Retrograde state dependent learning.

Sodium pentobarbital administered intravenously after acquisition in a one-trial passive avoidance task results in state dependent (drug dissociated) learning in male albino rats. Findings have methodological implications for drug-based research and theoretical implications for drug discrimination studies. Predictions based on a stimulus generalization hypothesis are not supported, whereas those based on an information storage hypothesis are supported.

JRK is a positive regulator of ß-catenin transcriptional activity commonly overexpressed in colon, breast and ovarian cancer.

The loss of ß-catenin inhibitory components is a well-established mechanism of carcinogenesis but ß-catenin hyperactivity can also be enhanced through its coactivators. Here we first interrogated a highly validated genomic screen and the largest repository of cancer genomics data and identified JRK as a potential new oncogene and therapeutic target of the ß-catenin pathway. We proceeded to validate the oncogenic role of JRK in colon cancer cells and primary tumors. Consistent with a ß-catenin activator function, depletion of JRK in several cancer cell lines repressed ß-catenin transcriptional activity and reduced cell proliferation. Importantly, JRK expression was aberrantly elevated in 21% of colorectal cancers, 15% of breast and ovarian cancers and was associated with increased expression of ß-catenin target genes and increased cell proliferation. This study shows that JRK is required for ß-catenin hyperactivity regardless of the adenomatous polyposis coli/ß-catenin mutation status and targeting JRK presents new opportunities for therapeutic intervention in cancer.

Chromosome microarray proficiency testing and analysis of quality metric data trends through an external quality assessment program for Australasian laboratories.

Chromosome microarrays are an essential tool for investigation of copy number changes in children with congenital anomalies and intellectual deficit. Attempts to standardise microarray testing have focused on establishing technical and clinical quality criteria, however external quality assessment programs are still needed. We report on a microarray proficiency testing program for Australasian laboratories. Quality metrics evaluated included analytical accuracy, result interpretation, report completeness, and laboratory performance data: sample numbers, success and abnormality rate and reporting times. Between 2009 and 2014 nine samples were dispatched with variable results for analytical accuracy (30-100%), correct interpretation (32-96%), and report completeness (30-92%). Laboratory performance data (2007-2014) showed an overall mean success rate of 99.2% and abnormality rate of 23.6%. Reporting times decreased from >90 days to <30 days for normal results and from >102 days to <35 days for abnormal results. Data trends showed a positive correlation with improvement for all these quality metrics, however only 'report completeness' and reporting times reached statistical significance. Whether the overall improvement in laboratory performance was due to participation in this program, or from accumulated laboratory experience over time, is not clear. Either way, the outcome is likely to assist referring clinicians and improve patient care.

Reduced cortical BACE1 content with one bout of exercise is accompanied by declines in AMPK, Akt, and MAPK signaling in obese, glucose-intolerant mice.

Obesity and type 2 diabetes are significant risk factors in the development of neurodegenerative diseases, such as Alzheimer's disease. A variety of cellular mechanisms, such as altered Akt and AMPK and increased inflammatory signaling, contribute to neurodegeneration. Exercise training can improve markers of neurodegeneration, but the underlying mechanisms remain unknown. The purpose of this study was to determine the effects of a single bout of exercise on markers of neurodegeneration and inflammation in brains from mice fed a high-fat diet. Male C57BL/6 mice were fed a low (LFD; 10% kcal from lard)- or a high-fat diet (HFD; 60% kcal from lard) for 7 wk. HFD mice underwent an acute bout of exercise (treadmill running: 15 m/min, 5% incline, 120 min) followed by a recovery period of 2 h. The HFD increased body mass and glucose intolerance (both P < 0.05). This was accompanied by an approximately twofold increase in the phosphorylation of Akt, ERK, and GSK in the cortex (P < 0.05). Following exercise, there was a decrease in beta-site amyloid precursor protein cleaving enzyme 1 (BACE1; P < 0.05) and activity (P < 0.001). This was accompanied by a reduction in AMPK phosphorylation, indicative of a decline in cellular stress (P < 0.05). Akt and ERK phosphorylation were decreased following exercise in HFD mice to a level similar to that of the LFD mice (P < 0.05). This study demonstrates that a single bout of exercise can reduce BACE1 content and activity independent of changes in adiposity. This effect is associated with reductions in Akt, ERK, and AMPK signaling in the cortex.

Postischemic glucose metabolism is modified in the hippocampal CA1 region depleted of excitatory input or pyramidal cells.

During early postischemic reperfusion, the vulnerable brain regions (e.g., hippocampal CA1) show a relatively high deoxyglucose accumulation. To investigate if this accumulation is a marker for the later-occurring regional cell death and to determine its cellular localization, we studied the glucose metabolism in the CA1 region post ischemia after removal of its pre- or postsynaptic components. A 20-min period of cerebral ischemia was used for selective removal of the main postsynaptic component in CA1 pyramidal cells, and a bilateral intraventricular injection of kainic acid for removal of the majority of presynaptic axon terminals in this region (and postsynaptic terminals and cell bodies in CA3). The glucose metabolism was studied in these two lesion types and in sham-operated animals before and after a period of ischemia. There was a 60% reduction of metabolism after ischemia in the nonvulnerable regions, whereas CA1 and sometimes CA3 showed a columnar pattern of high and low metabolism. CA1 and CA3 devoid of the postsynaptic component showed increased postischemic metabolism. The latter was due to the presence of macrophages, as demonstrated by an enzyme histochemical stain for nonspecific esterase. CA1 with no presynaptic component showed a postischemic depression of the glucose metabolism similar to the rest of the brain. It is suggested that the level of the postischemic glucose metabolism in the ischemia-vulnerable regions is determined by the presence of both synaptic components. The presence of macrophages in a region gives rise to apparently normal values of glucose metabolism.

Intravascular streaming and variable delivery to brain following carotid artery infusions in the Sprague-Dawley rat.

Intracarotid artery infusions in animals are commonly performed in studies of the blood-brain barrier and in chemotherapy trials. Implicit in the analysis of these experiments is that the infusate will be distributed to the territory of the internal carotid artery in a manner that is proportional to blood flow. Fifteen Sprague-Dawley rats were studied to determine if poor infusate mixing with blood due to intravascular streaming occurred during intracarotid artery drug infusions and if it could be eliminated with fast retrograde infusion. In three experimental groups, a radiolabeled flow tracer--14C-iodoantipyrine (IAP)--was infused retrograde through the external carotid artery into the common carotid artery at slow, medium, and fast rates (0.45, 1.5, and 5.0 ml/min). In a control group, IAP was injected intravenously (i.v.). Local isotope concentrations in the brain were determined by quantitative autoradiography, and the variability of isotope delivery was assessed in the frontoparietal cortex, temporal cortex, and caudate putamen of all animals. Streaming phenomena were manifest in all selected anatomic areas after the slow and medium rates of intraarterial infusion. After fast intracarotid infusion or i.v. injection, there was uniform distribution of isotope in the same brain regions.

Safety and immunogenicity of multiple conventional immunizations administered during early HIV infection.

Twenty-one asymptomatic adults who had recently received multiple polysaccharide, live viral, and protein-derived vaccines were identified as being infected with human immunodeficiency virus (HIV). The mean subject age was 24 years (range 18-33); 20 of 21 (95%) were male. The mean T4 count was 523/mm3 with a mean T4/T8 ratio of 0.6. Serologic responses to immunization with meningococcus group C, adenovirus types 4 and 7, tetanus, and diphtheria were evaluated for the HIV seropositive subjects and were compared with the responses of similarly vaccinated age-, sex-, and race-matched HIV-seronegative controls. Significantly fewer (p less than 0.03) HIV subjects responded to meningococcus C (bactericidal antibody) and adenovirus 4 (neutralizing antibody) vaccines than did normals; the HIV-infected subjects who did respond produced functional antibody comparable to that of normals. Booster responses of HIV subjects to tetanus and diphtheria were comparable to those of normals. HIV-infected vaccine nonresponders did not differ from HIV-infected responders in total white blood cell, T4, T4/T8, total serum IgG, or delayed-type hypersensitivity skin test reactivity. All HIV subjects had negative cultures for live vaccine viruses (rubella, measles, adenovirus, and poliovirus). Postimmunization, no clinically apparent adverse reactions to vaccination were detected.

Immunoenzymatic analysis by monoclonal antibodies of bacterial lipopolysaccharides after transfer to nitrocellulose.

A rapid, sensitive immunoenzymatic technique for the analysis of lipopolysaccharide (LPS) from gram-negative bacteria using monoclonal antibodies is described. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the LPS was either stained with silver or electrophoretically transferred to nitrocellulose. After reaction with anti-LPS monoclonal antibodies, the transferred antigens were visualized by reaction with alkaline-phosphatase-labelled anti-mouse antibodies and a substrate containing naphthol phosphoric acid and Fast Red.

125I interstitial brachytherapy for primary malignant brain tumors: technical aspects of treatment planning and implantation methods.

Use of interstitial radiation holds promise in the treatment of primary malignant brain tumors, but optimal technical factors have yet to be determined. We have developed a method of precise CT directed stereotactic placement of radioactive sources in a predetermined target volume. We use low activity (1-2 millicurie/speed) sources of 125I loaded in silastic catheters, which are positioned in a parallel array in the target. Positioning of such multiple sources toward the periphery of the volume enhances achievable dose homogeneity. Seeds of various activities can be differentially loaded into each catheter and the catheters can be positioned at various radii from the central target so that the treated volume corresponds to the identified (often irregular) target volume. Although the implant is designed to be permanent, the sources can be removed easily in a second procedure.

Differentiation of fetal pig endocrine cells after allografting into the thymus gland.

BACKGROUND: The thymus of large animals, such as the pig, is thought to be an appropriate site for transplanting adult islets, which contain numerous beta cells, for the purpose of reversing diabetes. Whether fetal islet-like cell clusters (ICCs), which contain few beta cells, will develop at this site, so that adequate amounts of insulin can be produced, is unknown. METHODS: Between 15,000 and 40,000 ICCs were injected into the thymus gland of six juvenile immunosuppressed pigs, and the animals were killed up to 30 days later. The graft was then examined histologically and comparisons made with untransplanted ICCs and those grafted into the omentum of immunosuppressed pigs. RESULTS: At transplantation, the percentage of cells in the ICCs containing insulin, glucagon, somatostatin, or pancreatic polypeptide was 9+/-1%, 13+/-2%, 9+/-1%, and 3+/-1% respectively. Within 9-30 days of transplantation into the thymus, the percentage of all endocrine cells increased, insulin to 41+/-3%, glucagon to 43+/-6%, somatostatin to 26+/-4%, and pancreatic polypeptide to 9+/-3%. There was co-localization of more than one hormone in some cells. Omental grafts contained a similar percentage of insulin and glucagon-containing cells, but significantly fewer somatostatin and pancreatic polypeptide-containing cells. CONCLUSIONS: Endocrine cells from the fetal pig pancreas will differentiate when transplanted into the thymus gland of the pig, making this a suitable site for grafting ICCs to test their ability to normalize blood glucose levels of diabetic recipients.

Lowering of blood glucose to nondiabetic levels in a hyperglycemic pig by allografting of fetal pig isletlike cell clusters.

BACKGROUND: Fetal pig isletlike cell clusters (ICCs) will differentiate when grafted into the thymus gland of outbred immunosuppressed nondiabetic pigs for up to 3 months. Whether these cells will survive for a similar period in a diabetic recipient and will mature with secretion of insulin to ameliorate the hyperglycemia is unknown. METHODS: Between 40,000 and 125,000 ICCs (7,000 to 11,400 ICCs/kg) were injected into the thymus gland of five juvenile pigs immunosuppressed with cyclosporine and deoxyspergualin, and the animals were subsequently made diabetic by the injection of streptozotocin. Insulin was administered subcutaneously, with one pig dying from hypoglycemia. The animal with the least number of ICCs transplanted was killed 81 days later, and the graft was analyzed histologically. Blood glucose levels and porcine C-peptide in the remaining animals were monitored for a median of 101 days. RESULTS: Histological analysis of the graft showed numerous epithelial cell clusters; the percentage of cells that contained insulin, glucagon, somatostatin, and pancreatic polypeptide were 61%, 64%, 25%, and 18%, respectively. Some cells contained more than one hormone. Porcine C-peptide was detected from 21 days after induction of diabetes but not before. In the pig receiving the most ICCs, blood glucose levels were lowered to nondiabetic levels 109 days after transplantation. Plasma C-peptide levels in response to glucagon in this pig steadily increased after grafting; peak levels were 0, 0.21, 0.45, and 0.52 ng/ml at 4, 21, 49, and 80 days after induction of diabetes compared to 0.09 ng/ml in control diabetic pigs. The secretion of C-peptide in response to oral and intravenous glucose and arginine also was greater than in untransplanted diabetic pigs, the pattern of secretion being consistent with developing fetal beta cells as the source of the C-peptide. Pancreatic insulin content was 0.1 mU/mg, 4% of that in nondiabetic pigs, and the number of beta cells per islet was 3 to 6 compared to 90 in nondiabetic controls. CONCLUSIONS: ICCs will differentiate and function for up to 111 days when transplanted into outbred immunosuppressed pigs rendered diabetic. Blood glucose levels can be lowered to nondiabetic levels when sufficient numbers of ICCs are grafted.

The NCI-atlas of dose distributions for regular 125I brain implants.

In connection with the development of an optimization method for 125I brain implants in irregularly shaped target volumes, a systematic study was conducted toward optimizing seed configurations for regular target volumes. The intention was to find basic rules for the positioning of strings of seeds in the cylindrical implant pattern of the stereotactic neurosurgical procedure in use, and in accordance with the following criteria: (i) steep dose fall-off outside the target volume; (ii) coverage of the target volume by making the prescribed dose surface coincident with the target volume surface within 1 mm; (iii) uniformity of the dose distribution in the target volume as far as achievable with a seed implant. As a result of this study, an atlas of optimized regular 125I brain implant configurations was compiled. Regular implants were understood as being cylindrical or spherical. Diameters and heights from 2 to 5 cm were covered.

A comparison of the sensitivity of pig and human peripheral blood mononuclear cells to the antiproliferative effects of traditional and newer immunosuppressive agents.

Difficulty in preventing rejection of fetal pig islet-like cell clusters (ICCs) transplanted into pigs using traditional forms of immunotherapy has been reported. An in vitro study of the efficacy of seven different immunosuppressive agents to inhibit proliferation of pig peripheral blood mononuclear cells (PBMC) was performed, and a comparison was made between the human and pig to determine if the efficacy of these agents differed between species. The efficacy of cyclosporine (CsA), azathioprine (Aza), methylprednisolone (MP), FK506, rapamycin (RAP), mycophenolate mofetil (MMF) and deoxymethylspergualin (MeDSG) to inhibit pig and human PBMC proliferation in mitogenic experiments using phytohaemagglutinin (PHA) as a stimulus was performed. Further, allogeneic pig mixed lymphocyte reactions (MLR) were used to determine the activity of these agents in a model more comparable to the allograft rejection process. It was found that pig PBMC stimulated with PHA or in a MLR were inhibited by the agents tested, with the exception of MeDSG that was ineffective in mitogenic experiments. The inhibitory effects of these agents differed between PHA and MLR, the respective (50% inhibitory concentration) IC50 values for pig PBMC being 1.7 and 0.08 microg/ml for CsA, 1.4 and 4.4 microg/ml for Aza, 0.11 and 0.002 microg/ml for MP, 3.0 and 2.8 ng/ml for FK506, 2.1 and 0.3 ng/ml for RAP and 10.8 and 454 ng/ml for MME Pig PBMC were less sensitive than human PBMC to the antiproliferative effects of CsA, Aza, FK506, RAP and MMF, but not MP on PHA stimulation, the ratio of the pig to human IC50 values being 19, 11, 13, 2.3, 1.4, and 0.4, respectively. These data suggest that the doses of most immunosuppressive agents administered to prevent rejection in pigs need to be higher than those used to achieve therapeutic benefit in humans.

The effects of phospholipase C inhibition on insulin-stimulated glucose transport in skeletal muscle.

Previous research has demonstrated that phospholipase C (PLC) is involved in insulin-stimulated glucose transport in 3T3-L1 adipocytes. The purpose of the current investigation was to determine if PLC is also involved in insulin-stimulated glucose uptake in rat skeletal muscle. To that end, we used an in vitro muscle preparation of the rat soleus muscle to test the effects of the PLC inhibitor, U73122, on glucose transport. The PLC inhibitor, U73122, led to a concentration-dependent inhibition of insulin (0.6 nmol/L)-stimulated glucose transport, whereas it had no effect on basal glucose transport. Specifically 10, 20, 50, and 150 micromol/L U73122 inhibited insulin (0.6 nmol/L)-stimulated glucose transport approximately 17%, 20%, 26%, and 38%, respectively, while an equal molar concentration of U73343 (inactive form of U73122) and/or carrier media (dimethyl sulfoxide [DMSO]) did not influence glucose uptake. A secondary aim of this investigation was to determine if increasing the concentration of insulin from a physiologic concentration (0.6 nmol/L) to a supraphysiologic concentration (6.0 nmol/L) could ameliorate the inhibitory effects of U73122. A 10-fold increase in insulin eliminated the inhibitory effects of U73122 on insulin-stimulated glucose uptake in soleus muscle. In summary, this preliminary report provides evidence to suggest that a PLC signaling mechanism modifies insulin-stimulated glucose uptake in skeletal muscle via its influence on insulin sensitivity.

Delayed c-fos proto-oncogene expression in the rat hippocampus induced by transient global cerebral ischemia: an in situ hybridization study.

The relative levels of c-fos mRNA in individual neurons of the hippocampal formation of rats is dramatically increased following 20 min of cerebral ischemia induced by 4-vessel occlusion. After 24 h of recirculation, a number of scattered neurons in the dentate hilus became hybridization positive. This effect appeared to peak between 24 and 48 h. A few neurons in the pyramidal cell layer of CA1 expressed c-fos as early as 24 h, but the most intense labeling in this region was seen at 72 h of recirculation. These results correlate well with the known distribution of delayed ischemic necrosis in the brain.

Near infrared spectrophotometry for the diagnosis of vasculogenic erectile dysfunction.

A specialized near infrared spectrophotometry instrument for noninvasive, continuous monitoring of the hemodynamic events of erection in the human penis has been developed. Its potential application for the diagnostic evaluation of erectile dysfunction was investigated. Thirty-eight patients and 18 volunteer subjects underwent penile near infrared spectrophotometry using an optical sensor probe with wavelength selectivity for hemoglobin absorption spectra. Penile blood volume changes and their time courses were measured following intracavernous pharmacostimulation in patients and visual sexual stimulation in volunteers. Spectrophotometric results were compared with results obtained simultaneously using color duplex ultrasonography, strain gauge penile circumference monitoring, penile tonometry, and clinical assessments. Spectrophotometric recordings of penile erection showed measurable blood volume changes consistent with the hemodynamic events of this biological function. Blood volume per cent (BV%) increase correlated with clinical ratings of erection quality (P < 0.001), penile rigidity measurements (P < 0.005), and penile circumference increases (P < 0.0001), and it correlated with mean peak systolic velocity measurements when BV% increase was restricted to values less than 50% (P < 0.001). The time to reach half the maximum blood volume change (BV T1) correlated directly with the time to reach half the maximum penile circumference size increase (P < 0.001), whereas BV T4 correlated inversely with mean resistive index measurements only when BV T(1/2) was restricted to values greater than 120 s (P < 0.05). Spectrophotometric criteria consisting of BV % less than 35% and BV T(1/2) greater than 120 s affirmed the diagnosis of severe erectile impairment with a similar degree of accuracy as standard ultrasonographic criteria (P < 0.002). Penile near infrared spectrophotometry is a safe, inexpensive and simply used biomedical optics technique that provides quantitative measurements of the vascular physiology of penile erection and appears to offer clinical utility in the diagnosis of vasculogenic erectile dysfunction.

The effect of unilateral and bilateral removal of the entorhinal cortex on the glucose utilization in various hippocampal regions in the rat.

In the present study lesion-induced changes in function of various hippocampal regions, as reflected by the metabolic rate of glucose, were measured by means of quantitative autoradiography, 4 days after unilateral or bilateral surgical removal of the entorhinal cortex. The greatest decrease (45%) was seen in the stratum lacunosum moleculare of the CA1, whereas a lesser decline (34%) was seen in the molecular layer of the dentate gyrus, stratum lucidum of the CA3 (31%) and the stratum radiatum of the CA1 (36%). These findings support the view that in addition to the indirect trisynaptic temporo-ammonic pathway, there is a functionally active direct pathway.

Binding of [3H]inositoltrisphosphate and [3H]phorbol 12,13-dibutyrate in rat hippocampus following transient global ischemia: a quantitative autoradiographic study.

An in vitro quantitative autoradiographic binding study of the phosphatidylinositol system ligands [3H]inositol 1,4,5-trisphosphate (IP3) and [3H]phorbol 12,13-dibutyrate (PDBU) to rat brain slices was performed at 6, 12, 24, 28 and 72 h following a 20 min ischemic injury. PDBU binding showed a transient 20% decrease in the dentate gyrus and the CA3 the first 24 h as well as a 50% decrease in the CA1 at 72 h. A 50% decline in IP3 binding was seen in all regions at 6-12 h except the pyramidal cell layer of the CA1. This downregulation of calcium mobilizing intracellular receptors is probably a defence against ischemic neuronal cell death.

Copper and silver corrosion activity in crown and bridge alloys.

Effects of decreased alloy nobility are measured with respect to copper and silver corrosion activity. A potentiodynamic technique is developed for this analysis. It is shown that cooper and silver have characteristic potentials for which individual current densities are maximal. Identification of characteristic potentials allows the definite analysis of 11 commercial crown and bridge alloys for copper and silver corrosion. It is shown that microstructure, as well as alloy chemistry, plays a major role in determining corrosion resistance.

Sequential MR studies of intracerebral hematomas in monkeys.

It is well recognized that the MR appearance of intracranial bleeding changes with the age of lesion. It is also well known that hemoglobin in stagnating blood undergoes oxidation to methemoglobin, a substance that lowers the relaxation times of surrounding water protons. To study these phenomena in a controlled way, about 3 ml of blood was injected into the right frontal lobe of two rhesus monkeys, and they were scanned sequentially for up to 2 months in a Picker NMR scanner (Bo = 0.25-0.5 T). The image intensity of the blood changed during the first week, consistent with the lowering of T1 and T2. On the inversion-recovery scans the initial appearance of the blood was less bright than was the contralateral white matter, reversing after 3-5 days. The opposite was true on spin-echo images. T1 and T2 values were calculated for all images. In parallel experiments, several milliliters of freshly drawn blood was placed in test tubes and relaxation times were measured in a bench-top analyzer at 0.25 T over a period of 10 days. The relaxation times dropped markedly, at a rate that depended on sterility, temperature, etc., closely approaching the expected result for complete conversion of hemoglobin to methemoglobin. Ten blood samples with different methemoglobin concentrations were prepared by adding varying doses of sodium nitrite. The change in 1/T1 was found to be roughly proportional to the methemoglobin concentration for values up to 40%, and the initial slope was consistent with published data.