Assessing the role of aromatic residues in the amyloid aggregation of human muscle acylphosphatase

Among the many parameters that have been proposed to promote amyloid fibril formation is the pi-stacking of aromatic residues. We have studied the amyloid aggregation of several mutants of human muscle acylphosphatase in which an aromatic residue was substituted with a non-aromatic one. The aggregation rate was determined using the Thioflavin T test under conditions in which the variants populated initially an ensemble of partially unfolded conformations. Substitutions in aggregation-promoting fragments of the sequence result in a dramatically decreased aggregation rate of the protein, confirming the propensity of aromatic residues to promote this process. Nevertheless, a statistical analysis shows that the measured decrease of aggregation rate following mutation arises predominantly from a reduction of hydrophobicity and intrinsic beta-sheet propensity. This suggests that aromatic residues favor aggregation because of these factors rather than for their aromaticity.     

In vivo effects of pentoxifylline on enzyme and non-enzyme antioxidant levels in rat liver after carrageenan-induced paw inflammation

The present study aimed to investigate the effects of pentoxifylline (PTX) on the carrageenan (CG)-induced paw oedema and on the endogenous levels of cell enzyme and non-enzyme antioxidants in rat liver, 4 and 24 h after CG injection. PTX (50 mg kg(-1) , i.p.), administered 30 min before CG, decreased the paw oedema, 2-4 h after CG administration. The drug protected CG-induced decrease of glutathione (non-enzyme antioxidant) and had no effect on CG-unchanged activities of superoxide dismutase, glutathione peroxidase (enzyme antioxidants) and glucose-6-phosphate dehydrogenase (enzyme, important for the activity of GSH-conjugated antioxidant enzymes). The drug showed a good antioxidant capacity in chemical systems, generating reactive oxygen species. The present results suggest that the antioxidant activity of PTX might contribute to its beneficial effects in liver injuries.     

The cardiovascular profile of soccer referees: an echocardiographic study

Background

Previous studies demonstrated a modest association between C-Reactive Protein (CRP), stenosis of carotid artery, and carotid Intima-Media Thickness (IMT) in general population. During present study, we aimed to evaluate the relationship between High Sensitivity C-Reactive Protein (hsCRP) and Common Carotid Intima-Media Thickness (CCIMT) in patients who candidate for Coronary Artery Bypass Grafting (CABG).

Methods

The study subjects were enrolled from patients with coronary arteries disease referred from Shahid Madani Hospital (Tabriz, Iran), who have been candidate for elective CABG from January 2005 to August 2007. The common carotid arteries were evaluated with high-resolution B-mode ultrasonography using a 7.5- MHz linear-array transducer to determine the IMT and grade of stenosis. Serum hsCRP level was measured using commercially available enzyme linked immunosorbent assay kit.

Results

Finally, information of 176 CABG candidates was analysed. The mean age of participants was 62.71 ± 9.45 years with 1.63 male to female ratio. The mean of CCIMT was 0.69 ± 0.54 mm. Although there was no significant correlation between serum hsCRP level and CCIMT in patients without carotid stenosis (p=0.113, r=0.186), participants with common carotid artery stenosis had higher levels of serum hsCRP than participants without stenosis (2.42+/-1.30 vs. 1.20+/-0.97 mg/dl; p=0.009).

Conclusion

Study results showed that there was no correlation between serum hsCRP level and CCIMT in patients without carotid stenosis, but patients with common carotid artery stenosis had higher levels of serum hsCRP than patients without stenosis.     

Pancreatic insulin content regulation by the estrogen receptor ER alpha

The function of pancreatic beta-cells is the synthesis and release of insulin, the main hormone involved in blood glucose homeostasis. Estrogen receptors, ER alpha and ER beta, are important molecules involved in glucose metabolism, yet their role in pancreatic beta-cell physiology is still greatly unknown. In this report we show that both ER alpha and ER beta are present in pancreatic beta-cells. Long term exposure to physiological concentrations of 17beta-estradiol (E2) increased beta-cell insulin content, insulin gene expression and insulin release, yet pancreatic beta-cell mass was unaltered. The up-regulation of pancreatic beta-cell insulin content was imitated by environmentally relevant doses of the widespread endocrine disruptor Bisphenol-A (BPA). The use of ER alpha and ER beta agonists as well as ER alphaKO and ER betaKO mice suggests that the estrogen receptor involved is ER alpha. The up-regulation of pancreatic insulin content by ER alpha activation involves ERK1/2. These data may be important to explain the actions of E2 and environmental estrogens in endocrine pancreatic function and blood glucose homeostasis.     

Small non-coding RNAs in animal development

The modulation of gene expression by small non-coding RNAs is a recently discovered level of gene regulation in animals and plants. In particular, microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) have been implicated in various aspects of animal development, such as neuronal, muscle and germline development. During the past year, an improved understanding of the biological functions of small non-coding RNAs has been fostered by the analysis of genetic deletions of individual miRNAs in mammals. These studies show that miRNAs are key regulators of animal development and are potential human disease loci.     

Neurophysiological Age Differences During Task-Performance in a Stereoscopic Virtual Environment

In today’s society, there is an increasing number of workplaces in virtual environments (VE). But, there are only a few reports dealing with occupational health issues or age effects. The question arises how VR generally interferes with cognitive processes. This interference might have relevant implications for workability and work-efficiency in virtual environments. Event-related potentials are known to reflect different stages of stimulus reception, evaluation, and response. We have established an electroencephalographic (EEG) monitoring, focussing on event-related potentials (N100; mismatch negativity, i.e., MMN) to obtain access to attention dependent and pre-attentive processing of sensory stimuli applied in VE. The MMN is known to be correlated with the ability of subjects to react to an unexpected event. The aim of the present study was to investigate cognitive responses to distracting auditory stimuli in two different age groups in a virtual environment (VE) and in a real environment (“real reality”), and to compare characteristic neurophysiological response patterns. Data show that stimulus detection as given by the N100 amplitude and latency does not differ in both age groups and task conditions. In contrast, the pre-attentive processing as given by the MMN is altered in the VR such as the non-VR condition in an age-related manner. A relevant finding of the present study was that the age related differences seen in the non-VR condition were not strengthened in VR.     

Neuropeptide neurotensin stimulates intestinal wound healing following chronic intestinal inflammation

Because neurotensin (NT) and its high-affinity receptor (NTR1) modulate immune responses, chloride secretion, and epithelial cell proliferation, we sought to investigate their role in the repair process that follows the development of mucosal injuries during a persistent inflammation. Colonic NT and NTR1, mRNA, and protein significantly increased only after dextran sodium sulfate (DSS)-induced inflammatory damage developed. Colitis-induced body weight loss, colonic myeloperoxidase activity, and histological damage were significantly enhanced by SR-48642 administration, a nonpeptide NTR1 antagonist, whereas continuous NT infusion ameliorated colitis outcome. To evaluate the NT and NTR1 role in tissue healing, mucosal inflammatory injury was established administering 3% DSS for 5 days. After DSS discontinuation, mice rapidly gained weight, ulcers were healed, and colonic NT, NTR1, and cyclooxygenase (COX)-2 mRNA levels were upregulated, whereas SR-48642 treatment caused a further body weight loss, ulcer enlargement, and a blunted colonic COX-2 mRNA upregulation. In a wound-healing model in vitro, NT-induced cell migration in the denuded area was inhibited by indomethacin but not by an antitransforming growth factor-beta neutralizing antibody. Furthermore, NT significantly increased COX-2 mRNA levels by 2.4-fold and stimulated PGE(2) release in HT-29 cells. These findings suggest that NT and NTR1 are part of the network activated after mucosal injuries and that NT stimulates epithelial restitution at least, in part, through a COX-2 dependent pathway.     

beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide

Endoproteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases generates the toxic amyloid beta-peptide (Abeta), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Abeta based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the beta-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the beta-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by alpha-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Abeta. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.     

Relative influence of hydrophobicity and net charge in the aggregation of two homologous proteins

A potentially amyloidogenic protein has to be at least partially unfolded to form amyloid aggregates. However, aggregation of the partially or totally unfolded state of a protein is modulated by at least three other factors: hydrophobicity, propensity to form secondary structure, and net charge of the polypeptide chain. We propose to evaluate the relative importance of net charge, as opposed to the other factors, on protein aggregation and amyloidogenicity. For this aim, we have used two homologous proteins that were previously shown to be able to form amyloid fibrils in vitro, the N-terminal domain of HypF from Escherichia coli (HypF-N) and human muscle acylphosphatase (AcP). The aggregation process from an ensemble of partially unfolded conformations is ca. 1000-fold faster for HypF-N than for AcP. This difference can mainly be attributed to a higher hydrophobicity and a lower net charge for HypF-N than for AcP. By using protein engineering methods, we have decreased the net charge of AcP to a value identical to that of wild-type HypF-N and increased the net charge of HypF-N to a value identical to that of wild-type AcP. Amino acid substitutions were selected to minimize changes in hydrophobicity and secondary structure propensities. We were able to estimate that the difference in net charge between the two wild-type proteins contributes to 20-25% of the difference in their aggregation rates. An understanding of the relative influences of these forces in protein aggregation has implications for elucidating the complexity of the aggregation process, for predicting the effect of natural mutations, and for accurate protein design.     

Tissue-specific regulation of Ca(2+) channel protein expression by sex hormones

The L-type Ca(2+) channel pore-forming alpha subunit, alpha(1C) can be detected in brain and heart as two proteins with molecular masses of approximately 240 kDa and approximately 190 kDa known as alpha(1C-long) and alpha(1C-short), respectively. In brain, the alpha(1C-short) is thought to be the product of a approximately 50 kDa C-terminus calpain-mediated proteolytic deletion. We now show that uterine smooth muscle also possesses alpha(1C-long) and alpha(1C-short) isoforms, and that the relative expression of these two forms is regulated by sex hormones in a tissue-specific manner. Protein expression of alpha(1C) L-type Ca(2+) channels was examined in uterine smooth muscle, brain and heart, comparing non-pregnant (NP) estrus vs. late-pregnant (21 days) rats. The two forms of alpha(1C) were detected in all studied tissues. In late-pregnant uterus, alpha(1C-long) doubled the expression of alpha(1C-short); in NP uterus the opposite occurred. However, these changes were restricted to the uterine muscle, with no changes in brain and heart. To investigate the mechanism of such regulation, ovariectomized rats were treated with sex hormones, progesterone (P4) and/or 17beta-estradiol (estrogen, E2). P4 treatment, which yielded P4 plasma levels of 5 +/- 1 ng/ml and a high P4/E2 ratio (3 +/- 1.5 x 10(3)) similar to the ratio in late-pregnant uterus (1.5 +/- 0.3 x10(3)), facilitated alpha(1C-long) expression. In contrast, E2 or E2+P4 treatment that increased E2 plasma levels to 60 +/- 8 pg/ml and 75 +/- 24 pg/ml, produced low P4/E2 ratios of 0.03 +/- 0.006 and 0.2 +/- 0.1, respectively. These low P4/E2 ratios also found in NP rats at estrus (0.3 +/- 0.1) favored the expression of alpha(1C-short) form in myometrium. Neither hormone treatment altered alpha(1C) expression in brain or heart. Our results indicate that expression of alpha(1C) isoforms depends on P4/E2 ratios. Plasma P4/E2 ratios <1 x 10(3) favor the expression of the alpha(1C-short); whereas ratios >1 x 10(3) facilitate the expression of the alpha(1C-long) form. This regulation is tissue-specific for myometrium since it did not occur in heart and brain tissues.