Proteomic analysis of activity-dependent synaptic plasticity in hippocampal neurons

Following long-term treatment with bicuculline and tetrodotoxin (TTX) aimed at modifying synaptic activity in cultured neurons, we used a proteomic approach to identify the associated changes in protein expression. The neurons were left untreated, or treated with bicuculline or TTX, and fractionated by means of differential detergent extraction, after which the proteins in each fraction were separated by means of two-dimensional (2D) gel electrophoresis, and 57 proteins of interest were identified by mass spectrometry. The proteins that showed altered expression and/or post-translational modifications include proteins or enzymes involved in regulating cell and protein metabolism, the cytoskeleton, or mitochondrial activity. These results suggest that extensive alterations in neuronal protein expression take place as a result of increased or decreased synaptic activity.     

Increased autophagic degradation of mitochondria in Alzheimer disease

Extensive literature exists supporting a role for mitochondrial dysfunction and oxidative damage in the pathogenesis of Alzheimer disease. Mitochondria are a major source of intracellular reactive oxygen species and are themselves particularly vulnerable to oxidative stress. It has been recently shown that the immunoreactivity of lipoic acid and cytochrome oxidase-1, two mitochondrial markers, is increased in the cytoplasm of pyramidal neurons in Alzheimer disease cases compared with controls. Furthermore, lipoic acid was found to be strongly associated with granular structures and, by ultrastructure analysis, shown to be localized in mitochondria, cytosol and, importantly, in organelles identified as autophagic vacuoles. Lipoic acid was also found associated with the electron dense core of lipofuscin in the brains of Alzheimer disease cases but not in controls, whereas cytochrome oxidase-1 immunoreactivity was limited to mitochondria and cytosol in both Alzheimer and control cases. These data suggest that mitochondria are key targets of increased autophagic degradation in Alzheimer disease. The study of autophagy in Alzheimer disease could clarify the mechanisms underlying this neurodegenerative disorder and, eventually, help in the development of new therapeutic strategies.     

Pilocarpine-induced status epilepticus in rats involves ischemic and excitotoxic mechanisms

The neuron loss characteristic of hippocampal sclerosis in temporal lobe epilepsy patients is thought to be the result of excitotoxic, rather than ischemic, injury. In this study, we assessed changes in vascular structure, gene expression, and the time course of neuronal degeneration in the cerebral cortex during the acute period after onset of pilocarpine-induced status epilepticus (SE). Immediately after 2 hr SE, the subgranular layers of somatosensory cortex exhibited a reduced vascular perfusion indicative of ischemia, whereas the immediately adjacent supragranular layers exhibited increased perfusion. Subgranular layers exhibited necrotic pathology, whereas the supergranular layers were characterized by a delayed (24 h after SE) degeneration apparently via programmed cell death. These results indicate that both excitotoxic and ischemic injuries occur during pilocarpine-induced SE. Both of these degenerative pathways, as well as the widespread and severe brain damage observed, should be considered when animal model-based data are compared to human pathology.     

Neuroacanthocytosis associated with a defect of the 4.1R membrane protein

Background

Neuroacanthocytosis (NA) denotes a heterogeneous group of diseases that are characterized by nervous system abnormalities in association with acanthocytosis in the patients' blood. The 4.1R protein of the erythrocyte membrane is critical for the membrane-associated cytoskeleton structure and in central neurons it regulates the stabilization of AMPA receptors on the neuronal surface at the postsynaptic density. We report clinical, biochemical, and genetic features in four patients from four unrelated families with NA in order to explain the cause of morphological abnormalities and the relationship with neurodegenerative processes.

Case presentation

All patients were characterised by atypical NA with a novel alteration of the erythrocyte membrane: a 4.1R protein deficiency. The 4.1R protein content was significantly lower in patients (3.40 ± 0.42) than in controls (4.41 ± 0.40, P < 0.0001), reflecting weakened interactions of the cytoskeleton with the membrane. In patients IV:1 (RM23), IV:3 (RM15), and IV:6 (RM16) the 4.1 deficiency seemed to affect the horizontal interactions of spectrin and an impairment of the dimer self-association into tetramers was detected. In patient IV:1 (RM16) the 4.1 deficiency seemed to affect the skeletal attachment to membrane and the protein band 3 was partially reduced.

Conclusion

A decreased expression pattern of the 4.1R protein was observed in the erythrocytes from patients with atypical NA, which might reflect the expression pattern in the central nervous system, especially basal ganglia, and might lead to dysfunction of AMPA-mediated glutamate transmission.     

p66SHC promotes T cell apoptosis by inducing mitochondrial dysfunction and impaired Ca2+ homeostasis

p66Shc, a redox enzyme that enhances reactive oxygen species (ROS) production by mitochondria, promotes T cell apoptosis. We have addressed the mechanisms regulating p66Shc-dependent apoptosis in T cells exposed to supraphysiological increases in [Ca2+]c. p66Shc expression resulted in profound mitochondrial dysfunction in response to the Ca2+ ionophore A23187, as revealed by dissipation of mitochondrial transmembrane potential, cytochrome c release and decreased ATP levels. p66Shc expression also caused a dramatic alteration in the cells' Ca2+-handling ability, which resulted in Ca2+ overload after A23187 treatment. The impairment in Ca2+ homeostasis was ROS dependent and caused by defective Ca2+ extrusion due at least in part to decreased plasma membrane ATPase (PMCA) expression. Both effects of p66Shc required Ca2+-dependent serine-36 phosphorylation. The mitochondrial effects of p66Shc were potentiated by but not strictly dependent on the rise in [Ca2+]c. Thus, Ca2+-dependent p66Shc phosphorylation causes both mitochondrial dysfunction and impaired Ca2+ homeostasis, which synergize in promoting T cell apoptosis.     

Cardiac reflections and natural vibrations: Force-frequency relation recording system in the stress echo lab

Background

The inherent ability of ventricular myocardium to increase its force of contraction in response to an increase in contraction frequency is known as the cardiac force-frequency relation (FFR). This relation can be easily obtained in the stress echo lab, where the force is computed as the systolic pressure/end-systolic volume index ratio, and measured for increasing heart rates during stress. Ideally, the noninvasive, imaging independent, objective assessment of FFR would greatly enhance its practical appeal.

Objectives

1 – To evaluate the feasibility of the cardiac force measurement by a precordial cutaneous sensor. 2 – To build the curve of force variation as a function of the heart rate. 3 – To compare the standard stress echo results vs. this sensor operator-independent built FFR.

Methods

The transcutaneous force sensor was positioned in the precordial region in 88 consecutive patients referred for exercise, dipyridamole, or pacing stress. The force was measured as the myocardial vibrations amplitude in the isovolumic contraction period. FFR was computed as the curve of force variation as a function of heart rate. Standard echocardiographic FFR measurements were performed.

Results

A consistent FFR was obtained in all patients. Both the sensor built and the echo built FFR identifiy pts with normal or abnormal contractile reserve. The best cut-off value of the sensor built FFR was 15.5 g * 10^-3 (Sensitivity = 0.85, Specificity = 0.77). Sensor built FFR slope and shape mirror pressure/volume relation during stress. This approach is extendable to daily physiological exercise and could be potentially attractive in home monitoring systems.     

Surgical outcomes for colon and rectal cancer over a decade: results from a consecutive monocentric experience in 902 unselected patients

Background

This study evaluates the surgical morbidity and long-term outcome of colorectal cancer surgery in an unselected group of patients treated over the period 1994–2003.

Methods

A consecutive series of 902 primary colorectal cancer patients (489 M, 413 F; mean age: 63 years ± 11 years, range: 24–88 years) was evaluated and prospectively followed in a university hospital (mean follow-up 36 ± 24 months; range: 3–108 months). Perioperative mortality, morbidity, overall survival, curative resection rates, recurrence rates were analysed.

Results

Of the total, 476 colorectal cancers were localized to the colon (CC, 53%), 406 to the rectum (RC, 45%), 12 (1%) were multicentric, and 8 were identified as part of HNPCC (1%). Combining all tumours, there were 186 cancers (20.6%) defined as UICC stage I, 235 (26.1%) stage II, 270 (29.9%) stage III and 187 (20.6%) stage IV cases. Twenty-four (2.7%) cases were of undetermined stage. Postoperative complications occurred in 38% of the total group (37.8% of CC cases, 37.2% of the RC group, 66.7% of the synchronous cancer patients and 50% of those with HNPCC, p = 0.19) Mortality rate was 0.8%, (1.3% for colon cancer, 0% for rectal cancer; p = 0.023). Multivisceral resection was performed in 14.3% of cases. Disease-free survival in cases resected for cure was 73% at 5-years and 72% at 8 years. The 5- and 8-year overall survival rates were 71% and 61% respectively (total cases). At 5-year analysis, overall survival rates are 97% for stage I disease, 87% for stage II, 73% for stage III and 22% for stage IV respectively (p < 0.0001). The 5-year overall survival rates showed a marked difference in R0, R1+R2 and non resected patients (82%, 35% and 0% respectively, p < 0.0001). On multivariate analysis, resection for cure and stage at presentation but not tumour site (colon vs. rectum) were independent variables for overall survival (p < 0.0001).

Conclusion

A prospective, uniform follow-up policy used in a single institution over the last decade provides evidence of quality assurance in colorectal cancer surgery with high rates of resection for cure where only stage at presentation functions as an independent variable for cancer-related outcome.     

Global 'worming'

A report on the 16th International Caenorhabditis elegans Meeting, Los Angeles, USA, 27 June-1 July 2007.     

Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies

The promyelocytic leukaemia (PML) tumour suppressor protein epitomizes the PML-nuclear body (PML-NB) and is crucially required for the proper assembly of this macromolecular nuclear structure. Unlike other, more specialized subnuclear structures such as Cajal and Polycomb group bodies, PML-NBs are functionally promiscuous and have been implicated in the regulation of diverse cellular functions. PML-NBs are dynamic structures that favour the sequestration and release of proteins, mediate their post-translational modifications and promote specific nuclear events in response to various cellular stresses. Recent data suggest that PML-NBs may be heterogeneous in composition, mobility and function.