Differential effects of voluntary physical exercise on behavioral and brain-derived neurotrophic factor expression deficits in Huntington's disease transgenic mice

Huntington's disease is a fatal neurodegenerative disorder caused by a mutation of the huntingtin gene and involves progressive motor abnormalities (including chorea), cognitive deficits (dementia) as well as psychiatric symptoms. We have previously demonstrated that environmental enrichment slows the onset and progression of Huntington's disease in transgenic mice. Here, we investigated the effects of enhanced physical exercise on disease progression and brain-derived neurotrophic factor expression. Standard-housed Huntington's disease mice developed phenotypic rear-paw clasping by 16 weeks of age, displayed abnormal rearing behavior, deficits in motor co-ordination and of spatial working memory. Huntington's disease mice with access to running wheels exhibited delayed onset of rear-paw clasping, normalized levels of rearing behavior and amelioration of the cognitive deficits. However, in contrast to our previous environmental enrichment studies, there was no rescue of motor coordination deficits in wheel-running Huntington's disease mice. An abnormal accumulation of brain-derived neurotrophic factor protein in the frontal cortex of Huntington's disease mice was unaffected by running. Striatal and hippocampal brain-derived neurotrophic factor protein levels were unchanged. Brain-derived neurotrophic factor mRNA levels were reduced in the anterior cortex, striatum and hippocampus of Huntington's disease mice, and only striatal deficits were ameliorated by running. Overall, we show that voluntary physical exercise delays the onset of Huntington's disease and the decline in cognitive ability. In addition, our results reveal that some aspects of hippocampal dependent memory are not entirely reliant on sustained hippocampal brain-derived neurotrophic factor expression.     

K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung

BACKGROUND. The capability of activated oncogenes to induce malignant transformation of immortalized cells in vitro has suggested that they have a similar role in the pathogenesis of human tumors. We previously found that activation of the K-ras oncogene by a point mutation in codon 12 occurs in about one third of human lung adenocarcinomas. METHODS. We studied the clinical importance of this oncogene-activation in 69 patients with lung adenocarcinoma in whom complete resection of the tumor was possible. The polymerase chain reaction was used to amplify ras-specific sequences of DNA isolated from frozen or paraffin-embedded tumor samples. Ras point mutations were subsequently detected and classified with the use of mutation-specific oligonucleotide probes. RESULTS. Nineteen of the tumors harbored a point mutation in codon 12 of the K-ras oncogene. There was no association between the K-ras point mutation and the age at diagnosis, sex, or presence of previous or concurrent neoplasms. Tumors positive for K-ras point mutations tended to be smaller and less differentiated than those without mutations. The K-ras codon-12 point mutation was a strong (and unfavorable) prognostic factor: 12 of the 19 patients with K-ras point-mutation-positive tumors died during the follow-up period, as compared with 16 of the 50 patients with no mutation in the K-ras oncogene (P = 0.002). This difference in prognosis was also reflected in the duration of disease-free survival (P = 0.038) and in the number of deaths due to cancer (P less than 0.001). CONCLUSIONS. The presence of K-ras point mutations defines a subgroup of patients with lung adenocarcinoma in whom the prognosis is very poor and disease-free survival is not usually long despite radical resection and a small tumor load.     

Involvement of lysosome-like particles in the metabolism of endogenous myocardial triglycerides during ischemia/reperfusion. Uptake and degradation of triglycerides by lysosomes isolated from rat heart

Summary The hormonal regulation and enzymatic basis of endogenous lipolysis in heart are not yet completely elucidated. The lysosomal fraction from rat heart appeared to be markedly enriched in triglycerides and a significant reduction in triglycerides in this fraction was found after prolonged perfusion or stimulation of lipolysis with glucagon. The enhanced rate of lipolysis, measured as glycerol release from the isolated perfused rat heart, was abolished 10–15 min after continuous glucagon administration. Omission of glucagon for another 60 min restored the ability of glucagon to stimulate lipolysis, indicating the limited availability of endogenous triglycerides and the presence of a transfer-system for triglycerides from a non-metabolically active pool to a metabolically active pool. The enhanced lipolysis induced by low-flow ischemia was found to be inhibited by the lysosomotropic agent methylamine (5 mM). Methylamine-perfusion during low-flow ischemia was accompanied by an increased recovery of myocardial triglycerides in the lysosomal fraction. The possible role of lysosome-like particles in myocardial triglyceride homeostasis was further investigated by studying the kinetics of uptake and degradation of labeled triglycerides by membrane-particles recovered in the subcellular fraction enriched with lysosomal marker enzymes. It appeared that isolated lysosomal membranes take up added triglycerides at an average rate of 30 nmoles/min/g protein. The bulk of these triglycerides taken up is stored whereas 20% is degraded to diglycerides and free fatty acids. More than 90% of the free fatty acids formed were released from the lysosomes into the supernatant. The uptake and degradation of triglyceride-filled liposomes by isolated myocardial lysosomes was inhibited during incubation with methylamine (5 mM). On the other hand, a lowering of pH during in vitro incubation increased the rate of uptake and degradation of added triglycerides by isolated lysosomes. These results indicate that lysosomes or lysosome-like particles are involved in the enhanced lipolysis during myocardial ischemia.     

Membrane expression of platelet calpain

Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin- activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme- linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.     

Effect of surfaces on fluid-phase prekallikrein activation

The activation of prekallikrein by factor XII fragments (XIIf), during incubation in plastic tubes was previously noted to be increased by high molecular weight (HMW) kininogen as well as other plasma proteins. In this report, we investigated the mechanism responsible for this increase. Although we confirmed that HMW kininogen, bovine serum albumin, fibrinogen, cold insoluble globulin, and mixed phospholipids apparently increased prekallikrein activation, we found that the product of prekallikrein activation (kallikrein) lost substantial activity in less than 0.5 min after exposure to a variety of fresh surfaces. This loss was partially prevented by the presence of various proteins and phospholipids. Similar protection against inactivation of XIIf, the enzyme in this reaction, was also found. In contrast, no loss of the substrate, prekallikrein, was observed during incubation. The loss of kallikrein activity was found to be proportional to the surface area of the incubation vessel as well as the concentration of kallikrein. Further loss of kallikrein activity could also be prevented by pretreating the vessel with kallikrein. We therefore conclude that various substances apparently affect prekallikrein activation in a purified system by preventing the enzyme and product in the reaction mixture from losing activity due to adsorption to a surface.     

Genome-wide analysis of CpG island methylation in bladder cancer identified TBX2, TBX3, GATA2, and ZIC4 as pTa-specific prognostic markers

Background

DNA methylation markers could serve as useful biomarkers, both as markers for progression and for urine-based diagnostic assays.

Objective

Identify bladder cancer (BCa)–specific methylated DNA sequences for predicting pTa-specific progression and detecting BCa in voided urine.

Design, setting, and participants

Genome-wide methylation analysis was performed on 44 bladder tumours using the Agilent 244K Human CpG Island Microarray (Agilent Technologies, Santa Clara, CA, USA). Validation was done using a custom Illumina 384-plex assay (Illumina, San Diego, CA, USA) in a retrospective group of 77 independent tumours. Markers for progression were identified in pTa (n = 24) tumours and validated retrospectively in an independent series of 41 pTa tumours by the SNaPshot method (Applied Biosystems, Foster City, CA, USA).

Measurements

The percentage of methylation in tumour and urine samples was used to identify markers for detection and related to the end point of progression to muscle-invasive disease with Kaplan-Meier models and multivariate analysis.

Results and limitations

In the validation set, methylation of the T-box 2 (TBX2), T-box 3 (TBX3), GATA binding protein 2 (GATA2), and Zic family member 4 (ZIC4) genes was associated with progression to muscle-invasive disease in pTa tumours (p = 0.003). Methylation of TBX2 alone showed a sensitivity of 100%, a specificity of 80%, a positive predictive value of 78%, and a negative predictive value of 100%, with an area under the curve of 0.96 (p < 0.0001) for predicting progression. Multivariate analysis showed that methylation of TBX3 and GATA2 are independent predictors of progression when compared to clinicopathologic variables (p = 0.04 and p = 0.03, respectively). The predictive accuracy improved by 23% by adding methylation of TBX2, TBX3, and GATA2 to the European Organisation for Research and Treatment of Cancer risk scores. We further identified and validated 110 CpG islands (CGIs) that are differentially methylated between tumour cells and control urine. The limitation of this study is the small number of patients analysed for testing and validating the prognostic markers.

Conclusions

We have identified four methylation markers that predict progression in pTa tumours, thereby allowing stratification of patients for personalised follow-up. In addition, we identified CGIs that will enable detection of bladder tumours in voided urine.     

Does sleep restore the topology of functional brain networks?

Previous studies have shown that healthy anatomical as well as functional brain networks have small‐world properties and become less optimal with brain disease. During sleep, the functional brain network becomes more small‐world‐like. Here we test the hypothesis that the functional brain network during wakefulness becomes less optimal after sleep deprivation (SD). Electroencephalography (EEG) was recorded five times a day after a night of SD and after a night of normal sleep in eight young healthy subjects, both during eyes‐closed and eyes‐open resting state. Overall synchronization was determined with the synchronization likelihood (SL) and the phase lag index (PLI). From these coupling strength matrices the normalized clustering coefficient C (a measurement of local clustering) and path length L (a measurement of global integration) were computed. Both measures were normalized by dividing them by their corresponding C‐s and L‐s values of random control networks. SD reduced alpha band C/C‐s and L/L‐s and theta band C/C‐s during eyes‐closed resting state. In contrast, SD increased gamma‐band C/C‐s and L/L‐s during eyes‐open resting state. Functional relevance of these changes in network properties was suggested by their association with sleep deprivation‐induced performance deficits on a sustained attention simple reaction time task. The findings indicate that SD results in a more random network of alpha‐coupling and a more ordered network of gamma‐coupling. The present study shows that SD induces frequency‐specific changes in the functional network topology of the brain, supporting the idea that sleep plays a role in the maintenance of an optimal functional network. Hum Brain Mapp, 2013. © 2011 Wiley Periodicals, Inc.     

Amidolytic assay of human factor XI in plasma: comparison with a coagulant assay and a new rapid radioimmunoassay

The traditional coagulant assay for plasma factor XI suffers from a relatively high coefficient of variation, the need for rare congenitally deficient plasma, and a poor correlation between precision and sensitivity. We have developed a simple functional amidolytic assay for factor XI in plasma using the chromogenic substrate PyrGlu-Pro-Arg- p-nitroanilide (S-2366). After inactivation of alpha 1-antitrypsin, CI inhibitor, and other plasma protease inhibitors with CHCI3, plasma was incubated with kaolin, in the absence of added calcium, which limited the enzymes formed to those dependent on contact activation. Soybean trypsin inhibitor was used to minimize the action of kallikrein on the substrate. Once the reaction was complete, corn trypsin inhibitor was used to inactive factor XIIa, the enzyme generated by exposure of plasma to negatively charged surfaces, which had activated the factor XI. The assay is highly specific for factor XI, since plasma totally deficient in that zymogen yielded only 1%-3% of the enzymatic activity in normal plasma under identical conditions. The requirements for complete conversion of factor XI to XIa in plasma within 60 min were, respectively, factor XII, 0.6 U/ml, and high molecular weight kininogen, 0.2 U/ml. Prekallikrein was not an absolute requirement for complete activation but did accelerate the reaction. The intraassay coefficient of variation was 3.4%, and the mean of 35 normal plasmas was 1.00 U +/- 0.24 SD. In addition, a new rapid radioimmunoassay was devised using staphylococcal protein A as the precipitating agent for a complex of factor XI antigen with monospecific rabbit antibody. The mean was 1.01 U +/- 0.30 SD. The correlation coefficients for amidolytic versus coagulant and amidolytic versus radioimmunoassay were r = 0.95 for the former and 0.96 for the latter. Thus, a simple, accurate amidolytic assay and a radioimmunoassay have been devised for measuring factor XI in plasma that correlate well with the coagulant activity of factor XI, as determined in our laboratory.     

Effects of monoclonal antibody therapy in patients with chronic lymphocytic leukemia

A phase I clinical trial was initiated to treat patients with stage IV B-derived chronic lymphocytic leukemia (CLL) with the IgG2a murine monoclonal antibody T101. This antibody binds to a 65,000-mol wt (T65) antigen found on normal T lymphocytes, malignant T lymphocytes, and B- derived CLL cells. All of the patients had a histologically confirmed diagnosis of advanced B-derived CLL and were refractory to standard therapy, and more than 50% of their leukemia cells reacted with the T101 antibody in vitro. The patients received T101 antibody two times per week, over two to 50 hours by intravenous administration in 100 mL of normal saline containing 5% human albumin. Twelve patients were treated with a fixed dosage of 1, 10, 50, or 100 mg, and one patient was treated with 140 mg of antibody. It was demonstrated that patients given two-hour infusions of 50 mg developed pulmonary toxicity, with shortness of breath and chest tightness. This toxicity was eliminated when infusions of 50 or 100 mg of T101 were prolonged to 50 hours. All dose levels caused a rapid but transient decrease in circulating leukemia cell counts. In vivo binding to circulating and bone marrow leukemia cells was demonstrated at all dose levels with increased binding at higher dosages. Antimurine antibody responses were not demonstrated in any patients at any time during treatment. Circulating free murine antibody was demonstrated in the serum of only the two patients treated with 100 mg of antibody as a 50-hour infusion and the patient treated with 140 mg of antibody over 30 hours. Antigenic modulation was demonstrated in patients treated at all dose levels but was particularly apparent in patients treated with prolonged infusions of 50 and 100 mg of antibody. We were also able to demonstrate antigenic modulation in lymph node cells, which strongly suggests in vivo labeling of these cells. Overall, T101 antibody alone appears to have a very limited therapeutic value for patients with CLL. The observations of in vivo labeling of tumor cells, antigenic modulation, antibody pharmacokinetics, toxicity, and antimurine antibody formation may be used in the future for more effective therapy when drugs or toxins are conjugated to the antibody.