PubMed Health⌕ Search

Biomedical subjects

Paul Anderson

Publications and source records attributed to Paul Anderson.

At least 37 records · Page 2Linked to original sources
PubMed2005

Scanning microradiographic study on the influence of diffusion in the external liquid on the rate of demineralization in hydroxyapatite aggregates.

Subsurface demineralization in enamel caries is known to entail diffusion of reagents and products both within the lesion and within the plaque biofilm external to the lesion. However, development of a predictive mathematical model for subsurface demineralization is hindered by limited quantitative understanding of the effects of these diffusion processes. The purpose of this quantitative study was to investigate and understand the effect of external diffusion length on the rate of demineralization in a simple model system. Ten, 500-microm thick sections cut from a porous hydroxyapatite (HAP) pellet were inserted in scanning microradiography (SMR) cells. The exposed thin edges of the sections were initially separated by columns of water (diffusion lengths) of 0-0.9 cm from a 1-l reservoir of demineralizing buffer (pH 4). Buffer was found to diffuse from the reservoir through the increasing diffusion lengths to the exposed HAP surface, whilst dissolved product diffused along the reverse path. Rates of HAP loss (from SMR measurements) decreased as the diffusion length increased. Experimental data were fitted to a general diffusion-reaction model. This showed that the solution near the HAP surface was almost completely saturated with HAP, and that the diffusion of dissolution products, rather than of buffer species, was rate limiting.

Acetic Acid↗
PubMed2004

Stress granule assembly is mediated by prion-like aggregation of TIA-1.

TIA-1 is an RNA binding protein that promotes the assembly of stress granules (SGs), discrete cytoplasmic inclusions into which stalled translation initiation complexes are dynamically recruited in cells subjected to environmental stress. The RNA recognition motifs of TIA-1 are linked to a glutamine-rich prion-related domain (PRD). Truncation mutants lacking the PRD domain do not induce spontaneous SGs and are not recruited to arsenite-induced SGs, whereas the PRD forms aggregates that are recruited to SGs in low-level-expressing cells but prevent SG assembly in high-level-expressing cells. The PRD of TIA-1 exhibits many characteristics of prions: concentration-dependent aggregation that is inhibited by the molecular chaperone heat shock protein (HSP)70; resistance to protease digestion; sequestration of HSP27, HSP40, and HSP70; and induction of HSP70, a feedback regulator of PRD disaggregation. Substitution of the PRD with the aggregation domain of a yeast prion, SUP35-NM, reconstitutes SG assembly, confirming that a prion domain can mediate the assembly of SGs. Mouse embryomic fibroblasts (MEFs) lacking TIA-1 exhibit impaired ability to form SGs, although they exhibit normal phosphorylation of eukaryotic initiation factor (eIF)2alpha in response to arsenite. Our results reveal that prion-like aggregation of TIA-1 regulates SG formation downstream of eIF2alpha phosphorylation in response to stress.

Amino Acid Sequence↗
PubMed2004

The crab-eating frog, Rana cancrivora, up-regulates hepatic carbamoyl phosphate synthetase I activity and tissue osmolyte levels in response to increased salinity.

The crab-eating frog Rana cancrivora is one of only a handful of amphibians worldwide that tolerate saline waters. They typically inhabit brackish water of mangrove forests of Southeast Asia, but live happily in freshwater and can be acclimated to 75% seawater (25 ppt) or higher. We report here that after transfer of juvenile R. cancrivora from freshwater (1 ppt) to brackish water (10 -->20 or 20 -->25 ppt; 4-8 d) there was a significant increase in the specific activity of the key hepatic ornithine urea cycle enzyme (OUC), carbamoyl phosphate synthetase I (CPSase I). At 20 ppt, plasma, liver and muscle urea levels increased by 22-, 21-, and 11-fold, respectively. As well, muscle total amino acid levels were significantly elevated by 6-fold, with the largest changes occurring in glycine and beta-alanine levels. In liver, taurine levels were 5-fold higher in frogs acclimated to 20 ppt. There were no significant changes in urea or ammonia excretion rates to the environment. As well, the rate of urea influx (J(in) (urea)) and efflux (J(out) (urea)) across the ventral pelvic skin did not differ between frogs acclimated to 1 versus 20 ppt. Taken together, these findings suggest that acclimation to saline water involves the up-regulation of hepatic urea synthesis, which in turn contributes to the dramatic rise in tissue urea levels. The lack of change in urea excretion rates, despite the large increase in tissue-to-water gradients further indicates that mechanisms must be in place to prevent excessive loss of urea in saline waters, but these mechanisms do not include cutaneous urea uptake. Also, amino acid accumulation may contribute to an overall rise in the osmolarity of the muscle tissue, but relative to urea, the contribution is small.

Amino Acids↗
PubMed2004

Bone formation in polymeric scaffolds evaluated by proton magnetic resonance microscopy and X-ray microtomography.

Magnetic resonance microscopy (MRM) and X-ray microtomography (XMT) were used to investigate de novo bone formation in porous poly(ethyl methacrylate) (PEMA) scaffolds, prepared by a novel co-extrusion process. PEMA scaffolds were seeded with primary chick calvarial osteoblasts and cultured under static conditions for up to 8 weeks. Bone formation within porous PEMA scaffolds was confirmed by the application of histologic stains to intact PEMA disks. Disks were treated with Alizarin red to visualize calcium deposits and with Sirius red to visualize regions of collagen deposition. DNA analysis confirmed that cells reached confluence on the scaffolds after 7 weeks in static culture. The formation of bone in PEMA scaffolds was investigated with water proton MRM. Quantitative MRM maps of the magnetization transfer ratio (MTR) yielded maps of protein deposition, and magnetic resonance (MR) relaxation times (T1 and T2) yielded maps of mineral deposition. The location of newly formed bone and local mineral concentrations were confirmed by XMT. By comparing MRM and XMT data from selected regions-of-interest in one sample, the inverse relationship between the MR relaxation times and mineral concentration was validated, and calibration curves for estimating the mineral content of cell-seeded PEMA scaffolds from quantitative MRM images were developed.

Animals↗
PubMed2004

FAST is a BCL-X(L)-associated mitochondrial protein.

The TIA-1-interacting protein Fas-activated serine/threonine phosphoprotein (FAST) is a component of a signaling cascade that is initiated by ligation of the Fas receptor. Immunofluorescence microscopy using affinity-purified antibodies raised against recombinant FAST reveals that the endogenous protein associates with mitochondria. Subcellular fractionation confirms that FAST is a component of mitochondria. FAST is tethered to mitochondria by a lysine/arginine-rich domain at its carboxyl terminus that is structurally similar to the mitochondrial tethering motifs of monoamine oxidase B and cytochrome b5. At the mitochondrial membrane, FAST interacts with BCL-X(L). The BCL-X(L) binding domain maps to a BCL-2-homology-3 (BH3)-related domain that is distinct from the mitochondrial-tethering domain (MTD). Although interactions between FAST and BCL-X(L) require both the BH3-related domain and the MTD, the requirement for mitochondrial tethering can be conferred by a heterologous MTD. Our results suggest that FAST-BCL-X(L) interactions are likely to regulate mitochondrial metabolism during Fas-induced apoptosis.

Amino Acid Sequence↗
PubMed2004

Post-transcriptional regulation of proinflammatory proteins.

Post-transcriptional mechanisms play a critical role in regulating the expression of numerous proteins that promote inflammatory arthritis. The mRNAs encoding a subset of these proteins possess adenine/uridine-rich elements (AREs) in their 3'-untranslated regions that profoundly influence the rate at which mRNA is degraded and translated into protein. Tristetraprolin (TTP) and T cell intracellular antigen-1 (TIA-1) are ARE-binding proteins that dampen the expression of this class of proteins by promoting mRNA degradation and protein translation, respectively. We have discovered that TIA-1 and TTP function as arthritis-suppressor genes: TIA-1-/- mice develop mild arthritis, TTP-/- mice develop severe arthritis, and TIA-1-/-TTP-/- mice develop very severe arthritis. Paradoxically, lipopolysaccharide (LPS)-activated macrophages derived from TIA-1-/-TTP-/- macrophages produce less tumor necrosis factor alpha (TNF-alpha) than TIA-1-/- or TTP-/- macrophages. The bone marrows of these mice exhibit increased cellularity, reflecting the presence of mature neutrophils that secrete TNF-alpha in response to LPS stimulation. We hypothesize that TIA-1-/-TTP-/- neutrophils are a source of arthritigenic TNF-alpha, which promotes severe erosive arthritis in these mice.

Animals↗
PubMed2004

MK2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and ARE-mRNA decay.

Stress granules (SGs) are dynamic cytoplasmic foci at which stalled translation initiation complexes accumulate in cells subjected to environmental stress. SG-associated proteins such as TIA-1, TIAR and HuR bind to AU-rich element (ARE)-containing mRNAs and control their translation and stability. Here we show that tristetraprolin (TTP), an ARE-binding protein that destabilizes ARE-mRNAs, is recruited to SGs that are assembled in response to FCCP-induced energy deprivation, but not arsenite-induced oxidative stress. Exclusion of TTP from arsenite-induced SGs is a consequence of MAPKAP kinase-2 (MK2)-induced phosphorylation at serines 52 and 178, which promotes the assembly of TTP:14-3-3 complexes. 14-3-3 binding excludes TTP from SGs and inhibits TTP-dependent degradation of ARE-containing transcripts. In activated RAW 264.7 macrophages, endogenous TTP:14-3-3 complexes bind to ARE-RNA. Our data reveal the mechanism by which the p38-MAPK/MK2 kinase cascade inhibits TTP-mediated degradation of ARE-containing transcripts and thereby contributes to lipopolysaccharide-induced TNFalpha expression.

14-3-3 Proteins↗
PubMed2004

Arthritis suppressor genes TIA-1 and TTP dampen the expression of tumor necrosis factor alpha, cyclooxygenase 2, and inflammatory arthritis.

TIA-1 and TTP are AU-rich element-binding proteins that prevent the pathological overexpression of tumor necrosis factor alpha (TNF-alpha). TIA-1 inhibits the translation of TNF-alpha transcripts, whereas TTP promotes the degradation of TNF-alpha transcripts. Here we show that TIA-1 and TTP function as arthritis suppressor genes: TIA-1(-/-) mice develop mild arthritis, TTP(-/-) mice develop severe arthritis, and TIA-1(-/-)TTP(-/-) mice develop very severe arthritis. Peritoneal macrophages derived from all three genotypes overexpress cyclooxygenase 2 and TNF-alpha. Surprisingly, lipopolysaccharide-activated TIA-1(-/-)TTP(-/-) macrophages secrete less TNF-alpha protein than either TIA-1(-/-) or TTP(-/-) macrophages. In these mice, arthritogenic cytokine may be produced by neutrophils that accumulate in the bone marrow and peripheral blood. Our results suggest that TIA-1 and TTP are genetic modifiers of inflammatory arthritis that can alter the spectrum of cells that produce arthritogenic cytokines.

Animals↗
PubMed2004

Guar seed beta-mannan synthase is a member of the cellulose synthase super gene family.

Genes for the enzymes that make plant cell wall hemicellulosic polysaccharides remain to be identified. We report here the isolation of a complementary DNA (cDNA) clone encoding one such enzyme, mannan synthase (ManS), that makes the beta-1, 4-mannan backbone of galactomannan, a hemicellulosic storage polysaccharide in guar seed endosperm walls. The soybean somatic embryos expressing ManS cDNA contained high levels of ManS activities that localized to Golgi. Phylogenetically, ManS is closest to group A of the cellulose synthase-like (Csl) sequences from Arabidopsis and rice. Our results provide the biochemical proof for the involvement of the Csl genes in beta-glycan formation in plants.

Amino Acid Sequence↗
PubMed2004

Regulation of gene expression by the CYP27B1 promoter-study of a transgenic mouse model.

The enzyme 25-hydroxyvitamin D 1-hydroxylase (CYP27B1) is the rate limiting enzyme in the two-step activation process of Vitamin D to its active form 1,25-dihydroxyvitamin D (1,25D) and is located in the mitochondrial fraction of the proximal tubular cells of the kidney. More recently CYP27B1 activity and expression have also been identified in a number of non-renal cells, which is suggestive of new, previously unidentified roles for Vitamin D in the human body. Although the regulation of CYP27B1 activity and expression has been a major focus of interest over the past decades, the exact molecular mechanism behind the regulation of CYP27B1 activity and expression and the role of the CYP27B1 promoter, herein, are still poorly understood. In this study, we created a transgenic mouse model that expresses the luciferase reporter gene under the control of the full-length, 1.5kb, human CYP27B1 promoter. This animal model allows us to study in vivo the tissue-specific, CYP27B1 promoter-controlled, regulation of the expression of the CYP27B1 gene.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2004

FAST is a survival protein that senses mitochondrial stress and modulates TIA-1-regulated changes in protein expression.

The Fas-activated serine/threonine phosphoprotein (FAST) is tethered to the outer mitochondrial membrane, where it interacts with BCL-X(L) (17). Here we show that RNA interference-mediated knockdown of endogenous FAST results in apoptosis, whereas overexpressed recombinant FAST inhibits Fas- and UV-induced apoptosis, indicating that FAST is a survival protein. The antiapoptotic effects of FAST are regulated by interactions with the translational silencer TIA-1: a FAST mutant lacking its TIA-1-binding domain does not inhibit apoptosis, and overexpressed recombinant TIA-1 inhibits the antiapoptotic effects of FAST. Because the antiapoptotic effects of FAST require ongoing protein synthesis, we hypothesized that FAST might function by preventing TIA-1-mediated silencing of mRNAs encoding inhibitors of apoptosis. Consistent with this hypothesis, FAST promotes the expression of cotransfected reporter proteins, a process that requires its TIA-1-binding domain and is inhibited by overexpressed recombinant TIA-1. More compellingly, recombinant FAST increases the expression of endogenous cIAP-1 and XIAP, but not GAPDH, in transfected HeLa cells. Because FAST is released from mitochondria in cells undergoing Fas- or UV-induced apoptosis, we propose that FAST serves as a sensor of mitochondrial stress that modulates a TIA-1-regulated posttranscriptional stress response program.

Animals↗
PubMed2003

Versatile 3-channel high-voltage power supply for microchip capillary electrophoresis.

The fabrication of a battery operated 3-channel high voltage power supply for microchip capillary electrophoresis is described. The power supply consists of two positive and one negative DC-DC converters, a microprocessor controlled timer, a battery and a transformer to recharge the battery and feed the high voltage relays. This arrangement allows the possibility to control the potentials applied in the 0 to +/-4000 V range to a variety of microchip setups. It can also be easily adapted to perform either gated or pinched injection. The inclusion of a rechargeable battery was adopted to feed the DC-DC converters to reduce noise levels and achieve portability.

Journal Article↗
PubMed2003

Regulation of cyclooxygenase-2 expression by the translational silencer TIA-1.

The cyclooxygenase-2 (COX-2) enzyme catalyzes the rate-limiting step of prostaglandin formation in inflammatory states, and COX-2 overexpression plays a key role in carcinogenesis. To understand the mechanisms regulating COX-2 expression, we examined its posttranscriptional regulation mediated through the AU-rich element (ARE) within the COX-2 mRNA 3'-untranslated region (3'UTR). RNA binding studies, performed to identify ARE-binding regulatory factors, demonstrated binding of the translational repressor protein TIA-1 to COX-2 mRNA. The significance of TIA-1-mediated regulation of COX-2 expression was observed in TIA-1 null fibroblasts that produced significantly more COX-2 protein than wild-type fibroblasts. However, TIA-1 deficiency did not alter COX-2 transcription or mRNA turnover. Colon cancer cells demonstrated to overexpress COX-2 through increased polysome association with COX-2 mRNA also showed defective TIA-1 binding both in vitro and in vivo. These findings implicate that TIA-1 functions as a translational silencer of COX-2 expression and support the hypothesis that dysregulated RNA-binding of TIA-1 promotes COX-2 expression in neoplasia.

Animals↗
PubMed2003

Geldanamycin inhibits the production of inflammatory cytokines in activated macrophages by reducing the stability and translation of cytokine transcripts.

OBJECTIVE: Heat-shock protein 90 (Hsp90) is critical in the intracellular signaling pathways that promote inflammatory cytokine production. Geldanamycin (GD) is a benzoquinone ansamycin that inhibits the function of Hsp90. GD inhibits the production of tumor necrosis factor alpha (TNFalpha) in activated macrophages and suppresses the progression of adjuvant-induced arthritis and experimental allergic encephalomyelitis in rodents. GD has been used to investigate the mechanisms by which Hsp90 regulates inflammatory cytokine production. METHODS: The macrophage cell line RAW264.7 (or primary peritoneal macrophages) was activated with lipopolysaccharide in the absence or presence of GD. The effect of GD on the transcription, stability, and translation of inflammatory cytokine messenger RNA (mRNA) was determined using nuclear run-on assays, mRNA decay assays, and sucrose gradient polysome profiles, respectively. RESULTS: Our data revealed that GD potently inhibits the production of TNFalpha, interleukin-6 (IL-6), and IL-1beta in activated macrophages. Although GD did not significantly reduce the transcription of inflammatory cytokine mRNA, it significantly decreased the stability of these transcripts. Polysome profiles indicated that GD also inhibited the translation of TNFalpha and IL-6 transcripts. These effects may be due, in part, to inhibition of p38 mitogen-activated protein kinase, a kinase known to regulate the stability and translation of inflammatory cytokine transcripts. CONCLUSION: These results indicate that the function of Hsp90 is important in the posttranscriptional control of inflammatory cytokine production.

Animals↗
PubMed2003

Granzyme B leakage-induced cell death: a new type of activation-induced natural killer cell death.

Activation-induced natural killer (NK) cell death is very rapid compared to activation-induced T or B cell death. Here we show that NK cell activation is accompanied by the leakage of granzymeB from intracellular granules into the cytoplasm. Evidence for granzyme B leakage includes the formation of granzyme B/serine proteinase inhibitor 9 (PI-9) complexes that are detected by immunoprecipitation as well as colocalization of granzyme B and PI-9 detected by immunocytochemistry. The pro-apoptotic molecule Bid, a specific substrate for granzyme B, was cleaved within 2 min following CD2-induced NK cell activation, suggesting that granzyme B triggers apoptosis by directing Bid to mitochondrial membranes. The granzyme B/PI-9 protein ratio was found to mirror the percentage of CD2-induced NK cell death, suggesting that an excess of leaked granzyme B over its inhibitor is a major determinant of cell death. We suggest that granzyme B leakage-induced cell death is an important determinant of activation-induced NK cell death and that this process may be important for the fate of NK cells which encounter malignant or virus-infected cells.

Apoptosis↗
PubMed2002

Human autoimmune sera as molecular probes for the identification of an autoantigen kinase signaling pathway.

Using human autoimmune sera as molecular probes, we previously described the association of phosphorylated serine/arginine splicing factors (SR splicing factors) with the U1-small nuclear ribonucleoprotein (U1-snRNP) and U3-small nucleolar RNP (snoRNP) in apoptotic cells. SR proteins are highly conserved autoantigens whose activity is tightly regulated by reversible phosphorylation of serine residues by at least eight different SR protein kinase kinases (SRPKs), including SRPK1, SRPK2, and the scleroderma autoantigen topoisomerase I. In this report, we demonstrate that only one of the known SRPKs, SRPK1, is associated with the U1-snRNP autoantigen complex in healthy and apoptotic cells. SRPK1 is activated early during apoptosis, followed by caspase-mediated proteolytic inactivation at later time points. SRPKs are cleaved in vivo after multiple apoptotic stimuli, and cleavage can be inhibited by overexpression of bcl-2 and bcl-x(L), and by exposure to soluble peptide caspase inhibitors. Incubation of recombinant caspases with in vitro-translated SRPKs demonstrates that SRPK1 and SRPK2 are in vitro substrates for caspases-8 and -9, respectively. In contrast, topoisomerase I is cleaved by downstream caspases (-3 and -6). Since each of these SRPKs sits at a distinct checkpoint in the caspase cascade, SRPKs may serve an important role in signaling pathways governing apoptosis, alternative mRNA splicing, SR protein trafficking, RNA stability, and possibly the generation of autoantibodies directed against splicing factors.

Apoptosis↗