Ectopic calcification is a driving force for a variety of diseases, including kidney stones and a... more Ectopic calcification is a driving force for a variety of diseases, including kidney stones and atherosclerosis, but initiating factors remain largely unknown. Given its importance in seemingly divergent disease processes, identifying fundamental principal actors for ectopic calcification may have broad translational significance. Here we establish a Drosophila melanogaster model for ectopic calcification by inhibiting xanthine dehydrogenase whose deficiency leads to kidney stones in humans and dogs. Micro X-ray absorption near edge spectroscopy (μXANES) synchrotron analyses revealed high enrichment of zinc in the Drosophila equivalent of kidney stones, which was also observed in human kidney stones and Randall's plaques (early calcifications seen in human kidneys thought to be the precursor for renal stones). To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation. Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation. Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.
NF-kappaB is a critical activator of genes involved in inflammation and immunity. Pro-inflammator... more NF-kappaB is a critical activator of genes involved in inflammation and immunity. Pro-inflammatory cytokines activate the IkappaB kinase (IKK) complex that phosphorylates the NF-kappaB inhibitors, triggering their conjugation with ubiquitin and subsequent degradation. Freed NF-kappaB dimers translocate to the nucleus and induce target genes, including the one for cyclo-oxygenase 2 (COX2), which catalyses the synthesis of pro-inflammatory prostaglandins, in particular PGE. At late stages of inflammatory episodes, however, COX2 directs the synthesis of anti-inflammatory cyclopentenone prostaglandins, suggesting a role for these molecules in the resolution of inflammation. Cyclopentenone prostaglandins have been suggested to exert anti-inflammatory activity through the activation of peroxisome proliferator-activated receptor-gamma. Here we demonstrate a novel mechanism of antiinflammatory activity which is based on the direct inhibition and modification of the IKKbeta subunit of IKK. A...
Journal of the American Academy of Dermatology, 1995
Diagnosis of the underlying dermatosis in erythroderma is often difficult. The cause of increased... more Diagnosis of the underlying dermatosis in erythroderma is often difficult. The cause of increased mortality in erythroderma, particularly in relation to infection, is incompletely understood. We investigated the potential diagnostic use of circulating intercellular adhesion molecule-1 (cICAM-1), vascular cell adhesion molecule-1 (cVCAM-1), and E-selectin (cE-selectin) levels in erythroderma. cICAM-1, cVCAM-1, and cE-selectin were measured by enzyme-linked immunosorbent assay in 14 patients with erythroderma of known cause and in 17 control subjects. Levels were correlated with other markers of the inflammatory response. In erythroderma median cICAM-1, cVCAM-1, and cE-selectin levels were significantly elevated, but no difference was found between values in patients with eczema and values in those with psoriasis. Circulating adhesion molecule levels did not correlate with erythrocyte sedimentation rate or total white blood cell count. cICAM-1, cVCAM-1, and cE-selectin were detectable in patients with erythroderma but were not differential diagnostic use in this study. Because in vitro these molecules may interfere with normal cell adhesion mechanisms, we speculate that they may contribute to the immunosuppressive state in these patients.
Arsenite is a potent environmental toxin that causes various pathologies including cancers and sk... more Arsenite is a potent environmental toxin that causes various pathologies including cancers and skin disorders. Arsenite is believed to exert its biological effects through reaction with exposed sulfhydryl groups, especially pairs of adjacent thiols. Here, we describe the mechanism by which arsenite affects the NF-kappaB signaling pathway. Activation of transcription factor NF-kappaB depends on the integrity of the IkappaB kinase (IKK) complex. We found that arsenite potently inhibits NF-kappaB and IKK activation by binding to Cys-179 in the activation loop of the IKK catalytic subunits, IKKalpha/beta. The affinity of IKKbeta for trivalent arsenic was verified in vitro by the ability of IKKbeta to enhance the fluorescence of an arsenic-substituted fluorescein dye. The addition of 1,2-dithiol antidotes or replacement of Cys-179 with an alanine residue abolished dye binding to and arsenite inhibition of IKKbeta. Overexpression of IKKbeta (C179A) protects NF-kappaB from inhibition by arsenite, indicating that despite the involvement of a large number of distinct gene products in this activation pathway, the critical target for inhibition by arsenite is on the IKK catalytic subunits.
The disposable soma theory suggests that longevity is determined through the setting of longevity... more The disposable soma theory suggests that longevity is determined through the setting of longevity assurance mechanisms so as to provide an optimal compromise between investments in somatic maintenance (including stress resistance) and in reproduction. A corollary is that species with low extrinsic mortality are predicted to invest relatively more effort in maintenance, resulting in slower intrinsic ageing, than species with high extrinsic mortality. We tested this prediction in a comparative study of stress resistance in primary skin fibroblasts and confirmed that cells from long-lived species are indeed more resistant to a variant of stressors. A widely studied example of within-species variation in lifespan is the rodent calorie restriction model. Food-restricted animals show elevations in a range of stress response mechanisms, and it has been suggested that this is an outcome of natural selection for life history plasticity. We have developed a theoretical model for dynamic optimisation of the allocation of effort to maintenance and reproduction in response to fluctuations in food availability. The model supports the suggestion that the response to calorie restriction may be an evolutionary adaptation, raising interesting questions about the hierarchy of genetic control of multiple stress response systems. The model identifies ecological factors likely to support such an adaptation that may be relevant in considering the likely relevance of a similar response to calorie restriction in other species. Comparative and theoretical studies support the role of somatic maintenance and stress response systems in controlling the rate of ageing.
Identifying the mechanisms determining species-specific life spans is a central challenge in unde... more Identifying the mechanisms determining species-specific life spans is a central challenge in understanding the biology of aging. Cellular stresses produce damage, that may accumulate and cause aging. Evolution theory predicts that long-lived species secure their longevity through investment in a more durable soma, including enhanced cellular resistance to stress. To investigate whether cells from long-lived species have better mechanisms to cope with oxidative and non-oxidative stress, we compared cellular resistance of primary skin fibroblasts from eight mammalian species with a range of life spans. Cell survival was measured by the thymidine incorporation assay following stresses induced by paraquat, hydrogen peroxide, tert-butyl hydroperoxide, sodium arsenite and alkaline pH (sodium hydroxide). Significant positive correlations between cell LD90 and maximum life span were found for all these stresses. Similar results were obtained when cell survival was measured by the MTT assay, and when lymphocytes from different species were compared. Cellular resistance to a variety of oxidative and non-oxidative stresses was positively correlated with mammalian longevity. Our results support the concept that the gene network regulating the cellular response to stress is functionally important in aging and longevity.
Sarcoidosis is a disease of unknown etiology characterized by non-caseating granulomata together ... more Sarcoidosis is a disease of unknown etiology characterized by non-caseating granulomata together with a number of systemic abnormalities. We have recently shown these include increased expression of the integrins CDI 1/CD 18 on peripheral blood leucocytes. Here we have measured serum levels of the adhesion molecules intercellular adhesion molecule-I (ICAM-1), E-selectin and vascular cell adhesion molecule-I (VCAM-1) in 23 patients and 14 normal controls using antigen capture sandwich ELISAs. Median circulating E-selectin levels in the patients were nearly three times those of the controls (P < 0-0001, Mann-Whitney U-test), whilst ICAM-I but not VCAM-l levels were only slightly elevated. These results show that endothelial cell activation and shedding of E-selectin into the circulation are additional features of the pathology of sarcoidosis.
We have developed a panel of MoAbs against four separate but overlapping epitopes on endothelial ... more We have developed a panel of MoAbs against four separate but overlapping epitopes on endothelial cell (EC) vascular cell adhesion molecule-I (VCAM-1). Two of the MoAbs (IGl 1 and 1E5) inhibited T cell adhesion to tumour necrosis factor (TNF)-activated EC, whilst two MoAbs (1.4C3 and 6D9) did not. Using these MoAbs we have identified a circulating form ofVCAM-I (cVCAM-1) which has identical epitope distribution to the EC form, and which is able to support the adhesion of the human lymphoblastoid cell line Jurkat J6 by a VLA-4-and VCAM-1-dependent mechanism when immobilized from plasma. cVCAM-l isolated by immunoaffinity and size-exclusion chromatographies was shown by SDS-PAGE to have an apparent mol. wt of 85-90 kD. Levels ofcVCAM-I were significantly raised (P < 0-001) in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) compared with normal individuals. It is possible that cVCAM-1 may be a useful plasma marker for the diagnosis and management of patients with inflammatory diseases. Furthermore, detection of elevated cVCAM-I levels may act as a guide to the importance of VCAM-I -dependent cell adhesion in different pathological settings.
Most inflammatory agents activate nuclear factor-kappaB (NF-kappaB), resulting in induction of ge... more Most inflammatory agents activate nuclear factor-kappaB (NF-kappaB), resulting in induction of genes coding for cytokines, chemokines, and enzymes involved in amplification and perpetuation of inflammation. Hypoestoxide (a bicyclo [9,3,1] pentadecane) is a diterpene from Hypoestes rosea, a tropical shrub in the family Acanthacea, several members of which are used in folk medicine in Nigeria. Here, we demonstrate that hypoestoxide (HE) abrogates the production of pro-inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) in lipopolysaccharide (LPS)-activated normal human peripheral blood mononuclear cells. Moreover, HE inhibits the production of nitric oxide (NO) by IL-1beta- or IL-17-stimulated normal human chondrocytes. In vivo, oral administration of HE to mice significantly ameliorated hind paw edema induced by antibodies to type II collagen plus LPS. Furthermore, topical administration of HE to mice also significantly inhibited phorbol ester-induced ear inflammation. The anti-inflammatory activity of HE may be due in part to its ability to inhibit NF-kappaB activation through direct inhibition of IkappaB kinase (IKK) activity. Thus, HE could be useful in treating various inflammatory diseases and may represent a prototype of a novel class of IKK inhibitors.
Two transcription factors, NF-κB and AP-1, have emerged in recent years as major targets for sign... more Two transcription factors, NF-κB and AP-1, have emerged in recent years as major targets for signaling pathways that are activated by a variety of proinflammatory stimuli. The same transcription factors were also found to be regulated by oxidative stress, although in this case, we find evidence for both positive and negative regulation (especially for NF-κB). After discussing how each of these transcription factors is regulated by proinflammatory stimuli, their regulation by oxidative stress and pro-oxidants will be addressed. Transcription factor NF-κB is regulated through interaction with inhibitory proteins, the IκBs. In response to cell stimulation with proinflammatory stimuli, the IκBs are rapidly phosphorylated at two conserved serines, followed by subsequent polyubiquitination and degradation through the 26S proteasome. Although many diverse stimuli including TNFα, IL-1, lipopolysaccharide (LPS) and other components of bacterial cell walls, double stranded (ds) RNA, viruses and ionizing radiation activate NF-κB through induction of IκB phosphorylation, they all operate through a common protein kinase-the IκB kinase (IKK). Purification of IKK indicates it is a large complex composed of at least three subunits, IKKα, IKKβ and IKKγ. The first two are closely related catalytic subunits (kinases) and the third is a regulatory subunit.
To compare the levels of circulating intercellular adhesion molecule 1 (cICAM-1) and vascular cel... more To compare the levels of circulating intercellular adhesion molecule 1 (cICAM-1) and vascular cell adhesion molecule 1 (cVCAM-1) in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Levels of cICAM-1 and cVCAM-1 were measured in both plasma and synovial fluid using monoclonal antibody sandwich enzyme-linked immunoassays. Levels of both cICAM-1 and cVCAM-1 were significantly increased (P &lt; 0.001) in RA patients compared with normal controls. In contrast, only cVCAM-1, and not cICAM-1, was increased in patients with SLE. Levels of cICAM-1 and cVCAM-1 were significantly elevated in synovial fluid compared with plasma in paired samples from patients with RA. There was no correlation between levels of cICAM-1 and levels of cVCAM-1, in either plasma or synovial fluid. Whereas levels of cVCAM-1 correlated significantly with the erythrocyte sedimentation rate (ESR) and C-reactive protein level in RA patients and with the ESR in SLE patients, no significant correlations were found between cICAM-1 and either of these indices of disease activity. These observations indicate that levels of cICAM-1 and cVCAM-1 reflect separate pathophysiologic processes. Both may be useful markers for the diagnosis and management of patients with rheumatic diseases.
Arsenite is a potent environmental toxin that causes various pathologies including cancers and sk... more Arsenite is a potent environmental toxin that causes various pathologies including cancers and skin disorders. Arsenite is believed to exert its biological effects through reaction with exposed sulfhydryl groups, especially pairs of adjacent thiols. Here, we describe the mechanism ...
Urolithiasis affects around 10% of the US population with an increasing rate of prevalence, recur... more Urolithiasis affects around 10% of the US population with an increasing rate of prevalence, recurrence and penetrance. The causes for the formation of most urinary calculi remain poorly understood, but obtaining the chemical composition of these stones might help identify key aspects of this process and new targets for treatment. The majority of urinary stones are composed of calcium that is complexed in a crystalline matrix with organic and inorganic components. Surprisingly, mitigation of urolithiasis risk by altering calcium homeostasis has not been very effective. Thus, studies to identify other therapeutic stone-specific targets, using proteomics, metabolomics and microscopy techniques, have been conducted, revealing a high level of complexity. The data suggest that numerous metals other than calcium and many nonmetals are present within calculi at measurable levels and several have distinct distribution patterns. Manipulation of the levels of some of these elemental components of calcium-based stones has resulted in clinically beneficial changes in stone chemistry and rate of stone formation. The elementome-the full spectrum of elemental content-of calcium-based urinary calculi is emerging as a new concept in stone research that continues to provide important insights for improved understanding and prevention of urinary stone disease.
The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin ... more The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation.
Reducing protein synthesis slows growth and development but can increase adult life span. We demo... more Reducing protein synthesis slows growth and development but can increase adult life span. We demonstrate that knockdown of eukaryotic translation initiation factor 4G (eIF4G), which is downregulated during starvation and dauer state, results in differential translation of genes important for growth and longevity in C. elegans. Genome-wide mRNA translation state analysis showed that inhibition of IFG-1, the C. elegans ortholog of eIF4G, results in a relative increase in ribosomal loading and translation of stress response genes. Some of these genes are required for life span extension when IFG-1 is inhibited. Furthermore, enhanced ribosomal loading of certain mRNAs upon IFG-1 inhibition was correlated with increased mRNA length. This association was supported by changes in the proteome assayed via quantitative mass spectrometry. Our results suggest that IFG-1 mediates the antagonistic effects on growth and somatic maintenance by regulating mRNA translation of particular mRNAs based, in part, on transcript length.
Extensive studies in model organisms in the last few decades have revealed that aging is subject ... more Extensive studies in model organisms in the last few decades have revealed that aging is subject to profound genetic influence. The conserved nutrient sensing TOR (Target of Rapamycin) pathway is emerging as a key regulator of lifespan and healthspan in various species from yeast to mammals. The TOR signaling pathway plays a critical role in determining how a eukaryotic cell
The antagonistic pleiotropy theory of aging proposes that aging takes place because natural selec... more The antagonistic pleiotropy theory of aging proposes that aging takes place because natural selection favors genes that confer benefit early on life at the cost of deterioration later in life. This theory predicts that genes that impact development would play a key role in shaping adult lifespan. To better understand the link between development and adult lifespan, we examined the genes previously known to be essential for development. From a pool of 57 genes that cause developmental arrest after inhibition using RNA interference, we have identified 24 genes that extend lifespan in Caenorhabditis elegans when inactivated during adulthood. Many of these genes are involved in regulation of mRNA translation and mitochondrial functions. Genetic epistasis experiments indicate that the mechanisms of lifespan extension by inactivating the identified genes may be different from those of the insulin/insulin-like growth factor 1 (IGF-1) and dietary restriction pathways. Inhibition of many of these genes also results in increased stress resistance and decreased fecundity, suggesting that they may mediate the trade-offs between somatic maintenance and reproduction. We have isolated novel lifespan-extension genes, which may help understand the intrinsic link between organism development and adult lifespan.
Ectopic calcification is a driving force for a variety of diseases, including kidney stones and a... more Ectopic calcification is a driving force for a variety of diseases, including kidney stones and atherosclerosis, but initiating factors remain largely unknown. Given its importance in seemingly divergent disease processes, identifying fundamental principal actors for ectopic calcification may have broad translational significance. Here we establish a Drosophila melanogaster model for ectopic calcification by inhibiting xanthine dehydrogenase whose deficiency leads to kidney stones in humans and dogs. Micro X-ray absorption near edge spectroscopy (μXANES) synchrotron analyses revealed high enrichment of zinc in the Drosophila equivalent of kidney stones, which was also observed in human kidney stones and Randall's plaques (early calcifications seen in human kidneys thought to be the precursor for renal stones). To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation. Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation. Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.
NF-kappaB is a critical activator of genes involved in inflammation and immunity. Pro-inflammator... more NF-kappaB is a critical activator of genes involved in inflammation and immunity. Pro-inflammatory cytokines activate the IkappaB kinase (IKK) complex that phosphorylates the NF-kappaB inhibitors, triggering their conjugation with ubiquitin and subsequent degradation. Freed NF-kappaB dimers translocate to the nucleus and induce target genes, including the one for cyclo-oxygenase 2 (COX2), which catalyses the synthesis of pro-inflammatory prostaglandins, in particular PGE. At late stages of inflammatory episodes, however, COX2 directs the synthesis of anti-inflammatory cyclopentenone prostaglandins, suggesting a role for these molecules in the resolution of inflammation. Cyclopentenone prostaglandins have been suggested to exert anti-inflammatory activity through the activation of peroxisome proliferator-activated receptor-gamma. Here we demonstrate a novel mechanism of antiinflammatory activity which is based on the direct inhibition and modification of the IKKbeta subunit of IKK. A...
Journal of the American Academy of Dermatology, 1995
Diagnosis of the underlying dermatosis in erythroderma is often difficult. The cause of increased... more Diagnosis of the underlying dermatosis in erythroderma is often difficult. The cause of increased mortality in erythroderma, particularly in relation to infection, is incompletely understood. We investigated the potential diagnostic use of circulating intercellular adhesion molecule-1 (cICAM-1), vascular cell adhesion molecule-1 (cVCAM-1), and E-selectin (cE-selectin) levels in erythroderma. cICAM-1, cVCAM-1, and cE-selectin were measured by enzyme-linked immunosorbent assay in 14 patients with erythroderma of known cause and in 17 control subjects. Levels were correlated with other markers of the inflammatory response. In erythroderma median cICAM-1, cVCAM-1, and cE-selectin levels were significantly elevated, but no difference was found between values in patients with eczema and values in those with psoriasis. Circulating adhesion molecule levels did not correlate with erythrocyte sedimentation rate or total white blood cell count. cICAM-1, cVCAM-1, and cE-selectin were detectable in patients with erythroderma but were not differential diagnostic use in this study. Because in vitro these molecules may interfere with normal cell adhesion mechanisms, we speculate that they may contribute to the immunosuppressive state in these patients.
Arsenite is a potent environmental toxin that causes various pathologies including cancers and sk... more Arsenite is a potent environmental toxin that causes various pathologies including cancers and skin disorders. Arsenite is believed to exert its biological effects through reaction with exposed sulfhydryl groups, especially pairs of adjacent thiols. Here, we describe the mechanism by which arsenite affects the NF-kappaB signaling pathway. Activation of transcription factor NF-kappaB depends on the integrity of the IkappaB kinase (IKK) complex. We found that arsenite potently inhibits NF-kappaB and IKK activation by binding to Cys-179 in the activation loop of the IKK catalytic subunits, IKKalpha/beta. The affinity of IKKbeta for trivalent arsenic was verified in vitro by the ability of IKKbeta to enhance the fluorescence of an arsenic-substituted fluorescein dye. The addition of 1,2-dithiol antidotes or replacement of Cys-179 with an alanine residue abolished dye binding to and arsenite inhibition of IKKbeta. Overexpression of IKKbeta (C179A) protects NF-kappaB from inhibition by arsenite, indicating that despite the involvement of a large number of distinct gene products in this activation pathway, the critical target for inhibition by arsenite is on the IKK catalytic subunits.
The disposable soma theory suggests that longevity is determined through the setting of longevity... more The disposable soma theory suggests that longevity is determined through the setting of longevity assurance mechanisms so as to provide an optimal compromise between investments in somatic maintenance (including stress resistance) and in reproduction. A corollary is that species with low extrinsic mortality are predicted to invest relatively more effort in maintenance, resulting in slower intrinsic ageing, than species with high extrinsic mortality. We tested this prediction in a comparative study of stress resistance in primary skin fibroblasts and confirmed that cells from long-lived species are indeed more resistant to a variant of stressors. A widely studied example of within-species variation in lifespan is the rodent calorie restriction model. Food-restricted animals show elevations in a range of stress response mechanisms, and it has been suggested that this is an outcome of natural selection for life history plasticity. We have developed a theoretical model for dynamic optimisation of the allocation of effort to maintenance and reproduction in response to fluctuations in food availability. The model supports the suggestion that the response to calorie restriction may be an evolutionary adaptation, raising interesting questions about the hierarchy of genetic control of multiple stress response systems. The model identifies ecological factors likely to support such an adaptation that may be relevant in considering the likely relevance of a similar response to calorie restriction in other species. Comparative and theoretical studies support the role of somatic maintenance and stress response systems in controlling the rate of ageing.
Identifying the mechanisms determining species-specific life spans is a central challenge in unde... more Identifying the mechanisms determining species-specific life spans is a central challenge in understanding the biology of aging. Cellular stresses produce damage, that may accumulate and cause aging. Evolution theory predicts that long-lived species secure their longevity through investment in a more durable soma, including enhanced cellular resistance to stress. To investigate whether cells from long-lived species have better mechanisms to cope with oxidative and non-oxidative stress, we compared cellular resistance of primary skin fibroblasts from eight mammalian species with a range of life spans. Cell survival was measured by the thymidine incorporation assay following stresses induced by paraquat, hydrogen peroxide, tert-butyl hydroperoxide, sodium arsenite and alkaline pH (sodium hydroxide). Significant positive correlations between cell LD90 and maximum life span were found for all these stresses. Similar results were obtained when cell survival was measured by the MTT assay, and when lymphocytes from different species were compared. Cellular resistance to a variety of oxidative and non-oxidative stresses was positively correlated with mammalian longevity. Our results support the concept that the gene network regulating the cellular response to stress is functionally important in aging and longevity.
Sarcoidosis is a disease of unknown etiology characterized by non-caseating granulomata together ... more Sarcoidosis is a disease of unknown etiology characterized by non-caseating granulomata together with a number of systemic abnormalities. We have recently shown these include increased expression of the integrins CDI 1/CD 18 on peripheral blood leucocytes. Here we have measured serum levels of the adhesion molecules intercellular adhesion molecule-I (ICAM-1), E-selectin and vascular cell adhesion molecule-I (VCAM-1) in 23 patients and 14 normal controls using antigen capture sandwich ELISAs. Median circulating E-selectin levels in the patients were nearly three times those of the controls (P < 0-0001, Mann-Whitney U-test), whilst ICAM-I but not VCAM-l levels were only slightly elevated. These results show that endothelial cell activation and shedding of E-selectin into the circulation are additional features of the pathology of sarcoidosis.
We have developed a panel of MoAbs against four separate but overlapping epitopes on endothelial ... more We have developed a panel of MoAbs against four separate but overlapping epitopes on endothelial cell (EC) vascular cell adhesion molecule-I (VCAM-1). Two of the MoAbs (IGl 1 and 1E5) inhibited T cell adhesion to tumour necrosis factor (TNF)-activated EC, whilst two MoAbs (1.4C3 and 6D9) did not. Using these MoAbs we have identified a circulating form ofVCAM-I (cVCAM-1) which has identical epitope distribution to the EC form, and which is able to support the adhesion of the human lymphoblastoid cell line Jurkat J6 by a VLA-4-and VCAM-1-dependent mechanism when immobilized from plasma. cVCAM-l isolated by immunoaffinity and size-exclusion chromatographies was shown by SDS-PAGE to have an apparent mol. wt of 85-90 kD. Levels ofcVCAM-I were significantly raised (P < 0-001) in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) compared with normal individuals. It is possible that cVCAM-1 may be a useful plasma marker for the diagnosis and management of patients with inflammatory diseases. Furthermore, detection of elevated cVCAM-I levels may act as a guide to the importance of VCAM-I -dependent cell adhesion in different pathological settings.
Most inflammatory agents activate nuclear factor-kappaB (NF-kappaB), resulting in induction of ge... more Most inflammatory agents activate nuclear factor-kappaB (NF-kappaB), resulting in induction of genes coding for cytokines, chemokines, and enzymes involved in amplification and perpetuation of inflammation. Hypoestoxide (a bicyclo [9,3,1] pentadecane) is a diterpene from Hypoestes rosea, a tropical shrub in the family Acanthacea, several members of which are used in folk medicine in Nigeria. Here, we demonstrate that hypoestoxide (HE) abrogates the production of pro-inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) in lipopolysaccharide (LPS)-activated normal human peripheral blood mononuclear cells. Moreover, HE inhibits the production of nitric oxide (NO) by IL-1beta- or IL-17-stimulated normal human chondrocytes. In vivo, oral administration of HE to mice significantly ameliorated hind paw edema induced by antibodies to type II collagen plus LPS. Furthermore, topical administration of HE to mice also significantly inhibited phorbol ester-induced ear inflammation. The anti-inflammatory activity of HE may be due in part to its ability to inhibit NF-kappaB activation through direct inhibition of IkappaB kinase (IKK) activity. Thus, HE could be useful in treating various inflammatory diseases and may represent a prototype of a novel class of IKK inhibitors.
Two transcription factors, NF-κB and AP-1, have emerged in recent years as major targets for sign... more Two transcription factors, NF-κB and AP-1, have emerged in recent years as major targets for signaling pathways that are activated by a variety of proinflammatory stimuli. The same transcription factors were also found to be regulated by oxidative stress, although in this case, we find evidence for both positive and negative regulation (especially for NF-κB). After discussing how each of these transcription factors is regulated by proinflammatory stimuli, their regulation by oxidative stress and pro-oxidants will be addressed. Transcription factor NF-κB is regulated through interaction with inhibitory proteins, the IκBs. In response to cell stimulation with proinflammatory stimuli, the IκBs are rapidly phosphorylated at two conserved serines, followed by subsequent polyubiquitination and degradation through the 26S proteasome. Although many diverse stimuli including TNFα, IL-1, lipopolysaccharide (LPS) and other components of bacterial cell walls, double stranded (ds) RNA, viruses and ionizing radiation activate NF-κB through induction of IκB phosphorylation, they all operate through a common protein kinase-the IκB kinase (IKK). Purification of IKK indicates it is a large complex composed of at least three subunits, IKKα, IKKβ and IKKγ. The first two are closely related catalytic subunits (kinases) and the third is a regulatory subunit.
To compare the levels of circulating intercellular adhesion molecule 1 (cICAM-1) and vascular cel... more To compare the levels of circulating intercellular adhesion molecule 1 (cICAM-1) and vascular cell adhesion molecule 1 (cVCAM-1) in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Levels of cICAM-1 and cVCAM-1 were measured in both plasma and synovial fluid using monoclonal antibody sandwich enzyme-linked immunoassays. Levels of both cICAM-1 and cVCAM-1 were significantly increased (P &lt; 0.001) in RA patients compared with normal controls. In contrast, only cVCAM-1, and not cICAM-1, was increased in patients with SLE. Levels of cICAM-1 and cVCAM-1 were significantly elevated in synovial fluid compared with plasma in paired samples from patients with RA. There was no correlation between levels of cICAM-1 and levels of cVCAM-1, in either plasma or synovial fluid. Whereas levels of cVCAM-1 correlated significantly with the erythrocyte sedimentation rate (ESR) and C-reactive protein level in RA patients and with the ESR in SLE patients, no significant correlations were found between cICAM-1 and either of these indices of disease activity. These observations indicate that levels of cICAM-1 and cVCAM-1 reflect separate pathophysiologic processes. Both may be useful markers for the diagnosis and management of patients with rheumatic diseases.
Arsenite is a potent environmental toxin that causes various pathologies including cancers and sk... more Arsenite is a potent environmental toxin that causes various pathologies including cancers and skin disorders. Arsenite is believed to exert its biological effects through reaction with exposed sulfhydryl groups, especially pairs of adjacent thiols. Here, we describe the mechanism ...
Urolithiasis affects around 10% of the US population with an increasing rate of prevalence, recur... more Urolithiasis affects around 10% of the US population with an increasing rate of prevalence, recurrence and penetrance. The causes for the formation of most urinary calculi remain poorly understood, but obtaining the chemical composition of these stones might help identify key aspects of this process and new targets for treatment. The majority of urinary stones are composed of calcium that is complexed in a crystalline matrix with organic and inorganic components. Surprisingly, mitigation of urolithiasis risk by altering calcium homeostasis has not been very effective. Thus, studies to identify other therapeutic stone-specific targets, using proteomics, metabolomics and microscopy techniques, have been conducted, revealing a high level of complexity. The data suggest that numerous metals other than calcium and many nonmetals are present within calculi at measurable levels and several have distinct distribution patterns. Manipulation of the levels of some of these elemental components of calcium-based stones has resulted in clinically beneficial changes in stone chemistry and rate of stone formation. The elementome-the full spectrum of elemental content-of calcium-based urinary calculi is emerging as a new concept in stone research that continues to provide important insights for improved understanding and prevention of urinary stone disease.
The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin ... more The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation.
Reducing protein synthesis slows growth and development but can increase adult life span. We demo... more Reducing protein synthesis slows growth and development but can increase adult life span. We demonstrate that knockdown of eukaryotic translation initiation factor 4G (eIF4G), which is downregulated during starvation and dauer state, results in differential translation of genes important for growth and longevity in C. elegans. Genome-wide mRNA translation state analysis showed that inhibition of IFG-1, the C. elegans ortholog of eIF4G, results in a relative increase in ribosomal loading and translation of stress response genes. Some of these genes are required for life span extension when IFG-1 is inhibited. Furthermore, enhanced ribosomal loading of certain mRNAs upon IFG-1 inhibition was correlated with increased mRNA length. This association was supported by changes in the proteome assayed via quantitative mass spectrometry. Our results suggest that IFG-1 mediates the antagonistic effects on growth and somatic maintenance by regulating mRNA translation of particular mRNAs based, in part, on transcript length.
Extensive studies in model organisms in the last few decades have revealed that aging is subject ... more Extensive studies in model organisms in the last few decades have revealed that aging is subject to profound genetic influence. The conserved nutrient sensing TOR (Target of Rapamycin) pathway is emerging as a key regulator of lifespan and healthspan in various species from yeast to mammals. The TOR signaling pathway plays a critical role in determining how a eukaryotic cell
The antagonistic pleiotropy theory of aging proposes that aging takes place because natural selec... more The antagonistic pleiotropy theory of aging proposes that aging takes place because natural selection favors genes that confer benefit early on life at the cost of deterioration later in life. This theory predicts that genes that impact development would play a key role in shaping adult lifespan. To better understand the link between development and adult lifespan, we examined the genes previously known to be essential for development. From a pool of 57 genes that cause developmental arrest after inhibition using RNA interference, we have identified 24 genes that extend lifespan in Caenorhabditis elegans when inactivated during adulthood. Many of these genes are involved in regulation of mRNA translation and mitochondrial functions. Genetic epistasis experiments indicate that the mechanisms of lifespan extension by inactivating the identified genes may be different from those of the insulin/insulin-like growth factor 1 (IGF-1) and dietary restriction pathways. Inhibition of many of these genes also results in increased stress resistance and decreased fecundity, suggesting that they may mediate the trade-offs between somatic maintenance and reproduction. We have isolated novel lifespan-extension genes, which may help understand the intrinsic link between organism development and adult lifespan.
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Papers by Pankaj Kapahi