Stroke, myocardial infarction, acute and chronic inflammatory diseases: caspases and other apoptotic molecules as targets for drug development

Mapping of the human and other eukaryotic genomes has provided the pharmacological industry with excellent models for drug discovery. Control of cell proliferation, differentiation, activation and cell removal is crucial for the development and existence of multicellular organisms. Each cell cycle progression, with sequences of DNA replication, mitosis, and cell division, is a tightly controlled and complicated process that, when deregulated, may become dangerous not only to a single cell, but also to the whole organism. Regulation and the proper control of the cell cycle and of programmed cell death (apoptosis) is therefore essential for mammalian development and the homeostasis of the immune system. The molecular networks that regulate these processes are critical targets for drug development, gene therapy, and metabolic engineering. In addition to the primary, intracellular apoptotic suicide machinery, components of the immune system can detect and remove cells and tissue fragments that no longer serve their defined functions. In this review we will focus on apoptotic pathways converging on caspase family proteases, summarizing pharmacological attempts that target genes, proteins, and intermolecular interactions capable of modulating apoptosis and the inflammatory response. The upcoming pharmacological development for treatment of acute pathologies, such as sepsis, SIRS, stroke, traumatic brain injury, myocardial infarction, spinal cord injury, acute liver failure, as well as chronic disorders such as Huntington's disease, Parkinson's disease, ALS, and rheumatoid arthritis, will be discussed in details. We also suggest new potential molecular targets that may prove to be effective in controlling apoptosis and the immune response in vivo.     

Clinical evaluation of a compomer and an amalgam primary teeth class II restorations: a 2-year comparative study

PURPOSE: This study was performed to compare the clinical performance between the compomer F2000 and amalgam Dispersalloy in Class II restorations in primary molars over a 2-year period. METHODS: Seventy-five amalgam and 75 compomer restorations were placed in 75 children based on a split-mouth design. The restorations were evaluated after 1 week and after 6, 12, 18, and 24 months of oral function. The evaluation consisted of a clinical assessment according to modified Ryge criteria, a radiographic examination using bite-wing radiographs, and an observation of epoxy casts under scanning electron microscopy. RESULTS: The results showed statistically significant differences in the marginal adaptation and anatomic form between amalgam and compomer restorations. A higher number of compomer restorations were rated as Bravo, while a higher number of amalgam restorations were rated as Alpha at 24 months. Significant differences in the failure of the restoration and development of secondary caries were not found between the materials. CONCLUSIONS: The use of compomer F2000 in Class II resorations in primary molars, although it presents a significantly higher number of restorations rated as Bravo regarding the marginal adaptation and anatomic form vs the amalgam, does not increase the risks of developing secondary caries and failure of the restoration over a period of 2 years.     

Detection of Anaplasma phagocytophilum in animals by real-time polymerase chain reaction

The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animals and to identify the phylogenetic relationships of different strains of this bacterium. We adapted six published conventional methods targeting 16S fragments for real-time polymerase chain reaction. Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed with several different primers and a TaqMan probe. We also performed DNA quantification and melting curve analysis. The nucleic acid of Anaplasma sp. was detected in a higher percentage of cases in members of the deer family, hares, bank voles and mice (12.5 approximately 15%) than in foxes, boars, cows, and horses (around 4 approximately 6%). We also performed blood analysis of cows, horses, mice, and ticks removed from animals, evaluating the presence of antibodies against granulocytic Anaplasma sp. Finally, we subjected 11 randomly selected PCR amplified products to direct sequencing and we constructed the corresponding phylogenetic tree with respect to the Ehrlichia equi sequence, homologous to the human granulocytic ehrlichiosis agent. Mutual identity of the sequencing ranged from 99% to 100%.     

Greek Orthodox fasting rituals: a hidden characteristic of the Mediterranean diet of Crete

The longevity and excellent health status of the population of Crete has been attributed to its lifestyle and dietary habits. The impact of Greek Orthodox Christian Church fasting on these dietary habits has never been studied. One hundred and twenty Greek Orthodox Christians living in Crete participated in a 1-year prospective study. One half of the subjects, who fasted regularly (fasters), and sixty non-faster controls were followed longitudinally for the three main fasting periods over 1 year; Christmas (40 d), Lent (48 d) and the Assumption (15 d). Pre- and end-holy days measurements were performed in each fasting period including: 24 h dietary recall, blood collection and anthropometric measurements. Based on the 24 h recall, fasters as compared with controls had lower intakes of end-holy days dietary cholesterol, total fat, saturated fatty acids, trans-fatty acids and protein (P < 0.001). Fasters presented a decrease of 753 kJ (180 kcal) in end-holy days energy intake (P < 0.05) compared with an increase of 573 kJ (137 kcal) in the controls (P < 0.05). Fasters had a decrease in end-holy days Ca intake (P < 0.001) and an increase in end-holy days total dietary fibre (P < 0.001) and folate (P < 0.05), attributed to their higher consumption of fruit and vegetables in end-holy periods (P < 0.001). There were no differences for other vitamins or minerals between pre- and end-holy periods in both groups except for vitamin B2. The Orthodox Christian dietary regulations are an important component of the Mediterranean diet of Crete characterised by low levels of dietary saturated fatty acids, high levels of fibre and folate, and a high consumption of fruit, vegetables and legumes.     

'Designer' tumors in mice

We have developed and tested successfully a general method based on Cre-mediated recombination that can be used for ubiquitous or tissue-specific expression of protein products, including tumor-inducing oncoproteins. Depending on the specificity of a chosen promoter driving cre expression, tumors develop by design in bitransgenic mouse progeny derived by crossing Cre-producing mice with partners carrying a dormant oncogenic transgene (targeted into the 3' noncoding region of the cytoplasmic beta-actin locus) that becomes functional after excision of a 'floxed' DNA segment. To provide proof-of-principle, we have used as models transgenes encoding the polyomavirus middle T antigen (PVMT) and the T antigens of the SV40 early region (SVER). Cre-dependent activation of widespread SVER expression resulted in hyperplasias or invasive tumors affecting particular visceral smooth muscles, whereas Cre-dependent, mammary gland-specific expression of PVMT-induced adenocarcinomas, according to plan. Unexpectedly, we also encountered spontaneous (Cre-independent) oncogene expression occurring as a rare event, which simulates the initiation of sporadic tumors and leads to PVMT-induced hemangiomas and mammary carcinomas or SVER-induced disseminated sarcomas, thus, revealing particular tissue susceptibilities to the actions of these oncoproteins.     

Cytokines, nitric oxide, and cGMP modulate the permeability of an in vitro model of the human blood-brain barrier

The endothelial cells (EC) of the microvasculature in the brain form the anatomical basis of the blood-brain barrier (BBB). In the present study, the effects of agents that modify the permeability of a well-established in vitro model of the human BBB were studied. The monolayers formed by confluent human brain microvessel endothelial cell (HBMEC) cultures are impermeable to the macromolecule tracer horseradish peroxidase (HRP) and have high electrical resistance. Exposure of HBMEC to various cytokines including TNF-alpha, IL-1beta, interferon gamma (IFN-gamma), or lipopolysaccharide (LPS) decreased transendothelial electrical resistance (TEER) mainly by increasing the permeability of the tight junctions. Primary cultures of HBMEC express endothelial nitric oxide synthase (eNOS) and produce low levels of NO. Treatment with the NO donors sodium nitroprusside (SNP) and DETA NONOate or the cGMP agonist 8-Br-cGMP significantly increased monolayer resistance. Conversely, inhibition of soluble guanylyl cyclase with ODQ rapidly decreased the resistance, and pretreatment of HBMEC with Rp-8-CPT-cGMPS, an inhibitor of cGMP-dependent protein kinase, partially prevented the 8-Br-cGMP-induced increase in resistance. Furthermore, NO donors and 8-Br-cGMP could also reverse the increased permeability of the monolayers induced by IL-1beta, IFN-gamma, and LPS. These results indicate that NO can decrease the permeability of the human BBB through a mechanism at least partly dependent on cGMP production and cGMP-dependent protein kinase activation.     

Altered long chain fatty acids composition in Duchenne muscular dystrophy erythrocytes

BACKGROUND: Biochemical abnormalities, increased efflux of soluble enzymes and muscle proteins, and altered permeability of muscle membranes imply the presence of a disorganized erythrocyte membrane in Duchenne muscular dystrophy (DMD). The purpose of the present study was to investigate this hypothesis of a generalized membrane defect. MATERIALS AND METHODS: Twenty-five patients with the disease were analyzed for their erythrocyte lipid composition and for alterations in their fatty acid content compared to twenty-five healthy subjects. RESULTS: DMD patients showed a decreased concentration of total phospholipids compared to healthy volunteers, with striking fluctuations in concentrations of erythrocyte long chain fatty acids. Specifically, the unsaturated fatty acids such as oleic, linoleic and arachidonic acids were significantly decreased in the disease, whereas the saturated fatty acid, palmitic acid was increased in DMD patients compared to healthy controls. CONCLUSION: Our findings suggest an abnormal fatty acid composition and disorganization of erythrocyte membrane in patients with DMD associated with possible functional alterations.     

Effect of maltose-containing sports drinks on exercise performance

This study examined the effect of maltose-containing sports drinks on exercise performance. Ten subjects completed 4 trials. Each trial consisted of a glycogen depletion protocol, followed by a 15-min refueling, after which subjects performed an 1-h performance test while consuming one of the experimental drinks (HGlu, glucose; HMal, maltose; MalMix, sucrose, maltose, and maltodextrin; Plac, placebo). Drinks provided 0.65 g/kg body weight carbohydrates during refueling and 0.2 g/kg every 15 min during the performance test. Although no significant differences were found in performance (HGlu: 67.2 +/- 2.0; HMal: 68.6 +/- 1.7; MalMix: 66.7 +/- 2.0; Plac: 69.4 +/- 3.0 min, P> 0.05), subjects completed the MalMix trial 3.9%; faster than the Plac. Carbohydrate drinks caused comparable plasma glucose values that were significantly higher during refueling and at the end of exercise, compared to Plac. The data suggest that although carbohydrate drinks help to maintain plasma glucose at a higher level, no differences in performance could be detected after glycogen-depleting exercise.