Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor.     

Adventitia-derived hydrogen peroxide impairs relaxation of the rat carotid artery via smooth muscle cell p38 mitogen-activated protein kinase

The role of adventitia-derived reactive oxygen species (ROS) in vascular disease and impaired vascular relaxation is not clear. Based on robust adventitial ROS generation and effects on MAPK involvement in vascular dysfunction, we hypothesized that adventitia-derived ROS hydrogen peroxide (H(2)O(2)) impairs vascular relaxation through activation of medial smooth muscle p38 MAPK. By using a novel in vivo model, the adventitial surface of rat carotid arteries was bathed in situ for 90 min with vehicle, angiotensin II (AngII; 500 nM), AngII+H(2)O(2)-scavenger catalase (3,000 U/ml), AngII+p38 MAPK inhibitor SB203580 (10 μM), or AngII+superoxide dismutase (SOD; 150 U/ml). After these in vivo treatments, ex vivo tone measurements on isolated vessels revealed that periadventitial application of AngII impaired both acetylcholine-induced (endothelium-dependent) and sodium nitroprusside-induced (endothelium-independent) relaxations. In vivo coincubation with catalase or SB203580 significantly improved, but SOD exacerbated AngII-induced impairment of in vitro endothelium-dependent and -independent vascular relaxations. Western blots of vascular media, separated from the adventitia, demonstrated increased medial p38 MAPK activation and decreased medial phosphatase SHP-2 activity in AngII-treated vessels. These effects were reversed by in vivo periadventitial addition of catalase. These findings provide the first evidence that adventitia-derived H(2)O(2) participates in vascular dysfunction through p38 MAPK activation and SHP-2 inhibition.     

Narrow-band imaging endoscopy to assess mucosal angiogenesis in inflammatory bowel disease: A pilot study

AIM: To investigate whether narrow band imaging (NBI) is a useful tool for the in vivo detection of angiogenesis in inflammatory bowel disease (IBD) patients. METHODS: Conventional and NBI colonoscopy was performed in 14 patients with colonic inflammation (8 ulcerative colitis and 6 Crohn’s disease). Biopsy samples were taken and CD31 expression was assayed immuno- histochemically; microvascular density was assessed by vessel count. RESULTS: In areas that were endoscopically normal but positive on NBI, ther...     

Expression and modulation of IFN-gamma-inducible chemokines (IP-10, Mig, and I-TAC) in human brain endothelium and astrocytes: possible relevance for the immune invasion of the central nervous system and the pathogenesis of multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) mediated by blood-derived immune cells invading the CNS. This invasion could be determined by chemokines, and their role within the MS-affected brain is still poorly defined. We investigated the expression by RT-PCR and protein release by ELISA of the interferon-gamma (IFN-gamma)-inducible chemokines in human brain microvascular endothelial cells (HBMECs) and astrocytes. The monokine induced by IFN-gamma (Mig) behaves as a homing chemokine constitutively expressed in HBMECs and astrocytes, whereas the IFN-gamma-inducible 10-kDa protein (IP-10) and IFN-inducible T cell alpha-chemoattractant (I-TAC) are induced only after inflammatory stimuli. The biologic activity of IFN-gamma-inducible chemokines from an endothelial source was analyzed, and the transendothelial migration of activated lymphocytes was partly antagonized by specific antibodies, especially anti-Mig antibody. Our data highlight the capability of cells of the CNS to activate the chemoattractant machinery in a proinflammatory environment and in MS.     

Impact of aging on cardiac sympathetic innervation measured by <Superscript>123</Superscript>I-mIBG imaging in patients with systolic heart failure

Purpose

Sympathetic nervous system (SNS) hyperactivity is a salient characteristic of chronic heart failure (HF) and contributes to the progression of the disease. Iodine-123 meta-iodobenzylguanidine (¹²³I-mIBG) imaging has been successfully used to assess cardiac SNS activity in HF patients and to predict prognosis. Importantly, SNS hyperactivity characterizes also physiological ageing, and there is conflicting evidence on cardiac ¹²³I-mIBG uptake in healthy elderly subjects compared to adults. However, little data are available on the impact of ageing on cardiac sympathetic nerve activity assessed by ¹²³I-mIBG scintigraphy, in patients with HF.

Methods and results

We studied 180 HF patients (age?=?66.1?±?10.5 years [yrs]), left ventricular ejection fraction (LVEF?=?30.6?±?6.3 %) undergoing cardiac ¹²³I-mIBG imaging. Early and late heart to mediastinum (H/M) ratios and washout rate were calculated in all patients. Demographic, clinical, and echocardiographic data were also collected. Our study population consisted of 53 patients aged >75 years (age?=?77.7?±?4.0 year), 67 patients aged 62–72 years (age?=?67.9?±?3.2 years) and 60 patients aged ≤61 year (age?=?53.9?±?5.6 years). In elderly patients, both early and late H/M ratios were significantly lower compared to younger patients (p?<?0.05). By multivariate analysis, H/M ratios (both early and late) and washout rate were significantly correlated with LVEF and age.

Conclusions

Our data indicate that, in a population of HF patients, there is an independent age-related effect on cardiac SNS innervation assessed by ¹²³I-mIBG imaging. This finding suggests that cardiac ¹²³I-mIBG uptake in patients with HF might be affected by patient age.

     

PSD-95 in CA1 Area Regulates Spatial Choice Depending on Age

Cognitive processes that require spatial information rely on synaptic plasticity in the dorsal CA1 area (dCA1) of the hippocampus. Since the function of the hippocampus is impaired in aged individuals, it remains unknown how aged animals make spatial choices. Here, we used IntelliCage to study behavioral processes that support spatial choices of aged female mice living in a group. As a proxy of training-induced synaptic plasticity, we analyzed the morphology of dendritic spines and the expression of a synaptic scaffold protein, PSD-95. We observed that spatial choice training in young adult mice induced correlated shrinkage of dendritic spines and downregulation of PSD-95 in dCA1. Moreover, long-term depletion of PSD-95 by shRNA in dCA1 limited correct choices to a reward corner, while reward preference was intact. In contrast, old mice used behavioral strategies characterized by an increased tendency for perseverative visits and social interactions. This strategy resulted in a robust preference for the reward corner during the spatial choice task. Moreover, training decreased the correlation between PSD-95 expression and the size of dendritic spines. Furthermore, PSD-95 depletion did not impair place choice or reward preference in old mice. Thus, our data indicate that while young mice require PSD-95-dependent synaptic plasticity in dCA1 to make correct spatial choices, old animals observe cage mates and stick to a preferred corner to seek the reward. This strategy is resistant to the depletion of PSD-95 in the CA1 area. Overall, our study demonstrates that aged mice combine alternative behavioral and molecular strategies to approach and consume rewards in a complex environment.SIGNIFICANCE STATEMENT It remains poorly understood how aging affects behavioral and molecular processes that support cognitive functions. It is, however, essential to understand these processes to develop therapeutic interventions that support successful cognitive aging. Our data indicate that while young mice require PSD-95-dependent synaptic plasticity in dCA1 to make correct spatial choices (i.e., choices that require spatial information), old animals observe cage mates and stick to a preferred corner to seek the reward. This strategy is resistant to the depletion of PSD-95 in the CA1 area. Overall, our study demonstrates that aged mice combine alternative behavioral and molecular strategies to approach and consume rewards in a complex environment. Second, the contribution of PSD-95-dependent synaptic functions in spatial choice changes with age.     

Serum human glandular kallikrein (hK2) and insulin-like growth factor 1 (IGF-1) improve the discrimination between prostate cancer and benign prostatic hyperplasia in combination with total and %free PSA

BACKGROUND: There is growing evidence describing an association of hK2 and IGFs with cancer. The aim of this study is to investigate the differences in serum levels of hK2 and IGFs in a large group of patients with benign prostatic hyperplasia (BPH) or prostatic carcinoma (CaP) and to examine the value of these variables, as well as their various combinations with PSA, for discriminating between these two clinical entities. METHODS: Human glandular kallikrein 2 (hK2), insulin-like growth factor-1 (IGF-1), free and total PSA concentrations were measured with non-competitive immunological procedures. Receiver operating characteristic (ROC) analysis as well as univariate and multivariate logistic regression analysis were performed to investigate the potential utility of the various markers and their combinations for discriminating between BPH and CaP. RESULTS: hK2 and IGF-1 concentrations were increased in CaP patients, in comparison to BPH patients. hK2/free PSA and free/total PSA ratios (area under the curve, AUC = 0.70) were stronger predictors of prostate cancer than the IGF-1/total PSA ratio (AUC = 0.56) in the group of patients with total PSA <4 microg/L. The hK2/free PSA ratio (AUC = 0.74) was found to have significant discriminatory value in patients with total PSA within the "gray zone" (4-10 microg/L). Multivariate logistic regression models confirmed the observed relationships and identified IGF-1/free PSA and hK2/free PSA as independent predictors of CaP. CONCLUSIONS: hK2/free PSA and IGF-1/free PSA ratios may be useful adjuncts in improving patient selection for prostate biopsy.