Background-Since its discovery in the early 1980s, O-linked-β-N-acetylglucosamine (OGlcNAc), a single sugar modification on the hydroxyl group of serine or threonine residues, has changed our views of protein glycosylation. While other forms of protein glycosylation modify proteins on the cell surface or within luminal compartments of the secretory machinery, O-GlcNAc modifies myriad nucleocytoplasmic proteins. GlcNAcylated proteins are involved in transcription, ubiquitination, cell cycle, and stress responses. GlcNAcylation is similar to protein phosphorylation in terms of stoichiometry, localization and cycling. To date, only two enzymes are known to regulate GlcNAcylation in mammals: O-GlcNAc transferase (OGT), which catalyzes the addition of OGlcNAc, and β-N-acetylglucosaminidase (O-GlcNAcase), a neutral hexosaminidase responsible for O-GlcNAc removal. OGT and O-GlcNAcase are regulated by RNA splicing, by nutrients, and by post-translational modifications. Their specificities are controlled by many transiently associated targeting subunits. As methods for detecting O-GlcNAc have improved our understanding of OGlcNAc's functions has grown rapidly.

SUMMARY Primary sensory neurons in the DRG play an essential role in initiating pain by detecting painful stimuli in the periphery. Tissue injury can sensitize DRG neurons, causing heightened pain sensitivity, often leading to chronic pain. Despite the functional importance, how DRG neurons function at a population level is unclear due to the lack of suitable tools. Here we developed an imaging technique that allowed us to simultaneously monitor the activities of >1,600 neurons/DRG in live mice and discovered a striking neuronal coupling phenomenon that adjacent neurons tend to activate together following tissue injury. This coupled activation occurs among various neurons and is mediated by an injury-induced upregulation of gap junctions in glial cells surrounding DRG neurons. Blocking gap junctions attenuated neuronal coupling and mechanical hyperalgesia. Therefore, neuronal coupling represents a new form of neuronal plasticity in the DRG and contributes to pain hypersensitivity by “hijacking” neighboring neurons through gap junctions.

SUMMARY The peripheral terminals of primary nociceptive neurons play an essential role in pain detection mediated by membrane receptors like TRPV1, a molecular sensor of heat and capsaicin. However, the contribution of central terminal TRPV1 in the dorsal horn to chronic pain has not been investigated directly. Combining primary sensory neuron-specific GCaMP3 imaging with a trigeminal neuropathic pain model, we detected robust neuronal hyperactivity in injured and uninjured nerves in the skin, soma in trigeminal ganglion, and central terminals in the spinal trigeminal nucleus. Extensive TRPV1 hyperactivity was observed in central terminals innervating all dorsal horn laminae. The central terminal TRPV1 sensitization was maintained by descending serotonergic (5-HT) input from the brainstem. Central blockade of TRPV1 or 5-HT/5-HT3A receptors attenuated central terminal sensitization, excitatory primary afferent inputs, and mechanical hyperalgesia in the territories of injured and uninjured nerves. Our results reveal new central mechanisms facilitating central terminal sensitization underlying chronic pain.

Recent findings suggest a pivotal role for mitochondria-associated hexokinase in the regulation of apoptosis in animal cells. In this study, virus-induced gene silencing (VIGS) of a hexokinase-encoding Hxk1 caused necrotic lesions on leaves, abnormal leaf morphology, and retarded plant growth in Nicotiana benthamiana. Hxk1 was associated with the mitochondria, and this association required the N-terminal membrane anchor. VIGS of Hxk1 reduced the cellular glucose-phosphorylating activity to ;31% of control levels without changing the fructose-phosphorylating activity and did not alter hexose phosphate content severely. The affected cells showed programmed cell death (PCD) morphological markers, including nuclear condensation and DNA fragmentation. Similar to animal cell apoptosis, cytochrome c was released into the cytosol and caspase-9-and caspase-3-like proteolytic activities were strongly induced. Furthermore, based on flow cytometry, Arabidopsis thaliana plants overexpressing Arabidopsis HXK1 and HXK2, both of which are predominantly associated with mitochondria, exhibited enhanced resistance to H 2 O 2 -and a-picolinic acid-induced PCD. Finally, the addition of recombinant Hxk1 to mitochondriaenriched fractions prevented H 2 O 2 /clotrimazole-induced cytochrome c release and loss of mitochondrial membrane potential. Together, these results show that hexokinase critically regulates the execution of PCD in plant cells, suggesting a link between glucose metabolism and apoptosis.

OBJECTIVE-O-linked N-acetylglucosamine (O-GlcNAc) is upregulated in diabetic tissues and plays a role in insulin resistance and glucose toxicity. Here, we investigated the extent of GlcNAcylation on human erythrocyte proteins and compared site-specific GlcNAcylation on erythrocyte proteins from diabetic and normal individuals.RESEARCH DESIGN AND METHODS-GlcNAcylated erythrocyte proteins or GlcNAcylated peptides were tagged and selectively enriched by a chemoenzymatic approach and identified by mass spectrometry. The enrichment approach was combined with solid-phase chemical derivatization and isotopic labeling to detect O-GlcNAc modification sites and to compare site-specific O-GlcNAc occupancy levels between normal and diabetic erythrocyte proteins. RESULTS-The enzymes that catalyze the cycling (addition and removal) of O-GlcNAc were detected in human erythrocytes. Twenty-five GlcNAcylated erythrocyte proteins were identified. Protein expression levels were compared between diabetic and normal erythrocytes. Thirty-five O-GlcNAc sites were reproducibly identified, and their site-specific O-GlcNAc occupancy ratios were calculated. CONCLUSIONS-GlcNAcylation is differentially regulated at individual sites on erythrocyte proteins in response to glycemic status. These data suggest not only that site-specific O-GlcNAc levels reflect the glycemic status of an individual but also that O-GlcNAc site occupancy on erythrocyte proteins may be eventually useful as a diagnostic tool for the early detection of diabetes. Diabetes 58:309-317, 2009

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Bcl-2 is involved in the progression of human malignancies, but the precise role and mechanism of Bcl-2 for tumor invasion and metastasis remains unclear. In this study, we have investigated the role and mechanism of Bcl-2 on tumor cell invasion and metastasis by using Bcl-2 overexpressing non-small cell lung cancer cells. Matrix metalloproteinases (MMPs) are important proteins involved in the processes of tumor invasion and metastasis. In vitro Matrigel invasion assays showed that Bcl-2 overexpression increased tumor cell invasion by 15-fold. Moreover, Bcl-2 overexpression enhanced in vivo lung metastasis by 4-fold. Consistent with its effect on invasion and metastasis, Bcl-2 overexpression induced not only MMP-2 mRNA and its protein expression, but this also activated the pro-MMP-2 protein to its active form. To explore the induction mechanism of MMP-2 by Bcl-2, we investigated the effects of Bcl-2 overexpression on MMP-2 transcriptional regulation. Nuclear run-on assays showed a 6-fold increase in the transcription rate of MMP-2 mRNA in the Bcl-2 transfectants (H157/Bcl-2) compared with that of the H157/vector control cells (H157/C). Overexpression of Bcl-2 induced the nuclear transcription factor activator protein 1 family, including the c-Jun, JunD, c-Fos, FosB, and Fra-1 proteins. Reporter assays combined with deletion mutagenesis analysis and gel shift assays showed the involvement of activator protein 1 in the activation of MMP-2 promoter activity by Bcl-2. Taken together, we have shown that Bcl-2 promotes tumor invasion and lung metastasis by inducing MMP-2 gene expression through the combined action of transcriptional and posttranslational mechanisms. (Cancer Res 2005; 65(13): 5554-60)

Induction of glucose-regulated proteins (GRPs) is a ubiquitous intracellular response to stresses such as hypoxia, glucose starvation and acidosis. The induction of GRPs offers some protection against these stresses in vitro, but the specific role of GRPs in vivo remains unclear. Hibernating bats present a good in vivo model to address this question. The bats must overcome local high oxygen demand in tissue by severe metabolic stress during arousal thermogenesis. We used brain tissue of a temperate bat Rhinolopus ferrumequinum to investigate GRP induction by high metabolic oxygen demand and to identify associated signaling molecules. We found that during 30 min of arousal, oxygen consumption increased from nearly zero to 11.9/kg/h, which was about 8.7-fold higher than its active resting metabolic rate. During this time, body temperature rose from 7°C to 35°C, and levels of TNF-a and lactate in brain tissue increased 2-2.5-fold, indicating a high risk of oxygen shortage. Concomitantly, levels of GRP75, GRP78 and GRP94 increased 1.5-1.7-fold. At the same time, c-Jun N-terminal protein kinase (JNK) activity increased 6.4-fold, and extracellular signal-regulated protein kinase (ERK) activity decreased to a similar degree (6.1-fold). p38 MAPK activity was very low and remained unchanged during arousal. In addition, survival signaling molecules protein kinase B (Akt) and protein kinase C (PKC) were activated 3-and 5-fold, respectively, during arousal. Taken together, our results showed that bat brain undergoes high oxygen demand during arousal from hibernation. Up-regulation of GRP proteins and activation of JNK, PKCc and Akt may be critical for neuroprotection and the survival of bats during the repeated process. Keywords: arousal from hibernation, metabolic oxygen demand, stress protein, survival signaling kinase.