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1.  Medial Septum-Diagonal Band of Broca (MSDB) GABAergic Regulation of Hippocampal Acetylcholine Efflux Is Dependent on Cognitive Demands 
The Journal of Neuroscience  2014;34(2):506-514.
The septohippocampal pathway contains cholinergic, GABAergic, and glutamatergic projections and has an established role in learning, memory, and hippocampal theta rhythm. Both GABAergic and cholinergic neurons in the medial septum-diagonal band of Broca (MSDB) have been associated with spatial memory, but the relationship between the two neuronal populations is not fully understood. The present study investigated the effect of selective GABAergic MSDB lesions on hippocampal acetylcholine (ACh) efflux and spatial memory during tasks that varied in memory demand. Male Sprague Dawley rats were given GABAergic lesions of the MSDB using GAT1-saporin (GAT1-SAP) and examined on spontaneous exploration (Experiment 1) and non-matching to position without (NMTP; Experiment 2) and with a delay (DNMTP; Experiment 3), while concurrently using in vivo microdialysis to measure hippocampal ACh efflux. Intraseptal GAT1-SAP treatment did not alter baseline or behaviorally stimulated hippocampal ACh efflux or maze exploration (Experiment 1). Moreover, GAT1-SAP did not alter evoked hippocampal ACh efflux related to NMTP nor did it impair working memory in NMTP (Experiment 2). In contrast, both ACh efflux and performance in DNMTP were impaired by intraseptal GAT1-SAP. Thus, GABAergic MSDB neurons are important for spatial working memory and modulate hippocampal ACh efflux under conditions of high memory load. The relationship between the septohippocampal cholinergic and GABAergic systems and working memory will be discussed.
PMCID: PMC3870934  PMID: 24403150
GAT1-saporin; hippocampus; learning; microdialysis; T-maze; working memory
2.  Glial Fibrillary Acidic Protein as a Biomarker for Periventricular White Matter Injury 
Periventricular white matter injury (PWMI), a precursor of cerebral palsy, is traditionally not diagnosed until 6 weeks of life by head ultrasound. We sought to determine if early neonatal glial fibrillary acidic protein (GFAP) levels could identify PWMI in low birth weight (< 2500 grams) infants.
Each case with PWMI on head ultrasound at 6 weeks from 4/09-4/11 was matched by gestational age and mode of delivery to 2 subsequent neonates with a normal head ultrasound. GFAP was measured in cord blood at birth, at neonatal intensive care unit (NICU) admission, and daily on days 1–4 of life.
During this 2 year period, 21 cases with PWMI with gestational age 27.4±3.3 weeks were compared to 42 controls. The incidence of cesarean delivery was 61.9% in both groups. GFAP was not significantly different in cord blood or at NICU admission, but was significantly elevated on day 1 (median, 5%–95%; 0, 0–0.98 ng/mL cases; 0,0–0.06 ng/mL controls, P=0.03), day 2 (0, 0–1.21 ng/mL; 0, 0–0.05 ng/mL; P=0.02), day 3 (0.05, 0–0.33 ng/mL; 0, 0–0.04 ng/mL, P=0.004) and day 4 (0.02, 0–1.03 ng/mL; 0, 0–0.05 ng/mL, P<0.001). The odds of developing PWMI significantly increased with increasing levels of GFAP from day 1 to day 4 of life adjusting for preeclampsia, antenatal steroid administration and neonatal chronic lung disease.
The ability to predict PWMI with a blood test for GFAP shortly after birth opens the possibility for rapid identification of infants for early intervention and provides a benchmark for qualifying new therapies to improve neurodevelopmental outcomes.
PMCID: PMC3708978  PMID: 23467054
Periventricular white matter injury; Glial fibrillary acidic protein; Cerebral palsy
3.  The Role of the Hippocampus in Avoidance Learning and Anxiety Vulnerability 
The hippocampus has been implicated in anxiety disorders and post-traumatic stress disorder (PTSD); human studies suggest that a dysfunctional hippocampus may be a vulnerability factor for the development of PTSD. In the current study, we examined the effect of hippocampal damage in avoidance learning, as avoidance is a core symptom of all anxiety disorders. First, the effect of hippocampal damage on avoidance learning was investigated in outbred Sprague Dawley (SD) rats. Second, the function of the hippocampus in Wistar-Kyoto (WKY) rats was compared to SD rats. The WKY rat is an animal model of behavioral inhibition, a risk factor for anxiety, and demonstrates abnormal avoidance learning, marked by facilitated avoidance acquisition and resistance to extinction. The results of the current study indicate that hippocampal damage in SD rats leads to impaired extinction of avoidance learning similar to WKY rats. Furthermore, WKY rats have reduced hippocampal volume and impaired hippocampal synaptic plasticity as compared to SD rats. These results suggest that hippocampal dysfunction enhances the development of persistent avoidance responding and, thus, may confer vulnerability to the development of anxiety disorders and PTSD.
PMCID: PMC4125878  PMID: 25152721
hippocampus; avoidance; PTSD; anxiety; WKY; synaptic plasticity; LTP
4.  Effects of Psychotropic Agents on Extinction of Lever-Press Avoidance in a Rat Model of Anxiety Vulnerability 
Avoidance and its perseveration represent key features of anxiety disorders. Both pharmacological and behavioral approaches (i.e., anxiolytics and extinction therapy) have been utilized to modulate avoidance behavior in patients. However, the outcome has not always been desirable. Part of the reason is attributed to the diverse neuropathology of anxiety disorders. Here, we investigated the effect of psychotropic drugs that target various monoamine systems on extinction of avoidance behavior using lever-press avoidance task. Here, we used the Wistar-Kyoto (WKY) rat, a unique rat model that exhibits facilitated avoidance and extinction resistance along with malfunction of the dopamine (DA) system. Sprague Dawley (SD) and WKY rats were trained to acquire lever-press avoidance. WKY rats acquired avoidance faster and to a higher level compared to SD rats. During pharmacological treatment, bupropion and desipramine (DES) significantly reduced avoidance response selectively in WKY rats. However, after the discontinuation of drug treatment, only those WKY rats that were previously treated with DES exhibited lower avoidance response compared to the control group. In contrast, none of the psychotropic drugs facilitated avoidance extinction in SD rats. Instead, DES impaired avoidance extinction and increased non-reinforced response in SD rats. Interestingly, paroxetine, a widely used antidepressant and anxiolytic, exhibited the weakest effect in WKY rats and no effects at all in SD rats. Thus, our data suggest that malfunctions in brain catecholamine system could be one of the underlying etiologies of anxiety-like behavior, particularly avoidance perseveration. Furthermore, pharmacological manipulation targeting DA and norepinephrine may be more effective to facilitate extinction learning in this strain. The data from the present study may shed light on new pharmacological approaches to treat patients with anxiety disorders who are not responding to serotonin re-uptake inhibitors.
PMCID: PMC4163983  PMID: 25309372
avoidance perseveration; anxiolytic; behavioral inhibition; dopamine; norepinephrine; serotonin; transporter inhibitors
5.  Mixed Lineage Kinase 3 is Required for Matrix Metalloproteinase Expression and Invasion in Ovarian Cancer Cells 
Experimental Cell Research  2012;318(14):1641-1648.
Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP) -1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells.
PMCID: PMC3389280  PMID: 22652451
MLK3; MMP; invasion; ovarian cancer; MAPK; AP-1
6.  Nitric oxide-dependent killing of aerobic, anaerobic and persistent Burkholderia pseudomallei 
Burkholderia pseudomallei infections are fastidious to treat with conventional antibiotic therapy, often involving a combination of drugs and long-term regimes. Bacterial genetic determinants contribute to the resistance of B. pseudomallei to many classes of antibiotics. In addition, anaerobiosis and hypoxia in abscesses typical of melioidosis select for persistent populations of B. pseudomallei refractory to a broad spectrum of antibacterials. We tested the susceptibility of B. pseudomallei to the drugs hydroxyurea, spermine NONOate and DETA NONOate that release nitric oxide (NO). Our investigations indicate that B. pseudomallei are killed by NO in a concentration and time-dependent fashion. The cytoxicity of this diatomic radical against B. pseudomallei depends on both the culture medium and growth phase of the bacteria. Rapidly growing, but not stationary phase, B. pseudomallei are readily killed upon exposure to the NO donor spermine NONOate. NO also has excellent antimicrobial activity against anaerobic B. pseudomallei. In addition, persistent bacteria highly resistant to most conventional antibiotics are remarkably susceptible to NO. Sublethal concentrations of NO inhibited the enzymatic activity of [4Fe-4S]-cofactored aconitase of aerobic and anaerobic B. pseudomallei. The strong anti-B. pseudomallei activity of NO described herein merits further studies on the application of NO-based antibiotics for the treatment of melioidosis.
PMCID: PMC3517295  PMID: 22521523
antibiotics; antimicrobials; melioidosis; reactive nitrogen species; therapy; [4Fe-4S] clusters
7.  Adaptation and Antibiotic Tolerance of Anaerobic Burkholderia pseudomallei ▿ † 
The Gram-negative bacterium Burkholderia pseudomallei is the etiological agent of melioidosis and is remarkably resistant to most classes of antibacterials. Even after months of treatment with antibacterials that are relatively effective in vitro, there is a high rate of treatment failure, indicating that this pathogen alters its patterns of antibacterial susceptibility in response to cues encountered in the host. The pathology of melioidosis indicates that B. pseudomallei encounters host microenvironments that limit aerobic respiration, including the lack of oxygen found in abscesses and in the presence of nitric oxide produced by macrophages. We investigated whether B. pseudomallei could survive in a nonreplicating, oxygen-deprived state and determined if this physiological state was tolerant of conventional antibacterials. B. pseudomallei survived initial anaerobiosis, especially under moderately acidic conditions similar to those found in abscesses. Microarray expression profiling indicated a major shift in the physiological state of hypoxic B. pseudomallei, including induction of a variety of typical anaerobic-environment-responsive genes and genes that appear specific to anaerobic B. pseudomallei. Interestingly, anaerobic B. pseudomallei was unaffected by antibacterials typically used in therapy. However, it was exquisitely sensitive to drugs used against anaerobic pathogens. After several weeks of anaerobic culture, a significant loss of viability was observed. However, a stable subpopulation that maintained complete viability for at least 1 year was established. Thus, during the course of human infection, if a minor subpopulation of bacteria inhabited an oxygen-restricted environment, it might be indifferent to traditional therapy but susceptible to antibiotics frequently used to treat anaerobic infections.
PMCID: PMC3122399  PMID: 21537012
8.  Regulation of Mixed Lineage Kinase 3 is Required for Neurofibromatosis-2-Mediated Growth Suppression in Human Cancer 
Oncogene  2010;30(7):781-789.
The Neurofibromatosis-2 (NF2) tumor suppressor merlin negatively regulates cell proliferation in numerous cell types. We have previously shown that the NF2 protein (merlin/schwannomin) associates with mixed lineage kinase 3 (MLK3), a mitogen-activated protein kinase (MAPK) kinase kinase that is required for the proliferation of normal and neoplastic cells. In the current study, we show that merlin inhibits MLK3 activity as well as the activation of its downstream effectors, B-Raf, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). The ability of merlin to regulate MLK3 activity requires a direct association between MLK3 and residues in the C-terminal region of merlin. Merlin integrates Rho GTPase family signaling with MAPK activity by inhibiting the binding between MLK3 and its upstream activator, Cdc42. Furthermore, we demonstrate that MLK3 is required for merlin suppression of cell proliferation and invasion. Collectively, these results establish merlin as a potent inhibitor of MLK3, ERK and JNK activation in cancer, and provide a mechanistic link between deregulated MAPK and Rho GTPase signaling in NF2 growth control.
PMCID: PMC3017676  PMID: 20890305
MLK3; NF2; MAPK; B-Raf; Cdc42
9.  Characterization of herpes simplex virus clinical isolate Y3369 as a glycoprotein G variant and its bearing on virus typing 
Virology Journal  2011;8:290.
Herpes simplex viruses exist as two major serotypes, type 1 (HSV-1) and type 2 (HSV-2). Determination of type, either HSV-1 or HSV-2, is important in accurate diagnosis and clinical control of transmission. Several tests are available for typing HSV, including a monoclonal antibody specific for glycoprotein G and several PCR assays.
A clinical isolate was identified as herpes simplex virus, but tested negative for both HSV-1 and HSV-2 antigens using type-specific monoclonal antibody assays. The isolate was determined to be HSV-1 by PCR analysis. A mutation which likely caused the monoclonal antibody non-reactivity was found in glycoprotein G. Phylogenetic analysis revealed two groups of HSV, one with the mutation and one without. Three population studies examining mutations in HSV-1 glycoprotein G were analyzed by chi-squared test. To this point, the epitope which the monoclonal antibody recognizes was only found in HSV-1 isolates from human European populations (p < 0.0001).
These findings suggest that the PCR-based methods for HSV typing may be more useful than the standard monoclonal antibody test in areas of the world where the variant in glycoprotein G is more prevalent.
PMCID: PMC3118968  PMID: 21658271
Herpes Simplex Virus; serotyping; glycoprotein G
10.  Mycobacterium bovis BCG Vaccine Strains Lack narK2 and narX Induction and Exhibit Altered Phenotypes during Dormancy▿  
Infection and Immunity  2008;76(6):2587-2593.
Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that affects one-third of the world's population. The sole extant vaccine for tuberculosis is the live attenuated Mycobacterium bovis bacillus Calmette-Guerin (BCG). We examined 13 representative BCG strains from around the world to ascertain their ability to express DosR-regulated dormancy antigens. These are known to be recognized by T cells of M. tuberculosis-infected individuals, especially those harboring latent infections. Differences in the expression of these antigens could be valuable for use as diagnostic markers to distinguish BCG vaccination from latent tuberculosis. We determined that all BCG strains were defective for the induction of two dormancy genes: narK2 (Rv1737c) and narX (Rv1736c). NarK2 is known to be necessary for nitrate respiration during anaerobic dormancy. Analysis of the narK2/X promoter region revealed a base substitution mutation in all tested BCG strains and M. bovis in comparison to the M. tuberculosis sequence. We also show that nitrate reduction by BCG strains during dormancy was greatly reduced compared to M. tuberculosis and varied between tested strains. Several dormancy regulon transcriptional differences were also identified among the strains, as well as variation in their growth and survival. These findings demonstrate defects in DosR regulon expression during dormancy and phenotypic variation between commonly used BCG vaccine strains.
PMCID: PMC2423053  PMID: 18362135
11.  Inactivation of [Fe-S] Metalloproteins Mediates Nitric Oxide-Dependent Killing of Burkholderia mallei 
PLoS ONE  2008;3(4):e1976.
Much remains to be known about the mechanisms by which O2-dependent host defenses mediate broad antimicrobial activity.
Methodology/Principal Findings
We show herein that reactive nitrogen species (RNS) generated by inducible nitric oxide (NO) synthase (iNOS) account for the anti-Burkholderia mallei activity of IFNγ-primed macrophages. Inducible NOS-mediated intracellular killing may represent direct bactericidal activity, because B. mallei showed an exquisite sensitivity to NO generated chemically. Exposure of B. mallei to sublethal concentrations of NO upregulated transcription of [Fe-S] cluster repair genes, while damaging the enzymatic activity of the [Fe-S] protein aconitase. To test whether [Fe-S] clusters are critical targets for RNS-dependent killing of B. mallei, a mutation was constructed in the NO-induced, [Fe-S] cluster repair regulator iscR. Not only was the iscR mutant hypersusceptible to iNOS-mediated killing, but its aconitase pool was readily oxidized by NO donors as compared to wild-type controls. Although killed by authentic H2O2, which also oxidizes [Fe-S] clusters, B. mallei appear to be resilient to NADPH oxidase-mediated cytotoxicity. The poor respiratory burst elicited by this bacterium likely explains why the NADPH oxidase is nonessential to the killing of B. mallei while it is still confined within phagosomes.
Collectively, these findings have revealed a disparate role for NADPH oxidase and iNOS in the innate macrophage response against the strict aerobe B. mallei. To the best of our knowledge, this is the first instance in which disruption of [Fe-S] clusters is demonstrated as cause of the bactericidal activity of NO congeners.
PMCID: PMC2276317  PMID: 18398486
12.  Lack of Immune Responses to Mycobacterium tuberculosis DosR Regulon Proteins following Mycobacterium bovis BCG Vaccination▿  
Infection and Immunity  2007;75(7):3523-3530.
Mycobacterium bovis BCG is widely used as a vaccine against tuberculosis (TB), despite its variable protective efficacy. Relatively little is known about the immune response profiles following BCG vaccination in relation to protection against TB. Here we tested whether BCG vaccination results in immune responses to DosR (Rv3133c) regulon-encoded proteins. These so-called TB latency antigens are targeted by the immune system during persistent Mycobacterium tuberculosis infection and have been associated with immunity against latent M. tuberculosis infection. In silico analysis of the DosR regulon in BCG and M. tuberculosis showed at least 97% amino acid sequence homology, with 41 out of 48 genes being identical. Transcriptional profiling of 14 different BCG strains, under hypoxia and nitric oxide exposure in vitro, revealed a functional DosR regulon similar to that observed in M. tuberculosis. Next, we assessed human immune responses to a series of immunodominant TB latency antigens and found that BCG vaccination fails to induce significant responses to latency antigens. Similar results were obtained with BCG-vaccinated BALB/c mice. In contrast, responses to latency antigens were observed in individuals with suspected exposure to TB (as indicated by positive gamma interferon responses to TB-specific antigens ESAT-6 and CFP-10) and in mice vaccinated with plasmid DNA encoding selected latency antigens. Since immune responses to TB latency antigens have been associated with control of latent M. tuberculosis infection, our findings support the development of vaccination strategies incorporating DosR regulon antigens to complement and improve the current BCG vaccine.
PMCID: PMC1932964  PMID: 17502400

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