Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.     

The Balkan chamois,an archipelago or a peninsula? Insights from nuclear and mitochondrial DNA

The Balkan chamois (Rupicapra rupicapra balcanica) is widespread on the Balkan Peninsula, along mountain massifs from Croatia in the north to Greece in the south and Bulgaria in the east. Knowledge on the genetic structure of Balkan chamois populations is limited and restricted to local studies. Therefore, the main objective of this study was to use nuclear (16 microsatellites) and mitochondrial (partial 376 base pairs control region) markers to investigate the genetic structure of this chamois subspecies throughout its distribution range and to obtain information on the degree of connectivity of the different (sub)populations. We extracted DNA from bone, dried skin and muscle tissue and successfully genotyped 92 individuals of Balkan chamois and sequenced the partial control region in 44 individuals. The Bayesian analysis suggested 3 genetic clusters and assigned individuals from Serbia and Bulgaria to two separate clusters, while individuals from the other countries belonged to the same cluster. Thirty new haplotypes were obtained from partial mitochondrial DNA sequences, with private haplotypes in all analyzed populations and only two haplotypes shared among populations, indicating the possibility of past translocations. The subspecies genetic composition presented here provides the necessary starting point to assess the conservation status of the Balkan chamois and allows the development of conservation strategies necessary for its sustainable management and conservation.

     

The impact of aerobic and anaerobic training regimes on blood pressure in normotensive and hypertensive rats: focus on redox changes

Molecular and Cellular Biochemistry - Melatonin is a crucial neurohormone synthesized in the pineal gland that influences the physiology of animals. The molecular mechanism of norepinephrine...     

Use of blood outgrowth endothelial cells as virus-producing vectors for gene delivery to tumors

Cell-based delivery of therapeutic viruses has potential advantages over systemic viral administration, including attenuated neutralization and improved viral targeting. One of the exciting new areas of investigation is the potential ability of endothelial-lineage cells to deliver genes to the areas of neovascularization. In the present study, we compared two types of endothelial-lineage cells [outgrowth endothelial cells (OECs) and culture-modified mononuclear cells (CMMCs), also known as "endothelial progenitor cells"] for their ability to be infected with adenovirus and to home to the areas of neovascularization. Both cell types were isolated from peripheral blood of healthy human donors and expanded in culture. We demonstrate that OECs are more infectable and home better to tumors expressing VEGF on systemic administration. Furthermore, we used an adenoviral/retroviral chimeric system to convert OECs to retrovirus-producing cells. When injected systemically into tumor-bearing mice, OECs retain their ability to produce retrovirus and infect surrounding tumor cells. Our data demonstrate that OECs could be efficient carriers for viral delivery to areas of tumor neovascularization.     

Structure-activity relationships of piperazinebenzylamines as potent and selective agonists of the human melanocortin-4 receptor

SAR studies on a series of piperazinebenzenes directed toward the human melanocortin-4 receptor resulted in potent MC4R agonists. Replacement of the triazole moiety of an initial lead 4 by a basic nitrogen baring a lipophilic side-chain increased the binding affinities of these compounds. Analogs bearing an additional hetero-atom in the side-chain possessed good agonist potency. Thus, 11h had a Ki of 11 nM, and 13g exhibited an EC50 of 3.8 nM and a Ki of 6.4 nM.     

Influence of the extraction mode on the yield of some furanocoumarins from Pastinaca sativa fruits

Analysis of plant material is an important task in chemotaxonomical investigations, in search of plants with pharmacological activity or in standardisation of plant drugs. The choice of optimal conditions for the analysis of plant material and effect of extraction method on the yield of furanocoumarins from Pastinaca sativa fruits were examined. The following extraction methods were used in experiments: exhaustive extraction in Soxhlet apparatus, ultrasonification (USAE) at 25 and 60 degrees C, microwave-assisted solvent extraction in open and closed system (MASE) and accelerated solvent extraction (ASE). In most cases, the yield of furanocoumarins was highest by use of ASE method as well as by ultrasonification at 60 degrees C.     

The energetics of specific binding of AT-hooks from HMGA1 to target DNA

The interaction of the second and third AT-hooks of HMGA1 (formerly HMGI/Y), which bind selectively in the minor groove of an AT-rich DNA sequence, was studied at different temperatures and ionic strengths by spectropolarimetry, spectrofluorimetry, isothermal titration calorimetry and differential scanning calorimetry. The data show that binding of the ten amino acid core element of the two AT-hooks, which penetrates deep into the minor groove, is entropically driven: both the entropy and enthalpy of association of the peptides to the target DNA are positive up to 50 degrees C. The seven amino acid extension of the core in the second AT-hook, which extends out from the minor groove and loops over the phosphodiester backbone, adds a substantial negative enthalpic component into the binding of the 17 residue DBD2 peptide to DNA that corresponds in magnitude to the enthalpy of formation of two hydrogen bonds. The ionic strength dependence of the association constant allowed an estimation of the electrostatic component of binding and, by subtraction, the contribution of the non-electrostatic component, which results from dehydration of the contacting surfaces and makes up almost 70% of the total energy of complex formation. The exceptionally large positive entropy and enthalpy of association of the core AT-hook peptides with target DNA suggest that the water, which is removed from the minor groove of DNA upon binding, is in a highly ordered state. Acetylation of the lysine residue in the second AT-hook, which corresponds to Lys65 of HMGA1, has little effect on the DNA binding; so it appears that repression of the hIFNbeta gene, which follows this modification, is not a direct result of the abrogation of DNA binding.     

Rapid detection of the hypertension-associated A1166C polymorphism of the angiotensin II type 1 receptor (AGTR1)

Screening for polymorphisms in the human type 1 angiotensin II receptor locus (AGTR1) has led to the identification of an A1166C transversion in the 3'-untranslated region. This molecular variant, C(1166), has been linked to essential hypertension. We describe here a rapid method for the detection of this point mutation by a simple modification of PCR amplification with allele-specific oligonucleotides (ASO), so as to avoid a hybridization procedure involving either radioactive- or non-radioactive-labeled probes, labeled primers, or restriction typing. The procedure described is convenient for routine clinical laboratory use with manual sample processing and offers the potential for further automation, as well.     

BOB.1/OBF.1 deficiency affects marginal-zone B-cell compartment

Marginal-zone (MZ) B cells represent a first line of defense against particulate blood-borne antigens. Together with the B1 cells, they are responsible for the early response against type II T-independent antigens. The molecular pathways controlling the development of MZ B cells are only poorly understood. We found that these cells are virtually absent in mice deficient in the BOB.1/OBF.1 coactivator. Loss of these B cells was demonstrated by the lack of cells showing the appropriate cell surface phenotype but also by histological analyses and tri-nitro-phenol-Ficoll capturing. The lack of these cells is a B-cell-intrinsic defect, as shown by bone marrow complementation experiments. We also show that the expression of BOB.1/OBF.1 in peripheral B cells is required for the development of MZ B lymphocytes. Our analysis of BOB.1/OBF.1-deficient splenic B cells reveals alterations in cell motility, tumor necrosis factor receptor expression, and B-cell receptor (BCR) signaling. These changes could contribute to the loss of MZ B lymphocytes by altering the maturation of the cells. Interestingly, development of and BCR signaling in B1 B cells are completely normal in BOB.1/OBF.1 mutant mice.     

Fluctuations of Serum Neuron Specific Enolase and Protein S-100B Concentrations in Relation to the Use of Shunt during Carotid Endarterectomy

Objective

To evaluate the changes in serum neuron specific enolase and protein S-100B, after carotid endarterectomy performed using the conventional technique with routine shunting and patch closure, or eversion technique without the use of shunt.

Materials and Methods

Prospective non-randomized study included 43 patients with severe (>80%) carotid stenosis undergoing carotid endarterectomy in regional anesthesia. Patients were divided into two groups: conventional endarterectomy with routine use of shunt and Dacron patch (csCEA group) and eversion endarterectomy without the use of shunt (eCEA group). Protein S-100B and NSE concentrations were measured from peripheral blood before carotid clamping, after declamping and 24 hours after surgery.

Results

Neurologic examination and brain CT findings on the first postoperative day did not differ from preoperative controls in any patients. In csCEA group, NSE concentrations decreased after declamping (P<0.01), and 24 hours after surgery (P<0.01), while in the eCEA group NSE values slightly increased (P=ns), accounting for a significant difference between groups on the first postoperative day (P=0.006). In both groups S-100B concentrations significantly increased after declamping (P<0.05), returning to near pre-clamp values 24 hours after surgery (P=ns). Sub-group analysis revealed significant decline of serum NSE concentrations in asymptomatic patients shunted during surgery after declamping (P<0.05) and 24 hours after surgery (P<0.01), while no significant changes were noted in non-shunted patients (P=ns). Decrease of NSE serum levels was also found in symptomatic patients operated with the use of shunt on the first postoperative day (P<0.05). Significant increase in NSE serum levels was recorded in non-shunted symptomatic patients 24 hours after surgery (P<0.05).

Conclusion

Variations of NSE concentrations seemed to be influenced by cerebral perfusion alterations, while protein S-100B values were unaffected by shunting strategy. Routine shunting during surgery for symptomatic carotid stenosis may have the potential to prevent postoperative increase of serum NSE levels, a potential marker of brain injury.