Delayed Infiltration of Peripheral Monocyte Contributes to Phagocytosis and Transneuronal Degeneration in Chronic Stroke.

BACKGROUND: Mononuclear phagocytes, including monocyte-derived macrophages (MDMs) and microglia, contribute to infarct development as well as tissue repair in the postischemic brain. Here, we identify the origin and function of MDMs in the brain during poststroke repair processes. METHODS: Adult mice were subjected to transient middle cerebral artery occlusion. Longitudinal brain atrophy and secondary degeneration were evaluated during acute to recovery phases of stroke. Adoptive transfer of GFP+ splenocytes into asplenic mice was used to distinguish MDMs from resident microglia. Fluorescence beads were injected into stroked animals to examine phagocytic function. RESULTS: Progressive atrophy and neuronal degeneration in remote regions were observed in chronic stroke, which also was accompanied by MDM infiltration into the ipsilateral hemisphere. Compared with microglia, MDMs had significantly higher phagocytic activity. MDM trafficking and phagocytosis was spatiotemporally regulated with acute and prolonged infiltration into infarcted tissue, as well as delayed entry in remote areas such as the thalamus and substantia nigra. CONCLUSIONS: The stepwise and long-lasting involvement of MDMs at multiple poststroke stages shows that MDMs have a role in progressive stroke-induced injury and repair processes. These findings suggest that manipulating monocyte entry at different stroke stages may be an effective immune-based strategy to limit injury propagation in chronic stroke.

Phagocytosis converts infiltrated monocytes to microglia-like phenotype in experimental brain ischemia.

BACKGROUND: Monocyte-derived macrophages (MDMs) and microglia elicit neural inflammation and clear debris for subsequent tissue repair and remodeling. The role of infiltrating MDMs in the injured brain, however, has been controversial due to overlapping antigen expression with microglia. In this study, we define the origin and function of MDMs in cerebral ischemia. METHODS: Using adoptive transfer of GFP+ splenocytes into adult asplenic mice subjected to transient middle cerebral artery occlusion, we compared the role of CD11b+/CD45+/NK1.1-/Ly6G- MDMs and microglia in the ischemic brain. The phagocytic activities of MDMs and microglia were measured by the uptake of fluorescent beads both in vivo with mice infused with GFP+ splenocytes and ex vivo with cultures of isolated brain immune cells. RESULTS: Stroke induced an infiltration of MDMs [GFP+] into the ipsilateral hemisphere at acute (3 days) and sub-acute phases (7 days) of post-stroke. At 7 days, the infiltrating MDMs contained both CD45High and CD45Low subsets. The CD45High MDMs in the injured hemisphere exhibited a significantly higher proliferation capacity (Ki-67 expression levels) as well as higher expression levels of CD11c when compared to CD45Low MDMs. The CD45High and CD45Low MDM subsets in the injured hemisphere were approximately equal populations, indicating that CD45High MDMs infiltrating the ischemic brain changes their phenotype to CD45Low microglia-like phenotype. Studies with fluorescent beads reveal high levels of MDM phagocytic activity in the post-stroke brain, but this phagocytic activity was exclusive to post-ischemic brain tissue and was not detected in circulating monocytes. By contrast, CD45Low microglia-like cells had low levels of phagocytic activity when compared to CD45High cells. Both in vivo and ex vivo studies also show that the phagocytic activity in CD45High MDMs is associated with an increase in the CD45Low/CD45High ratio, indicating that phagocytosis promotes MDM phenotype conversion. CONCLUSIONS: This study demonstrates that MDMs are the predominant phagocytes in the post-ischemic brain, with the CD45High subset having the highest phagocytic activity levels. Upon phagocytosis, CD45High MDMs in the post-ischemic brain adopt a CD45Low phenotype that is microglia-like. Together, these studies reveal key roles for MDMs and their phagocytic function in tissue repair and remodeling following cerebral ischemia.

USP7-Dependent Regulation of TRAF Activation and Signaling by a Viral Interferon Regulatory Factor Homologue.

Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRFs 1 to 4), all of which are expressed during lytic replication and inhibit a variety of antiviral signaling pathways. Viral IRFs 1, 2, and 3 are also expressed during latency in primary effusion lymphoma (PEL) cells, and vIRF-1 and vIRF-3 have been reported to promote PEL cell viability. Viral IRFs 1, 3, and 4 are known to interact with ubiquitin-specific protease 7 (USP7); interactions of vIRF-1 and vIRF-3 with USP7 promote PEL cell viability and regulate productive replication. Here, we report that vIRF-2 also targets USP7, utilizing a PSTS motif matching the USP7 N-terminal domain-binding A/PxxS consensus, but uniquely requires catalytic domain residues for intracellular interaction. In functional and mechanistic analyses, tumor necrosis factor receptor-associated factor (TRAF)-mediated signaling and associated polyubiquitination of TRAFs 3 and 6, specifically, were regulated negatively by USP7 and positively by vIRF-2-USP7 interaction, the latter competing for USP7-TRAF association. Using depletion, depletion-complementation, and targeted mutagenesis approaches, vIRF-2 was determined to promote latent PEL cell viability, likely independently of USP7 interaction, while lytic replication was inhibited by vIRF-2, in part or in whole via USP7 interaction. Together, our data identify a new molecular determinant of USP7 recognition, TRAF3/6-specific targeting by the deubiquitinase, associated activation of these TRAFs by vIRF-2, and activities of vIRF-2 and vIRF-2-USP7 interaction in HHV-8 latent and lytic biology.IMPORTANCE Human herpesvirus 8-encoded IRF homologues were the first to be identified in a virus. Through inhibitory interactions with cellular IRFs and other mediators of antiviral signaling, the vIRFs are believed to be essential for productive replication and also for latency in particular cell types. The deubiquitinase USP7 is a regulator of key cellular pathways, modulates HHV-8 latent and lytic infection, and is targeted by vIRFs 1, 3, and 4. Here, we report that vIRF-2 also interacts with USP7, via a means distinguishable from USP7 interactions with other vIRFs and other proteins, that this interaction modulates antiviral signaling via disruption of USP7 interactions with innate immune signaling proteins TRAF3 and TRAF6, and that vIRF-2 targeting of USP7 regulates HHV-8 productive replication. The presented data are the first to identify vIRF-2 targeting of USP7 and its role in HHV-8 biology, expanding our understanding of the repertoire and importance of virus-host interactions.

Human Herpesvirus 8 Interferon Regulatory Factors 1 and 3 Mediate Replication and Latency Activities via Interactions with USP7 Deubiquitinase.

Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRF-1 to -4) that likely function to suppress innate immune and cellular stress responses through inhibitory interactions with various cellular proteins involved in these activities. It is notable that vIRF-1 and -4 have been reported to interact with the deubiquitinase ubiquitin-specific protease 7 (USP7), substrates of which include p53 and the p53-targeting and -destabilizing ubiquitin E3 ligase MDM2. Structural studies of vIRF-1 and vIRF-4 USP7 binding sequences in association with USP7 have been reported; both involve interactions with N-terminal-domain residues of USP7 via EGPS and ASTS motifs in vIRF-1 and vIRF-4, respectively, but vIRF-4 residues also contact the catalytic site. However, the biological activities of vIRF-1 and vIRF-4 via USP7 interactions are unknown. Here, we report that vIRF-3, which is latently, as well as lytically, expressed in HHV-8-infected primary effusion lymphoma (PEL) cells, also interacts with USP7-via duplicated EGPS motifs-and that this interaction is important for PEL cell growth and viability. The interaction also contributes to suppression of productive virus replication by vIRF-3, which we identify here. We further show that vIRF-1, which is expressed at low levels in PEL latency, promotes latent PEL cell viability and that this activity and vIRF-1-promoted productive replication (reported previously) involve EGPS motif-mediated USP7 targeting by vIRF-1. This study is the first to identify latent and lytic functions of vIRF-1 and vIRF-3, respectively, and to address the biological activities of these vIRFs through their interactions with USP7.IMPORTANCE HHV-8 is associated with Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease; both latent and lytic viral functions are believed to contribute. Viral interferon regulatory factors specified by HHV-8 are thought to be critically important for successful productive replication through suppression of innate immune and stress responses triggered by the lytic cycle. Latently expressed vIRF-3 contributes significantly to PEL cell survival. Here, we identify ubiquitin-specific protease 7 (USP7) deubiquitinase targeting by vIRF-3 (in addition to previously reported USP7 binding by vIRF-1 and vIRF-4); the importance of vIRF-1 and vIRF-3 interactions with USP7 for latent PEL cell growth and viability; and the positive and negative contributions, respectively, of USP7 targeting by vIRF-1 and vIRF-3 to HHV-8 productive replication. This is the first report of the biological importance of vIRF-1 in PEL cell latency, the modulation of productive replication by vIRF-3, and the contributions of vIRF-USP7 interactions to HHV-8 biology.

Metabolic Suppression by 3-Iodothyronamine Induced Muscle Cell Atrophy via Activation of FoxO-Proteasome Signaling and Downregulation of Akt1-S6K Signaling.

The homeostasis of muscle properties depends on both physical and metabolic stresses. Whereas physical stress entails metabolic response for muscle homeostasis, the latter does not necessarily involve the former and may thus solely affect the homeostasis. We here report that metabolic suppression by the hypometabolic agent 3-iodothyronamine (T1AM) induced muscle cell atrophy without physical stress. We observed that the oxygen consumption rate of C2C12 myotubes decreased 40% upon treatment with 75 µM T1AM for 6 h versus 10% in the vehicle (dimethyl sulfoxide) control. The T1AM treatment reduced cell diameter of myotubes by 15% compared to the control (p<0.05). The cell diameter was reversed completely by 9 h after T1AM was removed. The T1AM treatment also significantly suppressed the expression levels of heat shock protein 72 and αB-crystallin as well as the phosphorylation levels of Akt1, mammalian target of rapamycin (mTOR), S6K, forkhead box O1 (FoxO1) and FoxO3. In contrast, the levels of ubiquitin E3 ligase MuRF1 and chymotrypsin-like activity of proteasome were significantly elevated by T1AM treatment. These results suggest that T1AM-mediated metabolic suppression induced muscle cell atrophy via activation of catabolic signaling and inhibition of anabolic signaling.

Obesity-induced Ly6CHigh and Ly6CLow monocyte subset changes abolish post-ischemic limb conditioning benefits in stroke recovery.

Remote limb conditioning (RLC), performed by intermittent interruption of blood flow to a limb, triggers endogenous tolerance mechanisms and improves stroke outcomes. The underlying mechanism for the protective effect involves a shift of circulating monocytes to a Ly6CHigh proinflammatory subset in normal metabolic conditions. The current study investigates the effect of RLC on stroke outcomes in subjects with obesity, a vascular comorbidity. Compared to lean mice, obese stroke mice displayed significantly higher circulating monocytes (monocytosis), increased CD45High monocytes/macrophages infiltration to the injured brain, worse acute outcomes, and delayed recovery. Unlike lean mice, obese mice with RLC at 2 hours post-stroke failed to shift circulating monocytes to pro-inflammatory status and nullified RLC-induced functional benefit. The absence of the monocyte shift was also observed in splenocytes incubated with RLC serum from obese mice, while the shift was observed in the cultures with RLC serum from lean mice. These results showed that the alteration of monocytosis and subsets underlies negating RLC benefits in obese mice and suggest careful considerations of comorbidities at the time of RLC application for stroke therapy.

Endothelial cell CD36 mediates stroke-induced brain injury via BBB dysfunction and monocyte infiltration in normal and obese conditions.

CD36 expressed in multiple cell types regulates inflammation, vascular function, and innate immunity. Specifically, CD36 in microvascular endothelial cells (ECs) signals to elicit inflammation and causes EC death. This study investigated roles for EC-CD36 on acute stroke pathology in normal and obese conditions. Obesity induced by a high-fat diet (HD) selectively increased CD36 expression in ECs, not in monocytes/macrophages, in the post-ischemic brain. Mice deficient CD36 in ECs (ECCD36-/-) showed reduced injury size and vascular permeability in normal conditions. While control mice fed a HD developed obesity and aggravated stroke injury, ECCD36-/- mice were resistant to develop an obesity phenotype. Subjecting ECCD36-/- mice to stroke resulted in reduced injury size and BBB disruption. Moreover, the mice had reduced MCP-1 and CCR2 gene expression, resulting in reduced monocyte trafficking with improved survival and acute motor function. Reduced MCP-1 and CCR2 expression was still evident in ECCD36-/- mice subjected to severe stroke, suggesting that monocyte trafficking is an infarct-independent metabolic effect associated with specific EC-CD36 deletion. Our findings demonstrate the importance of EC-CD36 in developing vascular comorbidities and suggest that targeting EC-CD36 is a potential preventative strategy to normalize vascular risk factors, leading to improved acute stroke outcomes.

Molecular and physiological characterization of the Arabidopsis thaliana Oxidation-related Zinc Finger 2, a plasma membrane protein involved in ABA and salt stress response through the ABI2-mediated signaling pathway.

CCCH-type zinc finger proteins are important for developmental and environmental responses. However, the precise roles of these proteins in plant stress tolerance are poorly understood. Arabidopsis thaliana Oxidation-related Zinc Finger 2 (AtOZF2) (At4g29190) is an AtOZF1 homolog previously isolated from Arabidopsis, which confers oxidative stress tolerance on plants. The AtOZF2 protein is localized in the plasma membrane, as is AtOZF1. Disruption expression of AtOZF2 led to reduced root length and leaf size. AtOZF2 was implicated to be involved in the ABA and salinity responses. atozf2 antisense lines were more sensitive to ABA and salt stress during the seed germination and cotyledon greening processes. In contrast, AtOZF2-overexpressing plants were more insensitive to ABA and salt stress than the wild type. Interestingly, in the presence of ABA and salt stress, the transcript level of ABA insensitive 2 (ABI2), but not that of ABI1, in AtOZF2-overexpressing plants was lower than that in the wild type, whereas the expression of ABI2 in atozf2 was significantly enhanced. Thus, AtOZF2 is involved in the ABA and salt stress response through the ABI2-mediated signaling pathway. Taken together, these findings provide compelling evidence that AtOZF2 is an important regulator for plant tolerance to abiotic stress.

Sustained torpidity following multi-dose administration of 3-iodothyronamine in mice.

Despite significant medical benefits as in space exploration or emergency care, prolonged torpidity of non-hibernator mammals remains unexplored to date. Here, we report that male Institute of Cancer Research mice could sustain two separate 2-day torpor bouts and maintain body temperature of 28-33°C following repeated treatments of 3-iodothyronamine (T(1) AM), a natural derivative of thyroid hormone. A 1-day interbout arousal period, adopted to mimic the behavior of true hibernators, seemed critical for the subjects to restore physiological homeostasis. Molecular studies of neuron-specific enolase, S100 calcium binding protein B and heat shock protein 72 suggested that the brain maintains functional and cytoprotective activities during sustained torpidity. Together, the results of this study propose a practical protocol using a torpor-arousal cycle that can be applied to the extreme medical situations.

The R-R-type MYB-like transcription factor, AtMYBL, is involved in promoting leaf senescence and modulates an abiotic stress response in Arabidopsis.

Functional analysis of a putative novel transcription factor Arabidopsis MYB-like protein designated AtMYBL, which contains two predicted DNA-binding domains, was performed. The physiological role of the R-R-type MYB-like transcription factor has not been reported in any plant. Analyses of an AtMYBL promoter-ß-glucuronidase (GUS) construct revealed substantial gene expression in old leaves and induction of GUS activity by ABA and salt stress. AtMYBL-overexpressing plants displayed a markedly enhanced leaf senescence phenotype. Moreover, the ectopic expression of the AtMYBL gene was very significantly influential in senescence parameters including Chl content, membrane ion leakage and the expression of senescence-related genes. Although the seed germination rate was improved under ABA and saline stress conditions in the AtMYBL-overexpressing plants, decreased salt tolerance was evident compared with the wild type and atmybl RNA interference lines during later seedling growth when exposed to long-term salt stress, indicating that AtMYBL protein is able to developmentally regulate stress sensitivity. Furthermore, AtMYBL protein activated the transcription of a reporter gene in yeast. Green fluorescent protein-tagged AtMYBL was localized in the nuclei of transgenic Arabidopsis cells. Taken together, these results suggest that AtMYBL functions in the leaf senescence process, with the abiotic stress response implicated as a putative potential transcription factor.

Physiological characterization of the Arabidopsis thaliana oxidation-related zinc finger 1, a plasma membrane protein involved in oxidative stress.

The CCCH-type zinc finger proteins are a superfamily containing tandem zinc-binding motifs involved in many aspects of plant growth and development. However, the precise role of these proteins involved in plant stress tolerance is poorly understood. This study was to examine the regulatory and functional role of the CCCH-type zinc finger protein, AtOZF1 (At2g19810), under oxidative stress. Interestingly, the AtOZF1 protein was localized in the plasma membrane. The AtOZF1 transcripts were highly induced by treatment with hydrogen peroxide, abscisic acid and salinity. The AtOZF1-overexpressing plants were relatively resistant to oxidative stress than wild-type and T-DNA insertion mutant atozf1. Malondialdehyde, a decomposition product of lipid peroxidation, accumulated in atozf1 mutants more than in wild-type and AtOZF1-overexpressing plants. Furthermore, atozf1 mutants displayed lower activities of catalase and guaiacol peroxidase, higher chlorosis, and down-regulated expression of antioxidant genes under oxidative stress. Taken together, these observations demonstrate that AtOZF1 is required for the tolerance of Arabidopsis to oxidative stress.

Molecular mechanism underlying muscle mass retention in hibernating bats: role of periodic arousal.

Hibernators like bats show only marginal muscle atrophy during prolonged hibernation. The current study was designed to test the hypothesis that hibernators use periodic arousal to increase protein anabolism that compensates for the continuous muscle proteolysis during disuse. To test this hypothesis, we investigated the effects of 3-month hibernation (HB) and 7-day post-arousal torpor (TP) followed by re-arousal (RA) on signaling activities in the pectoral muscles of summer-active (SA) and dormant Murina leucogaster bats. The bats did not lose muscle mass relative to body mass during the HB or TP-to-RA period. For the first 30-min following arousal, the peak amplitude and frequency of electromyographic spikes increased 3.1- and 1.4-fold, respectively, indicating massive myofiber recruitment and elevated motor signaling during shivering. Immunoblot analyses of whole-tissue lysates revealed several principal outcomes: (1) for the 3-month HB, the phosphorylation levels of Akt1 (p-Akt1) and p-mTOR decreased significantly compared to SA bats, but p-FoxO1 levels remained unaltered; (2) for the TP-to-RA period, p-Akt1 and p-FoxO1 varied little, while p-mTOR showed biphasic oscillation; (3) proteolytic signals (i.e., atrogin-1, MuRF1, Skp2 and calpain-1) remained constant during the HB and TP-to-RA period. These results suggest that the resistive properties of torpid bat muscle against atrophy might be attained primarily by relatively constant proteolysis in combination with oscillatory anabolic activity (e.g., p-mTOR) corresponding to the frequency of arousals occurring throughout hibernation.

Kaposi's Sarcoma-Associated Herpesvirus Drives a Super-Enhancer-Mediated Survival Gene Expression Program in Primary Effusion Lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). The cellular transcription factor (TF) interferon (IFN) regulatory factor 4 (IRF4) is an essential oncogene in PEL, but its specific role in PEL and how KSHV deregulates IRF4 remain unknown. Here, we report that the KSHV latency protein viral interferon regulatory factor 3 (vIRF3) cooperates with IRF4 and cellular BATF (basic leucine zipper ATF-like TF) to drive a super-enhancer (SE)-mediated oncogenic transcriptional program in PEL. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) experiments demonstrated that IRF4, vIRF3, and BATF cooccupy the SEs of key survival genes, in a pattern that is distinct from those seen with other IRF4-driven malignancies. All three proteins cooperatively drive SE-mediated IRF4 overexpression. Inactivation of vIRF3 and, to a lesser extent, BATF phenocopies the gene expression changes and loss of cellular viability observed upon inactivation of IRF4. In sum, this work suggests that KSHV vIRF3 and cellular IRF4 and BATF cooperate as oncogenic transcription factors on SEs to promote cellular survival and proliferation in KSHV-associated lymphomas.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes the aggressive disease primary effusion lymphoma (PEL). Here, we show that a viral transcription factor (vIRF3) cooperates with the cellular transcription factor IRF4 to control an oncogenic gene expression program in PEL cells. These proteins promote KSHV-mediated B cell transformation by activating the expression of prosurvival genes through super-enhancers. Our report thus demonstrates that this DNA tumor virus encodes a transcription factor that functions with cellular IRF4 to drive oncogenic transcriptional reprogramming.

Stress and signaling responses of rat skeletal muscle to brief endurance exercise during hindlimb unloading: a catch-up process for atrophied muscle.

At times, exercise accompanied by its anabolic effects is not a tractable countermeasure to muscle atrophy. Instead, training is often attempted after the affected muscle has atrophied greatly as a result of unloading. This study was designed to elucidate stress and signaling mechanisms underlying a process of muscle catch-up growth as a result of transitory exercise during unloading. Rats were exercised daily with a routine of 20- or 40-minute treadmill running (at 60% of maximum oxygen uptake) during the second week of a two-week hindlimb suspension. We examined the expression and activation of heat shock proteins and anabolic and proteolytic markers in the rat soleus muscle. Muscle mass relative to body mass decreased 2.4-fold in the unloaded group (HU) with respect to controls but decreased only 1.7-fold in the 40-min trained group (HT40) (P < 0.05) - equivalent to a 1.4-fold increase in the relative muscle mass over HU. Immunoblotting analyses on whole-tissue lysates demonstrated the following: (1) HSP72 and alphaB-crystallin were upregulated 7- and 2.5-fold, respectively, in HT40 versus HU; (2) phosphorylation of Akt1 and p70/S6K decreased only slightly in HU; (3) when compared to HU, HT40 phosphorylation of Akt1, S6K, and FoxO1 increased 1.4- to 3.0-fold while phosphorylation of FoxO3 was unchanged; and (4) activities of the ubiquitin E3 ligases, calpain 1 and caspase-3 increased 2- to 4-fold in the unloaded groups regardless of exercise duration. These results suggest that the significant upregulation of chaperones and anabolic markers (e.g., HSP72, p-Akt1, p-S6K) in HT40, along with the lack of the training effect on proteolytic activity, is likely crucial for muscle mass catch-up in the unloaded muscle.

A basic helix-loop-helix 104 (bHLH104) protein functions as a transcriptional repressor for glucose and abscisic acid signaling in Arabidopsis.

Transduction of glucose (Glc) signaling is critical for plant development, metabolism, and stress responses. However, identifying initial Glc sensing and response stimulating mechanisms in plants has been difficult due to dual functions of glucose as energy sources and signaling component. A basic Helix-Loop-Helix 104 (bHLH104) protein is a homolog of bHLH34 previously isolated from Arabidopsis that functions as a transcriptional activator of Glc and abscisic acid (ABA) responses. In this study, we characterized bHLH104 as a transcription factor that binds to the regulatory region of Arabidopsis Plasma membrane Glc-responsive Regulator (AtPGR) gene. The bHLH104 binds to 5'-AANA-3' element of the promoter region of AtPGR in vitro and represses beta-glucuronidase (GUS) activity in AtPGR promoter-GUS transgenic plants. Genetic approaches show that bHLH104 positively regulates Glc and abscisic acid (ABA) response. These results suggest that bHLH104 is involved in Glc- and ABA-mediated signaling pathway. Taken together, these findings provide evidence that bHLH104 is an important transcription regulator in plant-sensitivity to Glc and ABA signaling.

Behavioral hypothermia of a domesticated lizard under treatment of the hypometabolic agent 3-iodothyronamine.

Ectothermic animals rely on behavioral thermoregulation due to low capacity of heat production and storage. Previously, lizards were shown to achieve 'fever' during microbial infection by increasing their preferred body temperature (PBT) behaviorally, thereby attaining a relatively high survival rate. The purpose of this study was to investigate whether domesticated lizards pursued 'behavioral hypothermia' induced by a hypometabolic agent 3-iodothyronamine (T1AM). We found that treatment with 8.0 mg/kg T1AM caused a lizard species, the leopard gecko (Eublepharis macularius), to decrease its ventilation and oxygen consumption rates 0.64- and 0.76-fold, respectively, compared to those of the control (P<0.05). The lizards, habituated at an ambient temperature of 30 ± 0.5°C, also showed a significant decrease in the PBT range over a freely accessible thermal gradient between 5°C and 45°C. The upper limit of the PBT in the treated lizards lowered from 31.9°C to 30.6°C, and the lower limit from 29.5°C to 26.3°C (P<0.001). These findings demonstrate that the treated lizards pursued behavioral hypothermia in conjunction with hypoventilation and hypometabolism. Because prior studies reported a similar hypometabolic response in T1AM-injected laboratory mice, the domesticated lizards, as a part of the vertebrate phylogeny, may be a useful laboratory model for biological and pharmacological researches such as drug potency test.

Arabidopsis Basic Helix-Loop-Helix 34 (bHLH34) Is Involved in Glucose Signaling through Binding to a GAGA Cis-Element.

The modulation of glucose (Glc) homeostasis and signaling is crucial for plant growth and development. Nevertheless, the molecular signaling mechanism by which a plant senses a cellular Glc level and coordinates the expression of Glc-responsive genes is still incompletely understood. Previous studies have shown that Arabidopsis thaliana plasma membrane Glc-responsive regulator (AtPGR) is a component of the Glc-responsive pathway. Here, we demonstrated that a transcription factor bHLH34 binds to 5'-GAGA-3' element of the promoter region of AtPGR in vitro, and activates beta-glucuronidase (GUS) activity upon Glc treatment in AtPGR promoter-GUS transgenic plants. Gain- and loss-of-function analyses suggested that the bHLH34 involved in the responses to not only Glc, but also abscisic acid (ABA) and salinity. These results suggest that bHLH34 functions as a transcription factor in the Glc-mediated stress responsive pathway as well as an activator of AtPGR transcription. Furthermore, genetic experiments revealed that in Glc response, the functions of bHLH34 are different from that of a bHLH104, a homolog of bHLH34. Collectively, our findings indicate that bHLH34 is a positive regulator of Glc, and may affect ABA or salinity response, whereas bHLH104 is a negative regulator and epistatic to bHLH34 in the Glc response.

Regulation of Arabidopsis thaliana plasma membrane glucose-responsive regulator (AtPGR) expression by A. thaliana storekeeper-like transcription factor, AtSTKL, modulates glucose response in Arabidopsis.

Biochemical, genetic, physiological, and molecular research in plants has demonstrated a central role of glucose (Glc) in the control of plant growth, metabolism, and development, and has revealed networks that integrate light, stresses, nutrients, and hormone signaling. Previous studies have reported that AtPGR protein as potential candidates for Glc signaling protein. In the present study, we characterized transcription factors that bind to the upstream region of the AtPGR gene isolated using the yeast one-hybrid screening with an Arabidopsis cDNA library. One of the selected genes (AtSTKL) appeared to confer elevated sensitivity to Glc response. Overexpression of AtSTKLs (AtSTKL1 and AtSTKL2) increased the sensitivity to Glc during the post-germination stages. In contrast, atstkl1 and atstkl2 antisense lines displayed reduced sensitivity to high Glc concentration during the early seedling stage. Furthermore, we showed that the two AtSTKLs bind to the 5'-GCCT-3' element of the upstream promoter region of the AtPGR gene in vitro and repress the beta-glucuronidase (GUS) activity in AtPGR promoter-GUS (P999-GUS) transgenic plants. Green fluorescent protein (GFP)-tagged AtSTKLs were localized in the nuclei of transgenic Arabidopsis cells. Collectively, these results suggest that AtSTKL1 and AtSTKL2 function both as repressors of AtPGR transcription and as novel transcription factors in the Glc signaling pathway.

Conessine Interferes with Oxidative Stress-Induced C2C12 Myoblast Cell Death through Inhibition of Autophagic Flux.

Conessine, a steroidal alkaloid isolated from Holarrhena floribunda, has anti-malarial activity and interacts with the histamine H3 receptor. However, the cellular effects of conessine are poorly understood. Accordingly, we evaluated the involvement of conessine in the regulation of autophagy. We searched natural compounds that modulate autophagy, and conessine was identified as an inhibitor of autophagic flux. Conessine treatment induced the formation of autophagosomes, and p62, an autophagic adapter, accumulated in the autophagosomes. Reactive oxygen species such as hydrogen peroxide (H2O2) result in muscle cell death by inducing excessive autophagic flux. Treatment with conessine inhibited H2O2-induced autophagic flux in C2C12 myoblast cells and also interfered with cell death. Our results indicate that conessine has the potential effect to inhibit muscle cell death by interfering with autophagic flux.

Heterologous expression of the gourd E3 ubiquitin ligase gene LsRZF1 compromises the drought stress tolerance in Arabidopsis thaliana.

Protein ubiquitination is one of the major regulatory processes used by eukaryotic cells. The ubiquitin E3 ligase acts as a main determinant of substrate specificity. However, the precise roles of E3 ligase in plants to drought stress are poorly understood. In this study, a gourd family (Lagenaria siceraria) ortholog of Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1) gene, designated LsRZF1, was identified and characterized. LsRZF1 was reduced by abscisic acid (ABA), osmotic stress, and drought conditions. Compared to wild type, transgenic Arabidopsis plants ectopic expressing LsRZF1 were hypersensitive to ABA and osmotic stress during early seedling development, indicating that LsRZF1 negatively regulates drought-mediated control of early seedling development. Moreover, the ectopic expression of the LsRZF1 gene was very influential in drought sensitive parameters including proline content, water loss, and the expression of dehydration stress-related genes. Furthermore, ubiquitin E3 ligase activity and genetic data indicate that AtRZF1 and LsRZF1 function in similar pathway to control proline metabolism in Arabidopsis under drought condition. Together, these results suggest that the E3 ligase LsRZF1 is an important regulator of water deficit stress during early seedling development.