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Brian Herman

Publications and source records attributed to Brian Herman.

At least 19 recordsLinked to original sources
PubMed2006

Caspase-2 deficiency enhances aging-related traits in mice.

Alteration of apoptotic activity has been observed in a number of tissues in aging mammals, but it remains unclear whether and/or how apoptosis may affect aging. Caspase-2 is a member of the cysteine protease family that plays a critical role in apoptosis. To understand the impact of compromised apoptosis function on mammalian aging, we conducted a comparative study on caspase-2 deficient mice and their wild-type littermates with a specific focus on the aging-related traits at advanced ages. We found that caspase-2 deficiency enhanced a number of traits commonly seen in premature aging animals. Loss of caspase-2 was associated with shortened maximum lifespan, impaired hair growth, increased bone loss, and reduced body fat content. In addition, we found that the livers of caspase-2 deficient mice had higher levels of oxidized proteins than those of age-matched wild-type mice, suggesting that caspase-2 deficiency compromised the animal's ability to clear oxidatively damaged cells. Collectively, these results suggest that caspase-2 deficiency affects aging in the mice. This study thus demonstrates for the first time that disruption of a key apoptotic gene has a significant impact on aging.

Adipose Tissue↗
PubMed2006

ARC protects rat cardiomyocytes against oxidative stress through inhibition of caspase-2 mediated mitochondrial pathway.

Apoptosis repressor with a CARD domain (ARC) has been demonstrated to protect heart cells against ischemia/reperfusion (I/R) injury. In this study, we investigated the mechanism by which ARC protects heart cells against oxidative stress. We monitored the extent of apoptosis and activity of multiple components of the intrinsic apoptotic pathway in rat cardiac myoblast cell line H9c2 with either reduced or increased expression of ARC during oxidative stress. Overexpression of ARC-inhibited oxidative stress-induced caspase-2/3 activation, cytochrome c release, and translocation of Bax to mitochondria. Furthermore, phosphorylation of ARC at threonine 149 was found to be critical to its function. ARC containing a T149A mutation failed to translocate to mitochondria, did not inhibit caspase-2 activation, and had a dominant negative effect against the protective effect of endogenous ARC during oxidative stress. In addition, wild-type ARC but not the T149A mutant inhibited cell death induced by overexpression of caspase-2. Using a yeast two-hybrid (YTH) screening approach and co-immunoprecipitation (Co-IP), we found that protein phosphatase 2C (PP2C) interacted with ARC and that PP2C mediated-dephosphorylation of ARC inhibited its anti-apoptotic activity. Eliminating either the N-terminal CARD domain or the C-terminal P/E domain also abolished the anti-apoptotic function of ARC, suggesting that full-length ARC is required for its apoptotic inhibition. These results indicate that ARC plays an important role in protection of H9c2 cells against oxidative stress-induced apoptosis by phosphorylation-dependent suppression of the mitochondria-mediated intrinsic pathway, partially initiated through the activation of caspase-2.

Animals↗
PubMed2006

Spatio-temporal kinetics of growth hormone receptor signaling in single cells using FRET microscopy.

The growth hormone (GH) receptor (R)-mediated JAK2 (Janus kinase-2)-STAT5 (signaling transducer and activator of transcription-5) pathway involves a cascade of protein-protein interactions and tyrosine phosphorylations that occur in a spatially and temporally sensitive manner in cells. To study GHR dimerization or GH-induced conformational change of predimerized GHRs and STAT5 activation kinetics in intact cells, fluorescence resonance energy transfer (FRET) and live-cell imaging methods were employed. FRET measurements at the membrane of HEK-293T cells co-expressing GHRs tagged at the C-terminus with cyan (C) and yellow (Y) fluorescent proteins (FPs) revealed transient GHR dimerization lasting 2-3 min, with a maximum at 3 min after GH stimulation, which was sufficient to induce STAT5 activation. The transient nature of the dimerization or GH-induced conformational change of predimerized GHRs kinetics was not a result of GHR internalization, as neither potassium- nor cholesterol-depletion treatments prolonged the FRET signal. YFP-tagged STAT5 recruitment to the membrane, binding to GHR-CFP, and phosphorylation, occurred within minutes of GH stimulation. Activated STAT5a-YFP did not show nuclear accumulation, despite nuclear pSTAT5 increase, suggesting high turnover of STAT5 nuclear shuttling. Although GHR dimerization and STAT5 activation have been reported previously, this is the first spatially resolved demonstration of GHR-signaling kinetics in intact cells.

Cells, Cultured↗
PubMed2005

Identification of the hydrophobic strand in the A-B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists.

Interaction of leptin with its receptors resembles that of interleukin-6 and granulocyte colony-stimulating factor, which interact with their receptors through binding sites I-III. Site III plays a pivotal role in receptors' dimerization or tetramerization and subsequent activation. Leptin's site III also mediates the formation of an active multimeric complex through its interaction with the IGD (immunoglobulin-like domain) of LEPRs (leptin receptors). Using a sensitive hydrophobic cluster analysis of leptin's and LEPR's sequences, we identified hydrophobic stretches in leptin's A-B loop (amino acids 39-42) and in the N-terminal end of LEPR's IGD (amino acids 325-328) that are predicted to participate in site III and to interact with each other in a beta-sheet-like configuration. To verify this hypothesis, we prepared and purified to homogeneity (as verified by SDS/PAGE, gel filtration and reverse-phase chromatography) several alanine muteins of amino acids 39-42 in human and ovine leptins. CD analyses revealed that those mutations hardly affect the secondary structure. All muteins acted as true antagonists, i.e. they bound LEPR with an affinity similar to the wild-type hormone, had no agonistic activity and specifically inhibited leptin action in several leptin-responsive in vitro bioassays. Alanine mutagenesis of LEPR's IGD (amino acids 325-328) drastically reduced its biological but not binding activity, indicating the importance of this region for interaction with leptin's site III. FRET (fluorescence resonance energy transfer) microscopy experiments have documented that the transient FRET signalling occurring upon exposure to leptin results not from binding of the ligand, but from ligand-induced oligomerization of LEPRs mediated by leptin's site III.

Amino Acid Sequence↗
PubMed2005

Fluorescence resonance energy transfer (FRET) microscopy in living cells as a novel tool for the study of cytokine action.

Fluorescence resonance energy transfer (FRET) microscopy was used to study interactions between proteins in intact cells. We showed that growth hormone (GH) causes transient homodimerization of GH receptors tagged with yellow or cyan fluorescent proteins. The peak of FRET signaling occurred 2 to 4 min after hormonal stimulation and was followed by a decrease in FRET signal. Repeating those experiments in cells pretreated with the inhibitor of internalization methyl-beta-cyclodextrin, or in potassium-depleted cells showed no difference in the kinetics of FRET signaling as compared with the non-treated cells, indicating that the decrease in FRET signal does not result from receptor internalization by the pathways inhibited by methyl-beta-cyclodextrin or potassium depleted but might occur by other pathways of internalization. Using a similar methodology, we also demonstrated that ovine placental lactogen (oPL) causes transient heterodimerization of GH and prolactin (PRL) receptors 2.5 to 3 min after oPL application. On the other hand, oGH or oPRL had no effect at all, further substantiating the finding the oPL, which lacks a specific receptor, acts in homologous systems by heterodimerization of GH and PRL receptors. We also demonstrated that both PRL and leptin (LEP) are capable of transactivation of the oncogenic receptors erbB2 and erbB3. Upon PRL or LEP stimulation of HEK-293T cells transfected with LEP or PRL receptors and erbB2 or erbB3, erbB proteins are first phosphorylated and then activate MAPK (erk1/erk2). However, the FRET experiments failed to document any evidence of a direct interaction between erbB2 and the PRL or LEP receptors, suggesting that erbB activation probably occurs via activated JAK2, translocated from the respective receptors to erbB2.

Animals↗
PubMed2005

Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells.

BACKGROUND INFORMATION: Leptin, an adipocyte-secreted hormone, signals through activation of its membrane-embedded receptor (LEPR). To study the leptin-induced events occurring in short (LEPRa) and long (LEPRb) LEPRs in the cell membrane, by FRET (fluorescence resonance energy transfer) methodology, the respective receptors, tagged at their C-terminal with CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein), were prepared. RESULTS: The constructs encoding mLEPRa (mouse LEPRa)-YFP and mLEPRa-CFP, mLEPRb-YFP and mLEPRb-CFP were tested for biological activity in transiently transfected CHO cells (Chinese-hamster ovary cells) and HEK-293T cells (human embryonic kidney 293 T cells) for activation of STAT3 (signal transduction and activators of transcription 3)-mediated LUC (luciferase) activity and binding of radiolabelled leptin. All four constructs were biologically active and were as potent as their untagged counterparts. The localization pattern of the fused protein appeared to be confined almost entirely to the cell membrane. The leptin-dependent interaction between various types of receptors in fixed cells were studied by measuring FRET, using fluorescence lifetime imaging microscopy and acceptor photobleaching methods. CONCLUSIONS: Both methods yielded similar results, indicating that (1) leptin receptors expressed in the cell membrane exist mostly as preformed LEPRa/LEPRa or LEPRb/LEPRb homo-oligomers but not as LEPRb/LEPRa hetero-oligomers; (2) the appearance of transient leptin-induced FRET in cells transfected with LEPRb/LEPRb reflects both a conformational change that leads to closer interaction in the cytosolic part and a higher FRET signal, as well as de novo homo-oligomerization; (3) in LEPRa/LEPRa, exposure to leptin does not lead to any increase in FRET signalling as the proximity of CFP and YFP fluorophores in space already gives maximal FRET efficiency of the preoligomerized receptors.

Animals↗
PubMed2004

Transgenic mice overexpressing glutathione peroxidase 4 are protected against oxidative stress-induced apoptosis.

Glutathione peroxidase 4 (Gpx4) is uniquely involved in the detoxification of oxidative damage to membrane lipids. Our previous studies showed that Gpx4 is essential for mouse survival and that Gpx4 deficiency makes cells vulnerable to oxidative injury. In the present study, we generated two lines of transgenic mice overexpressing Gpx4 (Tg(GPX4) mice) using a genomic clone containing the human GPX4 gene. Both lines of Tg-(GPX4) mice, Tg5 and Tg6, had elevated levels of Gpx4 (mRNA and protein) in all tissues investigated, and overexpression of Gpx4 did not cause alterations in activities of glutathione peroxidase 1, catalase, Cu/Zn superoxide dismutase, and manganese superoxide dismutase. The human GPX4 transgene rescued the lethal phenotype of null mutation of the mouse Gpx4 gene, indicating that the transgene can replace the essential role of mouse Gpx4 in mouse development. Cell death induced by t-butylhydroperoxide and diquat was significantly less in murine embryonic fibroblasts from Tg(GPX4) mice compared with wild type mice. Liver damage and lipid peroxidation induced by diquat were reduced significantly in Tg(GPX4) mice. In addition, diquat-induced apoptosis was decreased in Tg(GPX4) mice, as evidenced by attenuated caspase-3 activation and reduced cytochrome c release from mitochondria. These data demonstrate that Gpx4 plays a role in vivo in the mechanism of apoptosis induced by oxidative stress that most likely occurs through oxidative damage to mitochondrial phospholipids such as cardiolipin.

Alanine Transaminase↗
PubMed2004

Transactivation of erbB2 by short and long isoforms of leptin receptors.

We generated kinase-positive and kinase-negative erbB2 tagged with YFP and the long form of leptin receptor (LEPRb) tagged with CFP. Both were as active as their untagged analogs. Both short and long isoforms of leptin receptor phosphorylated and thereby activated erbB2 upon leptin binding and enhanced MAPK activity. Our results unveil a novel route by which leptin may provoke erbB2's phosphorylation and thus enhance its oncogenic potential independently of HER family ligands or its overexpression. Using FRET technology in living cells, we found no evidence of complex formation between erbB2 and prolactin or leptin receptors, indicating that the transactivation occurs through an indirect interaction.

Animals↗
PubMed2004

Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer.

Mitochondrial depolarization promotes apoptotic and necrotic cell death and possibly other cellular events. Polarized mitochondria take up cationic tetramethylrhodamine methylester (TMRM), which is released after depolarization. Thus, TMRM does not label depolarized mitochondria. To identify both polarized and depolarized mitochondria in living cells, cultured rat hepatocytes, and sinusoidal endothelial cells were co-loaded with green-fluorescing MitoTracker Green FM (MTG) and red-fluorescing TMRM for imaging by laser scanning confocal microscopy. Like TMRM, MTG is a cationic fluorophore that accumulates electrophoretically into polarized mitochondria. Unlike TMRM, MTG binds covalently to intramitochondrial protein thiols and remains bound after depolarization. In cells labeled only with MTG, excitation with blue (488 nm) light yielded green but almost no red fluorescence. After subsequent loading with TMRM, green MTG fluorescence became quenched. Instead, blue excitation yielded red fluorescence. Mitochondrial de-energization restored green fluorescence and abolished red fluorescence. Conversely, when MTG was added to TMRM-labeled cells, red fluorescence excited by blue light was enhanced, an effect again reversed by de-energization. These observations of reversible quenching of donor fluorescence and augmentation of acceptor fluorescence signify fluorescence resonance energy transfer (FRET). In undisturbed hepatocytes, spontaneous depolarization of a subfraction of mitochondria was an ongoing phenomenon. In conclusion, confocal FRET discriminates individual depolarized mitochondria against a background of hundreds of polarized mitochondria.

Aldehydes↗
PubMed2004

Oxidative stress-induced apoptosis is associated with alterations in mitochondrial caspase activity and Bcl-2-dependent alterations in mitochondrial pH (pHm).

Oxidative stress, the result of cellular production of reactive oxygen species (ROS), has been implicated in a number of diseases of the eye. Exposure of eye tissues (e.g. the cornea and retina) to oxidative stress over time has been hypothesized to underlie the development of age-related macular degeneration (AMD) and maturity onset cataract formation. Light-induced free radicals can damage the eye, and alterations in the antioxidant defenses of the eye have been suggested to play a role in the etiology of glaucoma. Mitochondria are both a major endogenous source and target of ROS, and oxidative stress has been shown to induce apoptotic cell death by targeting the mitochondria directly. Mitochondrial-dependent apoptosis has been shown to require release of cytochrome c from mitochondria and subsequent activation of a specific class of cytoplasmic proteases known as caspases. Bcl-2, an anti-apoptotic protein localized to mitochondria, has been shown to inhibit cytochrome c release and protect against oxidative stress-induced apoptosis. Here we demonstrate that oxidative stress causes activation of mitochondrial matrix caspase-2 and -9 activity that is associated with Bcl-2-inhibitable acidification of mitochondrial pH (pH(m)). In conjunction with recent reports that caspase activation is maximal at acidic pH, these findings have led us to hypothesize that Bcl-2 may modulate cytochrome c release following oxidative stress by modifying the pH-dependent activation of mitochondrial caspase activity. These studies provide an increased understanding of the mechanism(s) by which oxidative stress damages tissues, and may have important therapeutic implications for treatment of opthamological diseases.

Animals↗
PubMed2004

Detection of mitochondrial caspase activity in real time in situ in live cells.

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP-caspase 3 substrate-YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

Animals↗
PubMed2004

Microscopic analysis of fluorescence resonance energy transfer (FRET).

We describe here detailed practical procedures for implementing various approaches of fluorescence resonance energy transfer (FRET) in microscope-based measurements. A comprehensive theoretical formalism is developed and the different experimental procedures are outlined. A step-by-step protocol is provided for preparing the specimens for FRET measurements, data acquisition procedures, analysis, and quantification. Particular emphasis is given to exemplify the FRET applications in the study of protein-protein interactions.

Animals↗
PubMed2003

Caspases, apoptosis and aging.

Caspases are a group of cysteine dependent aspartate-specific proteases. Originally found as the homolog of Ced-3 in C. elegans, 14 caspases have now been identified in mammals to date. Caspases play important roles in both the intrinsic and extrinsic apoptotic pathways and interact with the non-caspase apoptotic pathways. A number of recent published observations have suggested a strong association between apoptosis, age-related diseases and aging. Findings from our group and others reveal a strong correlation between alterations in caspase activity and aging. In this view point, we summarize current knowledge of the connection between caspases and aging observed in a variety of model systems from cultured cells in vitro to the in vivo models of rodents and humans.

Aging↗
PubMed2003

Ovine placental lactogen-induced heterodimerization of ovine growth hormone and prolactin receptors in living cells is demonstrated by fluorescence resonance energy transfer microscopy and leads to prolonged phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3.

HEK-293T cells transiently transfected with ovine (o) GH receptor (GHR) and prolactin receptor (PRLR) constructs respectively tagged downstream with cyan or yellow fluorescent proteins were used to study ovine placental lactogen (oPL)-stimulated heterodimerization by fluorescence resonance energy transfer (FRET) microscopy. The oPL-stimulated transient heterodimerization of GHR and PRLR had a peak occurring 2.5-3 min after oPL application, whereas oGH or oPRL had no effect at all. The results indicate none or only little dimerization occurring before the hormonal stimulation. The effect of heterodimerization was studied by comparing activation of Janus kinase 2, signal transducer and activator of transcription (STAT)1, STAT3, STAT5, and MAPK in Chinese hamster ovary cells stably transfected with chimeric genes encoding receptors consisting of cytosolic and transmembrane parts of oGHR and oPRLR, extracellular domains of human granulocyte and macrophage colony-stimulating factor (hGM-CSF) receptor alpha or beta, and cells transfected with the two forms (alpha or beta) of PRLR and GHR. Functionality of those proteins was verified by hGM-CSF-induced phosphorylation of both intracellular PRLR and GHR domains and hGM-CSF-induced heterodimerization was documented by chimeric receptor coimmunoprecipitation. Homodimerization or heterodimerization of PRLRs and GHRs had no differential effect on activation of STAT5 and MAPK. However, heterodimerization resulted in a prolonged phosphorylation of STAT1 and in particular STAT3, suggesting that the heterodimerization of alpha-oGHR and beta-oPRLR is able to transduce a signal, which is distinct from that occurring on homodimeric associations.

Animals↗
PubMed2002

Polymorphism at codon 72 of p53, human papillomavirus, and cervical cancer in South India.

PURPOSE: It has been suggested that host genetic factors play a role in human papillomavirus (HPV)-associated tumorigenesis, although the issue continues to be a focus of much debate. Previous studies have reported that a common polymorphism of the wild type p53 gene at codon 72 of exon 4 (Arg/Arg) is associated with a sevenfold increased risk of HPV-associated cancer compared to Arg/Pro and Pro/Pro polymorphisms. In vitro studies also suggested that the Arg/Arg polymorphism was much more susceptible to HPV 16 E6-mediated degradation as compared to other allelic forms. Subsequent studies published since this initial report indicated geographical differences with respect to the role of Arg/Arg polymorphism in increasing the risk of HPV-associated cervical cancer. METHODS: In this study we analyzed leukocyte DNA from a total of 421 subjects for the Arg/Arg, Arg/Pro or Pro/Pro p53 polymorphisms at various stages of the cervical tumor progression spectrum, using allele-specific PCR. All subjects were from the Thiruvananthapuram District of South India. HPV genotyping was done for all subjects using either DNA extracted from cervical biopsies or exfoliated cervical cells. All subjects were grouped on the basis of both of cyto-pathology and HPV status. RESULTS: The distribution of p53 genotypes was not significantly different in all study groups (HPV positive vs HPV negative and cases vs controls comparisons). Homozygosity for Arg/Arg was not associated with increased risk for cervical cancer. CONCLUSION: We find no evidence for any association between homozygosity for p53 arginine with either cervical dysplasia, cervical carcinoma or HPV infection in the population from South India.

Arginine↗
PubMed2002

Human papillomavirus type 16 E6 and E7 gene variations in Indian cervical cancer.

OBJECTIVES: Human papillomavirus type 16 is a causative factor for development of cervical cancer. The E6 and E7 genes of HPV 16 are critical to the process of immortalization and transformation of host cells. Recent reports suggest that variants of these two genes may contribute to the risk of malignant progression of cancer in the uterine cervix. However, no data exist on sequence variations of HPV 16 E6 and E7 genes that may exist in India. Therefore, we examined intratype variations in the E6 and E7 viral genes in DNA isolated from HPV 16-positive cervical scrapes and biopsies. METHODS: The open reading frames of the E6 and E7 genes were amplified by PCR and then directly sequenced by the fluorescent dye dideoxy termination method.Results. In addition to the prototype E6 gene sequence, five sets of mutations of the E6 gene were identified. The European prototype (350T) was detected in 9.1% of the study group while the European variant (350G) was seen in 28% of patients. The remaining variants (a combination of the 350G mutation with 335T, 145T, or 419G) were significantly associated with cases compared to controls. The 350G + 145T variant was found at much higher incidence in cases in younger women, suggesting that this variant may be associated with aggressive tumor behavior. Interestingly the 350G + 419G combination was found only in controls. There was no significant association between the four genotypes of E7 and any stage of tumor progression or age. CONCLUSIONS: The results indicate that specific mutations in the E6 gene are found in young Indian women with high-grade squamous intraepithelial lesions and invasive cancer, suggesting that these mutations represent more oncogenically active HPV 16. Whether this increased oncogenecity is due to differences in p53 inactivation, ineffective keratinocyte differentiation, and/or altered response to the immune system by these oncogenic E6 mutants remains to be clarified.

Adult↗