Low oxygen levels have shown to promote self-renewal in many stem cells. In tumors, hypoxia is associated with aggressive disease course and poor clinical outcomes. Furthermore, many aggressive tumors have shown to display gene expression signatures characteristic of human embryonic stem cells (hESC). We now tested whether hypoxia might be responsible for the hESC signature observed in aggressive tumors. We show that hypoxia, through hypoxia inducible factor (HIF), can induce a hESC-like transcriptional program, including the iPSC inducers, OCT4, NANOG, SOX2, KLF4, cMYC and miRNA-302 in eleven cancer cell lines (from prostate, brain, kidney, cervix, lung, colon, liver and breast tumors). Further, non-degradable forms of HIFα, combined with the traditional iPSC inducers are highly efficient in generating A549 iPSC-like colonies that have high tumorigenic capacity. To test potential correlation between iPSC inducers and HIF expression in primary tumors, we analyzed primary prostate tumors and found a significant correlation between NANOG-, OCT4- and HIF1α-positive regions. Further, NANOG and OCT4 expression positively correlated with increased prostate tumor Gleason score. In primary glioma-derived CD133 negative cells neurospheres and hESC markers were induced in hypoxia but not in normoxia. Together, these findings suggest that HIF targets may act as key inducers of a dynamic state of stemness in pathological conditions.

Zinc transporter-3 (ZnT-3), a member of a growing family of mammalian zinc transporters, is expressed in regions of the brain that are rich in histochemically reactive zinc (as revealed by the Timm's stain), including entorhinal cortex, amygdala, and hippocampus. ZnT-3 protein is most abundant in the zinc-enriched mossy fibers that project from the dentate granule cells to hilar and CA3 pyramidal neurons. We show here by electron microscopy that ZnT-3 decorates the membranes of all clear, small, round synaptic vesicles (SVs) in the mossy fiber boutons of both mouse and monkey. Furthermore, up to 60-80% of these SVs contain Timm'sstainable zinc. The coincidence of ZnT-3 on the membranes of SVs that accumulate zinc, and its homology with known zinc transporters, suggest that ZnT-3 is responsible for the transport of zinc into SVs, and hence for the ability of these neurons to release zinc upon excitation.Most of the zinc in the mammalian brain is associated with metalloproteins; however, there is also a pool of histochemically reactive zinc that exists in synaptic vesicles (SVs) of a subset of glutamatergic neurons, which has led to classification of these neurons as zinc-containing or zinc-ergic (1-4). Pathways utilizing this vesicular form of zinc have been mapped using histochemical stains such as the neo-Timm's sulfidesilver method (5), selenium stain (6, 7), and the fluorescent compound, TSQ (8). One of the best described zinc-ergic systems is found in the rodent hippocampal formation, where vesicular zinc can be detected in each component of the trisynaptic circuit that includes (i) perforant path projections from the entorhinal cortex to the outer molecular layer of the dentate gyrus, (ii) mossy fiber (MF) projections from granule cells in the dentate gyrus to hilar neurons and pyramidal cells in the CA3 region (4, 9-11), (iii) projections from CA3 pyramidal neurons to neurons in the CA1 region, and (iv) projections from CA1 to subiculum (3, 4). Electron microscopy (EM) has revealed that the Timm's stain precipitate is present within SVs in the giant axonal boutons of the MFs in the hilus and stratum (s.) lucidum (1, 2, 12). However, only Ϸ10-15% of the SVs within a given bouton have been shown to contain Timm's precipitate (2); thus, it has been difficult to ascertain whether zinc is present in a subset of vesicles or if it is present in the same vesicles as glutamate.Accumulation of zinc within SVs presumably depends on the action of specific transporters, by analogy with the accumulation of other neurotransmitters in SVs (13). A gene, designated zinc transporter-3 (ZnT3), homologous to two established zinc transporters (14, 15) was recently cloned (16). ZnT-3 was shown by in situ hybridization to be expressed at high levels in hippocampus and neocortex. Immunocytochemical studies demonstrated its localization to the MFs, where the histochemical Timm's reaction has revealed zinc-containing SVs. This profile suggested that ZnT-3 might be the vesicular zinc transporter responsible for sequestration ...

No abstract

_Studies of the avian auditory sytem indicate that neurons in nucleus magnocellularis (NM) and nucleus laminaris of young animals are dramatically altered by changes in the auditory receptor. We examined the role of presynaptic activity on these transneuronal regulatory events. TTX was used to block action potentials in the auditory nerve. TTX injections into the perilymph reliably blocked all neuronal activity in the cochlear nerve and NM. Far-field recordings of sound-evoked potentials revealed that responses returned within 6-12 hr after a single TTX injection. Changes in protein synthesis by NM neurons were measured by determining the incorporation of 3H-leucine using autoradiography.NM neurons on the side of the brain ipsilateral to the TTX injection were compared to normally active cells on the other side of the same tissue section.Grain counts over individual neurons revealed that a single injection of TTX produced a 40% decrease in grain density in ipsilateral NM neurons within 1.5 hr after the TTX injection. However, by 24 hr after a single TTX injection, grain densities were not different on the 2 sides of the brain. Continuous activity blockade for 6 hr caused the cessation of amino acid incorporation in a portion of NM neurons and a 15-20% decrease in the remaining neurons. These changes in amino acid incorporation are comparable to those following complete removal of the cochlea (Steward and Rubel, 1965). We also examined NM for neuron loss and soma shrinkage after blocking eighth nerve action potentials.TTX injected every 12 hr for 46 hr caused a 20% neuron loss and an 6% shrinkage of the remaining neurons. Similar reductions were found following cochlea removal (Born and Rubel, 1965). It is concluded that neuronal activity plays a major role in the maintenance of normal NM neurons. Furthermore, these results

The zebrafish, a genetically tractable model vertebrate, is naturally susceptible to tuberculosis caused by Mycobacterium marinum, a close genetic relative of the causative agent of human tuberculosis, Mycobacterium tuberculosis. We previously developed a zebrafish embryo-M. marinum infection model to study host-pathogen interactions in the context of innate immunity. Here, we have constructed a flowthrough fish facility for the large-scale longitudinal study of M. marinum-induced tuberculosis in adult zebrafish where both innate and adaptive immunity are operant. We find that zebrafish are exquisitely susceptible to M. marinum strain M. Intraperitoneal injection of five organisms produces persistent granulomatous tuberculosis, while the injection of ϳ9,000 organisms leads to acute, fulminant disease. Bacterial burden, extent of disease, pathology, and host mortality progress in a time-and dose-dependent fashion. Zebrafish tuberculous granulomas undergo caseous necrosis, similar to human tuberculous granulomas. In contrast to mammalian tuberculous granulomas, zebrafish lesions contain few lymphocytes, calling into question the role of adaptive immunity in fish tuberculosis. However, like rag1 mutant mice infected with M. tuberculosis, we find that rag1 mutant zebrafish are hypersusceptible to M. marinum infection, demonstrating that the control of fish tuberculosis is dependent on adaptive immunity. We confirm the previous finding that M. marinum ⌬RD1 mutants are attenuated in adult zebrafish and extend this finding to show that ⌬RD1 predominantly produces nonnecrotizing, loose macrophage aggregates. This observation suggests that the macrophage aggregation defect associated with ⌬RD1 attenuation in zebrafish embryos is ongoing during adult infection.

Hippocampal GABA and glutamate transporter IR differ in TLE patients compared with autopsies. These data support the hypothesis that excitatory and inhibitory neurotransmission and seizure susceptibility could be altered by neuronal and glial transporters in TLE patients.

The lack of an adequate model of post-traumatic epilepsy (PTE), in which, similarly to the human condition, chronic spontaneous focal seizures follow a single episode of traumatic brain injury, has hampered the identification of clinically relevant epileptogenic mechanisms and the development of effective therapies. We studied the electrophysiological, behavioural and structural consequences of a clinically relevant model of closed head injury, the lateral fluid percussion injury (FPI), in the rat. We found that a single episode of severe FPI is sufficient to cause PTE. Chronic electrocorticography (ECoG) demonstrated spontaneous chronic seizures that were partial, originated from the neocortex at the site of injury, and progressively worsened and spread over time. The cases of epilepsy in the post-traumatic population increased over time following injury. Post-FPI epileptic rats exhibited pauses in their behaviour, facial automatisms and myoclonus at the time of epileptiform ECoG events. In vitro local field potential recordings demonstrated persistent hyperexcitability of the neocortex at and around the site of injury that was associated with intense glial reactivity. These results for the first time demonstrate persistent hyperexcitability of the injured neocortex and define a useful model for pathophysiological studies of basic mechanisms of spontaneous epileptogenesis and for preclinical screening of effective antiepileptogenic drugs.

We recently described an in vivo model of post-traumatic epilepsy (PTE) in the rat where chronic spontaneous recurrent seizures appear following a single episode of fluid percussion injury (FPI). PTE, studied during the first 2 months post-injury, was focal and seizures originated predominantly from the frontal-parietal neocortex at or around the injury site. However, rarer bilateral seizures originating from a different and undefined focus were also observed. To shed light on the Posttraumatic Epileptogenic mechanisms and on the generation of bilateral seizures, we studied rats up to 7 months post-injury. In vivo paired epidural and depth-electrode recordings indicated that the anterior hippocampus evolves into an epileptic focus which initiates bilateral seizures. The rate of frontal-parietal seizures remained constant over time after 2 weeks post-injury, while the rate of hippocampal seizures greatly increased over time, suggesting that different mechanisms mediate neocortical and hippocampal post-traumatic epileptogenesis. Because of different temporal evolution of these foci, the epileptic syndrome was characterized by predominant frontal-parietal seizures early after injury, but by predominant mesio-temporal seizures at later time points. Pathological analysis demonstrated progressive hippocampal and temporal cortex pathology that paralleled the increase in frequency and duration of bilateral seizures. These results demonstrate that FPI-induced frontal-parietal epilepsy (FPE) progresses to mesial-temporal lobe epilepsy (MTLE) with dual pathology. These observations establish numerous similarities between FPI-induced and human PTE and further validate it as a clinically relevant model of PTE.