our data suggest LRIG1 as a tumor suppressor for head and neck cancers; LRIG1 downregulation in cancer cells enhances EGFR-MAPK-SPHK1 signaling and ECM remodeling activity, leading to malignant phenotypes of head and neck cancers.

Porphyromonas gingivalis fimbriae-dependent interleukin-6 autocrine regulation by increase of gp130 in endothelial cells.

BACKGROUND AND OBJECTIVE: Local persistent infection by Porphyromonas gingivalis leads to inflammatory systemic diseases, such as atherosclerosis. We have reported previously that avirulent P. gingivalis fimbriae-dependent invasion into endothelial cells might be involved in progression of atherosclerosis. Although interleukin-6 (IL-6) regulates progression of atherosclerosis, little is known about the relationship of P. gingivalis fimbriae-dependent invasion to IL-6 regulation in endothelial cells. MATERIAL AND METHODS: We examined the secretion of IL-6 and the expression of the IL-6 signal transducer gp130 in human umbilical vein endothelial cells (HUVEC) infected with the wild-type FDC381 strain of P. gingivalisand a fimbriae-deficient mutant (fimA) by enzyme-linked immunosorbent assay, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry (fluorescence-activated cell sorting, FACS) analysis. RESULTS: Coculture of HUVEC with P. gingivalis resulted in increase of IL-6 secretion at 24 h postinfection. Interestingly, the increase was inhibited significantly in HUVEC infected with the P. gingivalis fimA mutant. In addition, the increase of IL-6 secretion induced by P. gingivalis infection was significantly impaired by the meiosis specific kinase 1 inhibitor, PD98059, or the nuclear factor kappaB inhibitor, Bay11-7082. Furthermore, we demonstrated that gp130 expression increased with P. gingivalis infection. Importantly, gp130 expression was significantly impaired by P gingivalis fimA mutant infection compared with wild-type P. gingivalis infection, as assessed by both quantitative RT-PCR and FACS analysis. CONCLUSION: Our findings indicate that P. gingivalis fimbriae are important factors in the autocrine regulation of IL-6, by increasing gp130 in endothelial cells.

AKT-1, -2, and -3 are expressed in both normal and tumor tissues of the lung, breast, prostate, and colon.

PURPOSE: The AKT/PKB kinase controls many of the intracellular processes that are dysregulated in human cancer, including the suppression of apoptosis and anoikis and the induction of cell cycle progression. Three isoforms of AKT have been identified: AKT-1, -2, and -3. Selective up-regulation of AKT-3 RNA expression has been reported in hormone-independent breast and prostate cancer cell lines suggesting that AKT-3 expression may be increased with breast or prostate tumor progression. To determine whether AKT-3 RNA expression is selectively up-regulated in human cancers and whether the patterns of AKT RNA expression may change with tumor development, we examined AKT isoform expression by RT-PCR in human cancer cell lines, primary human cancers, and normal human tissues. EXPERIMENTAL DESIGN: AKT-1, -2, and -3 RNA expression was examined by RT-PCR. Because up-regulated AKT-3 expression has been implicated in human breast and prostate cancer progression, we also examined AKT-3 expression levels by semiquantitative RT-PCR using matched normal/tumor first-strand cDNA pairs from colon, breast, prostate, and lung cancers. RESULTS: Our data reveal that the overwhelming majority of both normal and tumor tissues express all three of the AKT isoforms. Moreover, semiquantitative RT-PCR of matched normal/tumor pairs confirmed similar AKT-3 RNA expression levels in both normal and tumor tissue. CONCLUSIONS: Our data show that both normal and tumor tissues express all three of the AKT isoforms and indicate that tumorigenesis does not involve a dramatic shift in the RNA expression patterns of the three AKT isoforms.

The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain.

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4' Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k(cat)/K(m) of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4' Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

Ceramide induces the dephosphorylation and inhibition of constitutively activated Akt in PTEN negative U87mg cells.

In the present study, treatment of the PTEN negative U87MG human glioblastoma cell line with C2-ceramide resulted in a dose- and time-dependent decrease in the constitutive phosphorylation of Akt at threonine 308 and serine 473. The C2-ceramide induced dephosphorylation of Akt correlated with a 90-95% reduction in the Akt kinase activity. Exposure to C2-ceramide did not affect the basal or PDGF activated levels PtdIns-3,4-P(2) and PtdIns-3,4,5-P(3), indicating PI3-K activity was not inhibited. Additionally, treatment of cells with the PI3-K inhibitor wortmannin and C2-ceramide resulted in an enhanced rate of Akt dephosphorylation versus either agent alone. Finally, treatment of cells with the phosphatase inhibitors okadaic acid or calyculin A prevented the C2-ceramide induced dephosphorylation and inhibition of Akt activity. These data demonstrate the ability of C2-ceramide to inhibit the constitutive phosphorylation and activity of Akt in U87MG cells and implicate the activation of ceramide activated protein phosphatase, rather than decreased PI3-K activity, as the mechanism of inhibition.

Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1.

Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretases generates the amino and carboxy termini, respectively, of the A beta amyloidogenic peptides A beta40 and A beta42--the major constituents of the amyloid plaques in the brain parenchyma of Alzheimer's disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease increase the production of A beta42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a gamma-secretase cofactor, or helps to colocalize gamma-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent gamma-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of gamma-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.

Presenilin 1 is linked with gamma-secretase activity in the detergent solubilized state.

gamma-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two Abeta peptide isoforms, Abeta40 and Abeta42. Here we report the detergent solubilization and partial characterization of gamma-secretase. The activity of solubilized gamma-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the beta-secretase cleavage site. Cleavage of C100Flag by gamma-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Abeta40 or Abeta42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Abeta40 and Abeta42 termini. Recovery of catalytically competent, soluble gamma-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized gamma-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Abeta40 and Abeta42 in mammalian cells. Upon gel exclusion chromatography, solubilized gamma-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 x 10(6). Anti-PS1 antibody immunoprecipitates gamma-secretase activity from the solubilized gamma-secretase preparation. These data suggest that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex.

Activation of p38 mitogen-activated protein kinase and mitochondrial Ca(2+)-mediated oxidative stress are essential for the enhanced expression of grp78 induced by the protein phosphatase inhibitors okadaic acid and calyculin A.

We have reported that treatment with okadaic acid, a potent protein phosphatase inhibitor, has the ability to enhance the synthesis of the 78-kDa glucose-regulated protein (GRP78). This article reports our investigation of another protein phosphatase inhibitor, calyculin A, demonstrating the signaling pathways elicited by the protein phosphatase inhibitors that lead to the induction of grp78. Our data showed that the induction process is abolished by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38(MAPK)). Phosphorylation-activation of p38(MAPK) in the treated cells was indicated by its own phosphorylation, as shown by double Western blotting analyses and directly confirmed by the in vitro kinase assay using MAPK-activated protein kinase-2, a well-known downstream effector of p38(MAPK), as a substrate. The involvement of p38(MAPK) in this process is further substantiated by using transient transfection assays with a plasmid, pGRP78-Luc, which contains a 0.72-kbp stretch of the grp78 promoter. By exploiting the same transfection assay, we demonstrated that the up-regulation of the grp78 promoter by the protein phosphatase inhibitors is suppressed in the presence of the cytoplasmic calcium chelator bis(aminophenoxy)ethane N,N'-tetraacetic acid, the mitochondria calcium uniporter inhibitor ruthenium red as well as the antioxidants N-acetyl cysteine and pyrrolidinedithiocarbamate. Taken together, our results lead us to conclude that treatment with the protein phosphatase inhibitors would activate the signaling pathways involving p38(MAPK) and mitochondrial calcium-mediated oxidative stress and that these pathways must act in concert in order to confer the induction of grp78 by okadaic acid and calyculin A.

Congenital rhabdomyosarcoma--a case report.

A term female newborn was noted to have a tumor mass in the oral cavity soon after birth. Oral computer tomography revealed a well-enhanced soft tissue mass about 4 x 4 x 3 cm in size in the left buccal area. Group III embryonal type congenital rhabdomyosarcoma was diagnosed after biopsy (gross removal was not feasible). Respiratory distress exacerbated due to rapid tumor growth compressing airway with the result that endotracheal tube had to be intubated. Chemotherapy was done and complicated by two episodes of neutropenic fever and sepsis. Radiotherapy was suggested but refused by the family. Tumor size was slightly reduced and endotracheal tube could be removed four months later. She was taken home under regular chemotherapy. Radiotherapy, was, however, clearly indicated.

Diverse effects of tyrosine kinase inhibitors on follicle-stimulating hormone-stimulated estradiol and progesterone production from rat granulosa cells in serum-containing medium and serum-free medium containing epidermal growth factor.

Epidermal growth factor (EGF) has been shown to influence FSH-stimulated estradiol (E2) and progesterone (P4) production from granulosa cells. RG 50810, a tyrosine kinase inhibitor (TKI), has previously been shown to inhibit the EGF-receptor tyrosine kinase. RG 50810 has also been shown to inhibit FSH-stimulated increases in mRNA for steroidogenic enzymes, implying a functional role of tyrosine kinases in FSH action in granulosa cells. However, inhibition of FSH-stimulated steroidogenesis by TKIs has not been evaluated in connection with the effects of EGF in granulosa cells. In the present studies, FSH-stimulated E2 production was inhibited similarly by inhibitors of protein kinase A (H-89) and protein kinase C (calphostin C) and by TKIs, and none of the inhibitors were capable of reversing the EGF-induced inhibition of FSH-stimulated E2 production. FSH-stimulated P4 production was enhanced dramatically in serum-containing medium with concentrations of TKI that were near previously reported IC50s. The enhancing effect of TKIs was less evident in serum-free medium. Addition of EGF to serum-free medium enhanced FSH-stimulated P4 production, and the TKIs reversed EGF-enhanced P4 production, but in a manner similar to that of protein kinase A inhibitor H-89. Compared to results in serum-free medium, the potency of RG 50810 and genistein to inhibit the effects of EGF on P4 production was 3- to 8-fold greater relative to H-89. These studies have demonstrated that TKIs RG 50810 and genistein selectively inhibit the effects of EGF on FSH-stimulated P4 production in granulosa cell cultures. In contrast, these studies have demonstrated nonselective inhibition of FSH-stimulated E2 and P4 production by TKIs in serum-free medium, in which it is not clear which enzyme system is affected by the compounds tested.

Kinetic pathway for the slow to fast transition of thrombin. Evidence of linked ligand binding at structurally distinct domains.

The kinetic pathway for the Na+-induced slow --> fast transition of thrombin was characterized. The slow form was shown to consist of two conformers in a 3:1 ratio (ES2:ES1) at 5 degrees C, pH 7.4, Gamma/2 0.3. ES2 binds Na+ 3 orders of magnitude faster than does ES1. The small molecule active site-directed inhibitor L-371,912, and the exosite I binding ligand hirugen, like Na+, bind selectively to ES2 and induce the slow --> fast conversion of thrombin. The slow --> fast transition is limited by the rate of conversion of ES1 to ES2 (k approximately 28 s-1 at 5 degrees C). Replacement of Arg-221a or Lys-224 at the Na+ binding site with Ala appears to selectively alter the slow form and reduce the apparent affinity of the mutants for Na+ and L-371,912. This replacement, however, has little effect on the affinity for the inhibitor in the presence of saturating concentrations of Na+. The kinetically linked ligand binding at the Na+ binding site, exosite I, and the active site of thrombin characterized in the present study indicates the basis for the plasticity of this important enzyme, and suggests the possibility that the substrate specificity and, therefore, the procoagulant and anticoagulant activities of thrombin may be subject to allosteric regulation by as yet unidentified physiologically important effectors.

A BRCA1 mutant alters G2-M cell cycle control in human mammary epithelial cells.

Mutations in BRCA1 increase the risk of breast and ovarian cancer. Although the mechanism by which mutant BRCA1 alters growth regulation is unknown, the COOH terminus of BRCA1 appears to play a critical role. To examine this, we introduced a vector expressing BRCA1 COOH-terminal residues 1293-1863 (CT-BRCA1) into nontumorigenic human breast epithelial cells. Overexpression of CT-BRCA1 led to a reduction in the doubling time (from 64 to 44 h) and a decreased reliance on growth factors, suggesting that this CT-BRCA1 may function in a dominant-negative manner. Expression of CT-BRCA1 induced alterations in cell cycle control, mainly in G2-M, including a loss of G2-M block by colchicine. These results suggest that one function of BRCA1-related growth control occurs by governing checkpoint(s) between DNA replication and mitosis.

Electron spin resonance spin trapping assay and immunohistochemical localization of superoxide dismutases in the rat nasal mucosa.

The immunohistochemical method and electron spin resonance (ESR) spin trapping assay were employed to detect the localization and biochemical activity of superoxide dismutases (SODs) in the rat nasal mucosa. Manganese SOD and copper-zinc SOD were immunohistochemically illustrated to be richly expressed in the epithelial cells and the subepithelial glands of nasal mucosa. The olfactory vesicles also showed positive immunostaining for manganese SOD and copper-zinc SOD. ESR spin trapping assay revealed that SOD activity in the mucosa of olfactory areas was significantly higher than in the mucosa of respiratory areas; however, the ratio of SOD activity in the mitochondrial fraction to SOD activity in the cytosolic fraction was similar, approximating 17:83 in the mucosa of both the olfactory and respiratory areas. The predominant localization of SODs in epithelial cells of nasal mucosa suggests the importance of mucosal epithelium in protecting nasal mucosa against cytotoxic superoxide (O2-) radicals. Epithelial goblet cells and the connective tissue of lamina propria, which showed no positive immunostaining for SODs, are considered to be vulnerable to oxidative insults implicated in the generation of O2- radicals. The higher SODs activity in the mucosa of olfactory areas implies that there is a different requirement of SOD in mucosa of the respiratory and olfactory areas on scavenging microenvironmental O2- radicals.

Immunohistochemical localization of manganese superoxide dismutase in rat vestibular dark cell regions.

A modified immunoglobulin peroxidase bridge sequence method was used to detect the localization of manganese superoxide dismutase (MnSOD), a superoxide radical (O2-) scavenging enzyme locating in mitochondrial matrix, in the vestibular labyrinth of pigmented rats. Strong positive MnSOD immunostaining was demonstrated in the dark cell regions of the ampullae, utricle, and common crus. The result provides for the first time direct evidence demonstrating the existence of mitochondrial O2- scavengers in the vestibular labyrinth and illustrates that the specific sites for vestibular MnSOD immunolocalization are the dark cell regions. This site specificity of MnSOD immunolocalization suggests that dark cell regions may possess high metabolic activity and may encounter constant threat from O2-. We assume MnSOD is needed in protecting some physiologic functions of the dark cell regions. Cell types showing negative MnSOD immunostaining may conceivably be relatively vulnerable to acute O2- damage.

Immunohistochemical localization of manganese superoxide dismutase in the rat cochlea.

There has been recent increasing interest in the involvement of superoxide radicals (O2-) and their scavenging enzymes, the superoxide dismutases, in the patho-physiology of certain diseases. Since mitochondria are significant intracellular sources of O2- and important targets of oxidant injury, determining the intracochlear localization of mitochondrial O2- scavenging enzyme may provide important insight into the pathogenesis of injury due to cochlear oxidants. In order to locate the mitochondrial O2- scavenging enzyme, manganese superoxide dismutase (MnSOD), the authors used a modified immunoglobulin peroxidase bridge sequence method to detect MnSOD in paraffin-embedded, formalin-fixed rat cochleas. Site-specific immunolocalization of MnSOD could be demonstrated in the cochlear labyrinth, suggesting that the generation of intracochlear O2- was possibly implicated in the metabolically active sites and sites rich in vascularity. This study also provided a useful probe for detecting MnSOD immunohistochemically from ethylenediamine tetra-acetic acid-treated materials without requiring an antigen retrieval procedure.

Superoxide dismutases in human palatine tonsils.

In order to investigate the protective system of human palatine tonsils against the cytotoxic superoxide radicals (O(-)(2)) generated from the oxygen-related bactericidal system, immunohistochemistry and electron spin resonance (ESR) spectrometry were used to detect the distribution and activities of superoxide dismutases (SODs) in tonsils of different related systemic diseases. Immunohistochemistry showed that SODs distribute in extrafollicular lymphatic tissue and crypt epithelium. No distribution difference could be found between tonsils of different related systemic diseases. ESR revealed no significant difference between SODs activities in tonsils of different related systemic diseases. However, the mitochondrial SOD activity was found to constitute approximately 50%-60% of the total tonsillar cellular SODs activity. The results suggest: i)tonsils possess the ability to control cytotoxic O(-)(2), ii) crypt epithelium and extrafollicular lymphatic tissue may encounter more O(-)(2) threat, iii) SODs may be important in protecting germinal centers from O(-)(2) injury, and iv) systemic diseases are less related to the local expression of tonsillar SODs.