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1.  GABAB-mediated rescue of altered excitatory–inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR-hypofunction 
Translational Psychiatry  2012;2(7):e142-.
Reduced N-methyl-D-aspartate-receptor (NMDAR) signaling has been associated with schizophrenia, autism and intellectual disability. NMDAR-hypofunction is thought to contribute to social, cognitive and gamma (30–80 Hz) oscillatory abnormalities, phenotypes common to these disorders. However, circuit-level mechanisms underlying such deficits remain unclear. This study investigated the relationship between gamma synchrony, excitatory–inhibitory (E/I) signaling, and behavioral phenotypes in NMDA-NR1neo−/− mice, which have constitutively reduced expression of the obligate NR1 subunit to model disrupted developmental NMDAR function. Constitutive NMDAR-hypofunction caused a loss of E/I balance, with an increase in intrinsic pyramidal cell excitability and a selective disruption of parvalbumin-expressing interneurons. Disrupted E/I coupling was associated with deficits in auditory-evoked gamma signal-to-noise ratio (SNR). Gamma-band abnormalities predicted deficits in spatial working memory and social preference, linking cellular changes in E/I signaling to target behaviors. The GABAB-receptor agonist baclofen improved E/I balance, gamma-SNR and broadly reversed behavioral deficits. These data demonstrate a clinically relevant, highly translatable neural-activity-based biomarker for preclinical screening and therapeutic development across a broad range of disorders that share common endophenotypes and disrupted NMDA-receptor signaling.
doi:10.1038/tp.2012.69
PMCID: PMC3410621  PMID: 22806213
animal model; GABAergic signaling; gamma oscillation; neuropsychiatric disease; NMDA-receptor
2.  Increasing Age Is Associated with Worse Prognostic Factors and Increased Distant Recurrences despite Fewer Sentinel Lymph Node Positives in Melanoma 
Background. Advanced age is associated with a poorer prognosis in patients with melanoma. Despite this established finding, a decreased incidence of positive sentinel lymph nodes (SLNs) with advancing age has paradoxically been described. Methods. Using a single-institution database of melanoma patients between 1994 and 2009, the relationship between standard clinicopathologic variables and recurrence based on age was evaluated. Results. 1244 patients who underwent successful SLN biopsies were analyzed (mean followup 80.3 months). Increasing age was independently associated with worse survival on multivariable analysis (P = 0.02). SLN status was more likely to be negative if the patient was older (P = 0.01). Conclusions. Our data supports the paradox that increasing age is associated with a lower frequency of positive-SLN biopsies despite age itself being a poor prognostic factor. We propose that age-dependent variations in the primary tumor and the patient may predispose to a hematogenous route of spread for the older population, leading to worse survival.
doi:10.1155/2012/456987
PMCID: PMC3317190  PMID: 22523678
3.  Effective Semi-Automated Extraction of Intact Mitochondria from Solid Tissues Using Gentle Mechanical Homogenization and Pressure Cycling Technology 
Impaired mitochondrial function has been linked to many diseases, such as stroke, heart disease, cancer, Type II diabetes and Parkinson's disease. Mitochondria-enriched preparations are needed for proteomic and metabolomic studies that may provide crucial insights into tissue-specific mitochondrial function and dysfunction, and answer fundamental questions of cell energetic and oxidative stress. Mitochondria extractions from whole tissue samples are typically performed using Potter-Elvehjem homogenizers or similar labor intensive manual disruption methods that require extensive operator experience, and often result in damage to fragile organelles and high sample-to-sample variability. Here we describe a semi-automated method that uses a novel gentle mechanical homogenizer (The PCT Shredder) and hydrostatic pressure to release intact mitochondria from fresh rat tissues with minimal hands-on time. Pressure Cycling Technology (PCT)-based tissue homogenization is conducted under controlled thermodynamic conditions (time, temperature and pressure) leading to more reproducible results. The quality of mitochondria preparations was characterized by electron microscopy, 2D PAGE and respiration assays. Our data demonstrate that mitochondria extracted by the PCT sample preparation system (PCT-SPS) are intact, functional, and exhibit a protein pattern comparable to control samples isolated using a conventional Potter-Elvehjem homogenizer. The resulting mitochondria-enriched samples were also subjected to trypsin digestion followed by nanoLC-MS/MS analysis on an LTQ-Orbitrap. Proteomic and pathway profiles of mitochondria samples prepared using the novel extraction technique were compared to those extracted using a conventional manual method to demonstrate the purity of mitochondrial preparations extracted using the novel PCT-SPS method.
PMCID: PMC3186655
4.  How willing are parents to improve pedestrian safety in their community? 
Study objective: To determine how likely parents would be to contribute to strategies to reduce pedestrian injury risks and how much they valued such interventions.
Design: A single referendum willingness to pay survey. Each parent was randomised to respond to one of five requested contributions towards each of the following activities: constructing speed bumps, volunteering as a crossing guard, attending a neighbourhood meeting, or attending a safety workshop.
Setting: Community survey.
Participants: A sample of 723 Baltimore parents from four neighbourhoods stratified by income and child pedestrian injury risk. Eligible parents had a child enrolled in one of four elementary schools in Baltimore City in May 2001.
Main results: The more parents were asked to contribute, the less likely they were to do so. Parents were more likely to contribute in neighbourhoods with higher ratings of solidarity. The median willingness to pay money for speed bumps was conservatively estimated at $6.43. The median willingness to contribute time was 2.5 hours for attending workshops, 2.8 hours in community discussion groups, and 30 hours as a volunteer crossing guard.
Conclusions: Parents place a high value on physical and social interventions to improve child pedestrian safety.
doi:10.1136/jech.57.12.951
PMCID: PMC1732358  PMID: 14652260
5.  Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrate 
Gut  2003;52(2):224-230.
Background and aims: Factors that induce luminal bacteria to cross the intestinal epithelium following injury remain poorly defined. The aim of this study was to investigate the interaction between glutamine metabolism, energy supply, and inflammatory mediators in determining the translocation of non-pathogenic bacteria across cultured enterocytes.
Methods: The effect of tumour necrosis factor α (TNF-α) on translocation of Escherichia coli C25 across Caco-2 epithelial monolayers was studied in the presence of products and inhibitors of glutamine metabolism. Simultaneous measurements of transepithelial electrical resistance (TEER) and flux of lucifer yellow were used to assess effects on the paracellular pathway. Lactate dehydrogenase release was used to monitor enterocyte integrity. Imaging of monolayers in these experimental conditions was undertaken with transmission electron microscopy.
Results: Exposure to basolateral TNF-α (20 ng/ml) for six hours induced translocation of E coli across Caco-2 but only if accompanied by simultaneous glutamine depletion (p<0.01). Translocation was inhibited by addition of glutamine for two hours (p<0.01) but not by an isonitrogenous mixture of non-glutamine containing amino acids. Inhibition of glutamine conversion to α-ketoglutarate, but not blockade of glutathione or polyamine synthesis, also induced translocation in the presence of TNF-α. Manipulations that induced bacterial translocation were associated with a marked reduction in enterocyte ATP levels. No effect of these treatments on paracellular permeability or lactate dehydrogenase release was observed. Conditions in which translocation occurred were associated with the presence of bacteria within enterocyte vacuoles but not the paracellular space.
Conclusions: In inflammatory conditions, the availability of glutamine as an enterocyte fuel substrate is essential for the preservation of a functional barrier to microorganisms. In conditions of acute glutamine depletion, cytokine mediated bacterial translocation appears to be primarily a transcellular process.
PMCID: PMC1774948  PMID: 12524404
intestinal barrier; sepsis, cytokines; Caco-2 cells; glutamine; tumour necrosis factor
6.  Opiate withdrawal syndrome mimicking postoperative intestinal obstruction. 
Patients with complicated inflammatory bowel disease commonly undergo repeated surgical procedures, often against a background of chronic opiate use. We describe a case in which a postoperative attempt to withdraw opiate analgesia on two separate occasions led to a clinical syndrome strongly suggestive of intestinal obstruction, the signs and symptoms of which settled rapidly on re-introduction of opiates. Small bowel contrast studies indicated a level of obstruction which not only fluctuated, but occurred at an unusual site for mechanical obstruction. In patients with a history of long-standing opiate use, postoperative opiate withdrawal can cause a significant, functional bowel disorder and should be borne in mind in the differential diagnosis of postoperative intestinal obstruction. Patients can be treated effectively with clonidine.
doi:10.1308/003588404322827464
PMCID: PMC1964156  PMID: 15005926
8.  Intestinal failure after surgery for complicated radiation enteritis. 
Between 1983 and 1997, a total of 16 patients were referred to a tertiary Intestinal Failure Unit (IFU) following surgery elsewhere for complications of radiation enteritis. Eleven were female with a mean age of 43 years (range 21-71 years) and the most common primary site of malignancy was genitourinary (n = 13). Patients had undergone an average of two laparotomies (range 1-7 laparotomies) for complications of radiation enteritis prior to transfer to the IFU. On admission, the principal problem in eight patients was persisting intestinal fistulation, four patients had continuing intestinal obstruction and four had the short bowel syndrome after extensive intestinal resection. Only one patient had evidence of residual malignancy; this patient with short bowel syndrome was allowed home without invasive therapy. Of the remaining 15 patients, 12 required an abdominal surgical procedure, while three were discharged without further surgery after training for home parenteral nutrition (HPN). Following abdominal surgery, five patients died in hospital, but the remaining seven patients went home alive--including two further patients on HPN. Overall, of the 15 patients referred with intestinal failure after surgery for complications of radiation enteritis and actively treated, one-third died in hospital and a further third required institution of HPN before being able to be discharged home.
PMCID: PMC2503431  PMID: 10858685
9.  McLaughlin Research Institute. 
Molecular Medicine  1998;4(3):133-137.
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PMCID: PMC2230349  PMID: 9562971
10.  Failure to transmit disease from gray tremor mutant mice. 
Journal of Virology  1997;71(3):2342-2345.
Mice homozygous for mutant alleles at the gray tremor (gt) locus develop a marked non-intention tremor beginning at 8 days of age. Most homozygous mice die by 3 months. Homozygotes exhibit intense vacuolation of the central nervous system gray matter and vacuolation and hypomyelination of some white matter tracts. Based on neuropathological similarities with scrapie, other investigators inoculated wild-type mice with gray tremor brain homogenates to test the hypothesis of transmissibility. Published reports indicated that spongiform encephalopathy (R. L. Sidman, H. C. Kinney, and H. O. Sweet, Proc. Natl. Acad. Sci. USA 82:253-257, 1985) and disease, including hind limb paralysis in NFS mice (P. M. Hoffman, R. G. Rohwer, C. MacAuley, J. A. Bilello, J. W. Hartley, and H. C. Morse III, Proc. Natl. Acad. Sci. USA 84:3866-3870, 1987), were transmitted by inoculation of gt/gt brain homogenates. In our hands, however, no NFS/NCr animals inoculated intracerebrally with gt/gt or +/+ brain preparations showed any signs of disease or pathological changes in the brain. Positive transmission by other investigators may reflect the microbiological status of their donor or recipient mice.
PMCID: PMC191343  PMID: 9032370
12.  Regulation of the C/EBP-related gene gadd153 by glucose deprivation. 
Molecular and Cellular Biology  1993;13(8):4736-4744.
gadd153 encodes a CCAAT/enhancer-binding protein (C/EBP)-related protein that lacks a functional DNA-binding domain. Since the gadd153 protein is capable of heterodimerizing with other C/EBPs, gadd153 may function as a negative regulator of these transcription factors. Here we examined the role of glucose in regulating gadd153 expression. We found that glucose deprivation markedly induces gadd153 mRNA levels in both HeLa and 3T3-L1 cells and that addition of D-(+)-glucose resulted in a rapid decrease of gadd153 mRNA. Similar induction and reversal of gadd153 expression were observed at the protein level. Because C/EBP alpha appears to play an important role in regulating genes involved in adipogenesis and energy metabolism, we examined gadd153 expression during the differentiation of 3T3-L1 preadipocytes and as a function of glucose utilization in differentiated adipocytes. Using a standard differentiation protocol that consisted of hormonal stimulation for 2 days followed by medium changes every 2 days thereafter, we observed that both C/EBP alpha and gadd153 mRNAs were elevated. However, C/EBP alpha induction occurred on day 3, while gadd153 expression was not seen until day 4, when the cells were fully differentiated. Frequent addition of fresh medium to the cells during the differentiation process, as well as supplementation of medium with glucose, reduced gadd153 expression without preventing C/EBP alpha expression or interfering with cellular differentiation. Thus, gadd153 expression is not essential for the process of adipocyte differentiation but is significantly influenced by the availability of glucose to the cell.
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PMCID: PMC360099  PMID: 8336711
13.  Induction of HSP70 gene expression by the antiproliferative prostaglandin PGA2: a growth-dependent response mediated by activation of heat shock transcription factor. 
Molecular and Cellular Biology  1992;12(4):1528-1534.
Prostaglandins (PG) of the A series are potent inhibitors of cell proliferation. Recently, it was shown that PGA2-induced growth arrest was associated with the increased synthesis of stress proteins encoded by the HSP70 gene family. In this study, we have examined the molecular basis for this increases HSP70 expression. Northern (RNA) blot analysis and nuclear run-on assays demonstrated that induction of high levels of HSP70 mRNA results from an increase in the rate of transcription. High-level induction is specific to the HSP70 family of heat shock proteins and is rapid, reversible, dose dependent, and specific for PGs capable of growth-arresting HeLa cells. In addition, the response was found to be highly dependent on the growth state of the cells, as induction occurs in growing but not in confluent nongrowing cell populations. Induction is dependent on the activation of heat shock factor. Cycloheximide pretreatment, which inhibits the antiproliferative effects of PGA2, prevents activation of the heat shock factor and induction of HSP70 mRNA by PGA2. These results support a role for HSP70 in mediating the antiproliferative effects of PGA2.
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PMCID: PMC369594  PMID: 1549109
14.  Urinary retention and the lunisolar cycle: is it a lunatic phenomenon? 
BMJ : British Medical Journal  1989;299(6715):1560-1562.
OBJECTIVE--To determine whether a relation between urinary retention and temporal rhythms exists. DESIGN--Retrospective analysis of patients presenting over three years. SETTING--Urology departments in two hospitals. PATIENTS--815 Patients presenting as emergency admissions with urinary retention and requiring immediate decompression of the bladder. MAIN OUTCOME MEASURES--Calendar date of each admission to determine circadian, monthly, and seasonal periodicity. RESULTS--No association was found between urinary retention and circadian, monthly, or seasonal rhythms. A significantly higher (p less than 0.001) incidence of urinary retention was observed during the new moon in comparison with other phases of the lunar cycle. CONCLUSIONS--Urinary retention is periodic in nature. This should be considered when the workload of a specialist urological department is organised.
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PMCID: PMC1838760  PMID: 2514900
15.  Genetics and polymorphism of the mouse prion gene complex: control of scrapie incubation time. 
Molecular and Cellular Biology  1988;8(12):5528-5540.
The mouse prion protein (PrP) gene (Prn-p), which encodes the only macromolecule that has been identified in scrapie prions, is tightly linked or identical to a gene (Prn-i) that controls the duration of the scrapie incubation period in mice. Constellations of restriction fragment length polymorphisms distinguish haplotypes a to f of Prn-p. The Prn-pb allele encodes a PrP that differs in sequence from those encoded by the other haplotypes and, in inbred mouse strains, correlates with long scrapie incubation time (Westaway et al., Cell 51: 651-662, 1987). In segregating crosses of mice, we identified rare individuals with a divergent scrapie incubation time phenotype and Prn-p genotype, but progeny testing to demonstrate meiotic recombination was not possible because scrapie is a lethal disease. Crosses involving the a, d, and e haplotypes demonstrated that genes unlinked to Prn-p could modulate scrapie incubation time and that there were only two alleles of Prn-i among the mouse strains tested. All inbred strains of mice that had the Prnb haplotype were probably direct descendants of the I/LnJ progenitors. We established the linkage relationship between the prion gene complex (Prn) and other chromosome 2 genes; the gene order, proximal to distal, is B2m-II-1a-Prn-Itp-A. Recombination suppression in the B2m-Prn-p interval occurred during the crosses involved in transferring the I/LnJ Prnb complex into a C57BL/6J background. Transmission ratio distortion by Prna/Prnb heterozygous males was also observed in the same crosses. These phenomena, together with the founder effect, would favor apparent linkage disequilibrium between Prn-p and Prn-i. Therefore, transmission genetics may underestimate the number of genes in Prn.
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PMCID: PMC365657  PMID: 3149717
16.  The voluntary acceptance of HIV-antibody screening by intravenous drug users. 
Public Health Reports  1987;102(4):391-394.
Intravenous drug abusers in a methadone program in Minnesota were offered HIV-antibody screening to determine the degree of interest in screening and extent of infection. Thirty-nine (85 percent) were willing to be tested. Only seven refused. All patients were aware of acquired immunodeficiency syndrome (AIDS) and their high risk of exposure to the AIDS virus through sharing of injection paraphernalia. None reported exposure to additional risk factors, such as homosexual or bisexual activity or having received a blood transfusion. Of the patients tested, none was positive for HIV antibodies. The high degree of patient interest in screening was unanticipated as was the lack of positive laboratory findings for HIV antibodies. Factors associated with acceptance of testing included patient awareness of high seroprevalence rates, indifference to potential negative social consequences of positive HIV-antibody status, and the voluntary nature of the testing. These findings raise a cautious sense of optimism about HIV-antibody screening for similar risk groups.
PMCID: PMC1477865  PMID: 3112849
17.  Binding and distribution studies in the SENCAR mouse of compounds demonstrating a route-dependent tumorigenic effect. 
Previous investigators have determined that benzo(a)pyrene [B(a)P] was much more effective in causing skin papillomas if applied topically than when administered orally in the initiation-promotion assay in SENCAR mouse. Conversely, urethane and acrylamide caused a higher percentage of mice to develop papillomas and induced more tumors per mouse when given orally. In an attempt to understand the reason for this discrepancy in route dependency, 3H-benzo(a)pyrene, 14C-urethane and 14C-acrylamide were administered as single doses orally or topically to male SENCAR mice. Distribution in skin, stomach, liver, and lung was determined for time periods up to 48 hr. The binding of these compounds to DNA, RNA, and protein in these tissues was determined 6 and 48 hr after administration. For all three compounds, high concentrations were found in the skin following topical application, but very little material reached this target organ following oral administration. In contrast, the internal organs generally contained more material after oral administration. The binding of label compounds to DNA, RNA, and protein generally reflected the distribution data, thus more compound was bound in the stomach, liver, and lung after oral administration compared to topical application, whereas the opposite was true for the skin. This finding was particularly evident for B(a)P. The results suggest that differences in distribution to the skin and binding to macromolecules following oral or topical administration cannot explain the greater tumorigenicity of urethane and acrylamide after oral administration in the SENCAR mouse.
PMCID: PMC1474256  PMID: 3780633
18.  Diverticulum and Fistula of the Lower Cervical Esophagus in a Horse 
The Canadian Veterinary Journal  1983;24(12):385-387.
Esophageal diverticulum and perforation were diagnosed in a horse. The condition was characterized clinically by dysphagia, polypnea, diffuse swelling of the ventral aspect of neck and cutaneous fistula. Endoscopic examination revealed the presence of food material in the guttural pouch. A barium sulfate esophagram was performed to outline the diverticulum.
Exploratory surgery confirmed esophageal perforation and entrapment of food material between the muscular planes of the cervical region and the horse was euthanized. At necropsy a food-filled periesophageal tract was found extending from the esophageal rupture to the left guttural pouch.
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PMCID: PMC1790477  PMID: 17422340
19.  Warfarin: A Review with Emphasis on Its Use in the Horse 
The Canadian Veterinary Journal  1983;24(7):211-213.
Warfarin or dicoumarol prevents the production of functional clotting factors II, VII, IX and X. Navicular disease and thrombophlebitis are examples of equine thrombotic diseases in which warfarin has been used therapeutically. The initiation of anticoagulant therapy is relatively simple but attending veterinarians must be aware of the potential risks in order to minimize them. These risks include epistaxis, bleeding into the gastrointestinal tract and at the venipuncture site, and increased susceptibility to hematoma formation following local trauma. Vitamin K, especially vitamin K1 is a swift and specific antidote for warfarin toxicity.
PMCID: PMC1790352  PMID: 17422277
20.  Ricinoleic acid effect on the electrical activity of the small intestine in rabbits. 
Journal of Clinical Investigation  1978;61(3):640-644.
Using myoelectric recording techniques, we examined the myoelectric effects of castor oil; ricinoleic acid (cis isomer), the active ingredient of castor oil; and ricinelaidic acid (trans isomer) in the small intestine of New Zealand white rabbits. Ricinoleic acid, 2 microgram/kg per min (6mM), was perfused into a distal 12-cm ileal loop. An abnormal myoelectric pattern developed that was similar to the alteration in the electrical activity that has previously been reported for cholera enterotoxin. Castor oil, 0.85 ml/kg, had a similar effect. Ricinelaidic acid, 2 microgram/kg per min, induced no activity. A second preparation consisted of an intraluminal perfusion of ricinoleic acid, 2 microgram/kg per min, into the first section of the duodenum. The abnormal myoelectric pattern was observed in the jejunum and the ileum but not the duodenum. The mean onset time for the development of this altered myoelectric state for all experiments was 3.5 h. These studies suggest that an active motility component in addition to the secretory state exists throughout the small intestine that is exposed to castor oil or ricinoleic acid.
PMCID: PMC372576  PMID: 641145
21.  Intestinal myoelectric activity in response to live Vibrio cholerae and cholera enterotoxin. 
The myoelectric response of the rabbit ileum was studied in response to live Vibrio cholerae culture, a whole cell lysate of cholera, and the purified enterotoxin. Each cholera preparation produced a series of highly organized migrating action potential complexes (MAPC). An MAPC was defined as action potential discharge with a duration of 2.5 s or longer, followed by similar activity on at least one other consecutive electrode site. The mean and modal onset time of MAPC activity occurred 4 h after the infection with live Vibrio cholerae culture, the freeze-dried whole cell lysate preparation, or the purified enterotoxin. After the onset of activity this pattern persisted for the duration of the recording period (up to 12 h). The MAPC had a mean propagation velocity of 0.85+/-0.07 cm/s (mean+/-SEM), which remained constant with time. Direct visual observation of the loop revealed that the MAPC's resulted in contractions that propelled intraluminal contents in an aborad direction. The mean fluid output from the 12-cm ileal loops was 6.4+/-1.1 ml/h (mean+/-SEM). Control experiments consisted of recordings from: (a) a ligated ileal loop into which nothing was placed; (b) a ligated ileal loop into which either uninfected culture broth or 0.9% NaCl solution was injected; (c) a ligated ileal loop infused with 0.9% NaCl solution at a rate of 11.2 ml/h, and (d) rapid injection of 1.0, 2.5, 5.0, or 10.0-ml boluses of 0.9% NaCl into the proximal catheter. MAPC activity was not observed in any of the control experiments. These studies indicate that in addition to a secretory component to cholera, there exists a highly organized MAPC that results in contractions that propel intraluminal contents in an aborad direction.
PMCID: PMC333159  PMID: 932212

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