Chemical Diversity of Volatiles of Teucrium orientale L. var. orientale,var. puberulens,and var. glabrescens Determined by Simultaneous GC‐FID and GC/MS Techniques

In the present work, three varieties of Teucrium orientale, var. orientale, var. puberulens, and var. glabrescens, were collected and investigated for chemical composition of the oils. Subsequent gas chromatography (GC‐FID) and gas chromatography coupled to mass spectrometry (GC/MS) revealed high abundance of sesquiterpenes in the essential oils analyzed. All the oils contained β‐caryophyllene (22.6, 8.5, and 6.3%, resp.) and hexadecanoic acid (7.9, 12.8, and 13.1%). Germacrene D (24.6 and 33.4%) and bicyclogermacrene (6.7 and 8.5%) were found to be the main constituents of var. orientale and var. puberulens, respectively. The high percentages of β‐cubebene (26.9%), α‐cubebene (9.0%), and α‐copaene (7.2%) established the diversity of var. glabrescens. The qualitative difference between the essential oils allowed the differentiation between the varieties in agreement with the morphological observations described in Flora of Turkey for each variety studied. In addition, a cluster analysis of twelve Teucrium taxa based on the essential‐oil composition has been carried out. Hovewer, the analysis did not clearly reflect the infrageneric classification of the genus, it largely confirmed the relationships between the infraspecific taxa of Teucrium orientale and T. chamaedrys.     

Brain’s alpha activity is highly reduced in euthymic bipolar disorder patients

Brain’s alpha activity and alpha responses belong to major electrical signals that are related to sensory/cognitive signal processing. The present study aims to analyze the spontaneous alpha activity and visual evoked alpha response in drug free euthymic bipolar patients. Eighteen DSM-IV euthymic bipolar patients (bipolar I n = 15, bipolar II n = 3) and 18 healthy controls were enrolled in the study. Patients needed to be euthymic at least for 4 weeks and psychotrop free for at least 2 weeks. Spontaneous EEG (4 min eyes closed, 4 min eyes open) and evoked alpha response upon application of simple visual stimuli were analyzed. EEG was recorded at 30 positions. The digital FFT-based power spectrum analysis was performed for spontaneous eyes closed and eyes open conditions and the response power spectrum was also analyzed for simple visual stimuli. In the analysis of spontaneous EEG, the ANOVA on alpha responses revealed significant results for groups (F(1,34) = 8.703; P < 0.007). Post-hoc comparisons showed that spontaneous EEG alpha power of healthy subjects was significantly higher than the spontaneous EEG alpha power of euthymic patients. Furthermore, visual evoked alpha power of healthy subjects was significantly higher than visual evoked alpha power of euthymic patients (F(1,34) = 4.981; P < 0.04). Decreased alpha activity in spontaneous EEG is an important pathological EEG finding in euthymic bipolar patients. Together with an evident decrease in evoked alpha responses, the findings may lead to a new pathway in search of biological correlates of cognitive impairment in bipolar disorder.     

CHEK2 1100delC, IVS2+1G>A and I157T mutations are not present in colorectal cancer cases from Turkish population

BackgroundThe cell cycle checkpoint kinase 2 (CHEK2) protein participates in the DNA damage response in many cell types. Germline mutations in CHEK2 (1100delC, IVS2+1G>A and I157T) have been impaired serine/threonine kinase activity and associated with a range of cancer types. This hospital-based case–control study aimed to investigate whether CHEK2 1100delC, IVS2+1G>A and I157T mutations play an important role in the development of colorectal cancer (CRC) in Turkish population.MethodsA total of 210 CRC cases and 446 cancer-free controls were genotyped for CHEK2 mutations by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele specific-polymerase chain reaction (AS-PCR) methods.ResultsWe did not find the CHEK2 1100delC, IVS2+1G>A and I157T mutations in any of the Turkish subjects.ConclusionOur result demonstrate for the first time that CHEK2 1100delC, IVS2+1G>A and I157T mutations have not been agenetic susceptibility factor for CRC in the Turkish population. Overall, our data suggest that genotyping of CHEK2 mutations in clinical settings in the Turkish population should not be recommended. However, independent studies are need to validate our findings in a larger series, as well as in patients of different ethnic origins.     

Ezrin-radixin-moesin-binding sequence of PSGL-1 glycoprotein regulates leukocyte rolling on selectins and activation of extracellular signal-regulated kinases

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the capture (tethering) of free-flowing leukocytes and subsequent rolling on selectins. PSGL-1 interactions with endothelial selectins activate Src kinases and spleen tyrosine kinase (Syk), leading to α(L)β(2) integrin-dependent leukocyte slow rolling, which promotes leukocyte recruitment into tissues. In addition, but through a distinct pathway, PSGL-1 engagement activates ERK. Because ezrin, radixin and moesin proteins (ERMs) link PSGL-1 to actin cytoskeleton and because they serve as adaptor molecules between PSGL-1 and Syk, we examined the role of PSGL-1 ERM-binding sequence (EBS) on cell capture, rolling, and signaling through Syk and MAPK pathways. We carried out mutational analysis and observed that deletion of EBS severely reduced 32D leukocyte tethering and rolling on L-, P-, and E-selectin and slightly increased rolling velocity. Alanine substitution of Arg-337 and Lys-338 showed that these residues play a key role in supporting leukocyte tethering and rolling on selectins. Importantly, EBS deletion or Arg-337 and Lys-338 mutations abrogated PSGL-1-induced ERK activation, whereas they did not prevent Syk phosphorylation or E-selectin-induced leukocyte slow rolling. These studies demonstrate that PSGL-1 EBS plays a critical role in recruiting leukocytes on selectins and in activating the MAPK pathway, whereas it is dispensable to phosphorylate Syk and to lead to α(L)β(2)-dependent leukocyte slow rolling.     

Magnesium Sulfate in Emergency Department Patients with Hypertension

To compare the effect of IV magnesium with other antihypertensives in emergency department (ED) patients with hypertension. ED patients with a systolic BP > 135 mmHg or diastolic BP > 85 were approached for entry into the study. Those granting consent were randomly placed into one of three treatment groups: (1) 1.5 gm IV MgSO₄ (n = 42), (2) a parenteral or oral antihypertensive agent (n = 41), (3) both IV MgSO₄ and an antihypertensive agent (n = 44). Systolic and diastolic blood pressures were measured at entry into the study and at 15, 30, 45, and 60 min after magnesium or other antihypertensive medications were given. The main outcome measure was blood pressure at 60 min, and results were compared using one-way analysis of variance with the post hoc Tukey HSD test. Compared to systolic and diastolic blood pressures at time 0, both were lower at 15, 30, 45, and 60 min in all groups (p < 0.05). No significant difference in systolic or diastolic BP at any time point was observed when response to treatment was compared between the three groups. Intravenous MgSO₄ is as effective as antihypertensives at lowering BP in emergency department patients.     

Salicylic acid-induced changes to growth and phenolic metabolism in <Emphasis Type="Italic">Matricaria chamomilla</Emphasis> plants

The influence of salicylic acid (SA) doses of 50 and 250 μM, for a period of up to 7 days, on selected physiological aspects and the phenolic metabolism of Matricaria chamomilla plants was studied. SA exhibited both growth-promoting (50 μM) and growth-inhibiting (250 μM) properties, the latter being correlated with decrease of chlorophylls, water content and soluble proteins. In terms of phenolic metabolism, it seems that the higher SA dose has a toxic effect, based on the sharp increase in phenylalanine ammonia-lyase (PAL) activity (24 h after application), which is followed by an increase in total soluble phenolics, lignin accumulation and the majority of the 11 detected phenolic acids. Guaiacol-peroxidase activity was elevated throughout the experiment in 250 μM SA-treated plants. In turn, some responses can be explained by mechanisms associated with oxidative stress tolerance; these mitigate acute SA stress (which is indicated by an increase in malondialdehyde content). However, PAL activity decreased with prolonged exposure to SA, indicating its inhibition. Accumulation of coumarin-related compounds (umbelliferone and herniarin) was not affected by SA treatments, while (Z)- and (E)-2-β-d-glucopyranosyloxy-4-methoxycinnamic acids increased in the 250 μM SA-treated rosettes. Free SA content in the rosettes increased significantly only in the 250 μM SA treatment, with levels tending to decrease towards the end of the experiment and the opposite trend was observed in the roots.     

Leukotriene B<Subscript>4</Subscript> regulates proliferation and differentiation of cultured rat myoblasts via the BLT1 pathway

Skeletal muscle regeneration is a highly orchestrated process initiated by activation of adult muscle satellite cells. Upon muscle injury, the inflammatory process is always accompanied by muscle regeneration. Leukotriene B₄ is one of the essential inflammatory mediators. We isolated and cultured primary satellite cells. RT-PCR showed that myoblasts expressed mRNA for LTB₄ receptors BLT1 and BLT2, and LTB4 promoted myoblast proliferation and fusion. Quantitative real-time PCR and immunoblotting showed that LTB₄ treatment expedited the expression process of differentiation markers MyoD and M-cadherin. U-75302, a specific BLT1 inhibitor, but not LY2552833, a specific BLT2 inhibitor, blocked proliferation and differentiation of myoblasts induced by LTB₄, which implies the involvement of the BLT1 pathway. Overall, the data suggest that LTB₄ contributes to muscle regeneration by accelerating proliferation and differentiation of satellite cells. These authors contributed equally to this work.     

Application of Label-free Quantitative Peptidomics for the Identification of Urinary Biomarkers of Kidney Chronic Allograft Dysfunction

The advent of quantitative proteomics opens new opportunities in biomedical and clinical research. Although quantitative proteomics methods based on stable isotope labeling are in general preferred for biomolecular research, biomarker discovery is a case example of a biomedical problem that may be better addressed by using label-free MS techniques. As a proof of concept of this paradigm, we report the use of label-free quantitative LC-MS to profile the urinary peptidome of kidney chronic allograft dysfunction (CAD). The aim was to identify predictive biomarkers that could be used to personalize immunosuppressive therapies for kidney transplant patients. We detected (by LC-M/MS) and quantified (by LC-MS) 6000 polypeptide ions in undigested urine specimens across 39 CAD patients and 32 control individuals. Although unsupervised hierarchical clustering differentiated between the groups when including all the identified peptides, specific peptides derived from uromodulin and kininogen were found to be significantly more abundant in control than in CAD patients and correctly identified the two groups. These peptides are therefore potential biomarkers that might be used for the diagnosis of CAD. In addition, ions at m/z 645.59 and m/z 642.61 were able to differentiate between patients with different forms of CAD with specificities and sensitivities of 90% in a training set and, significantly, of ∼70% in an independent validation set of samples. Interestingly low expression of uromodulin at m/z 638.03 coupled with high expression of m/z 642.61 diagnosed CAD in virtually all cases. Multiple reaction monitoring experiments further validated the results, illustrating the power of our label-free quantitative LC-MS approach for obtaining quantitative profiles of urinary polypeptides in a rapid, comprehensive, and precise fashion and for biomarker discovery.A major goal of clinical proteomics is to identify biomarkers that can aid in the diagnosis and prognosis of different conditions. In their ideal form, these biomarkers will not only assist the clinician in the diagnosis of a disease, but they will also give directions as to which therapy may be more appropriate for each patient, thus contributing to the development of personalized medicine. In this regard, urine represents an ideal, but yet largely unexplored, source of biomarkers because of the presence of large numbers of small peptides in this biological fluid and because it can be obtained non-invasively.However, although proteomics techniques are instrumental for increasing our understanding of molecular cell biology (1) the impact of proteomics in clinical practice has not yet reached initial expectations perhaps because of technological limitations (2, 3). Using hyphenated methods such as novel LC-MS techniques for quantitative proteomics (4, 5) may prove advantageous for the identification and validation of biomarkers (3, 6). This is because LC-MS allows the detection of proteomes with greater depth, dynamic range, and enhanced accuracy of quantization than when using one-dimensional profiling techniques that record all ions in a single mass spectrum, such as MALDI-TOF MS or SELDI-TOF MS (7). On-line LC-ESI-MS is quantitative in nature because the initial LC separation step contributes to reducing the amount of analytes that are simultaneously ionized, thus reducing the possibility of ion suppression, and because ion formation by electrospray ionization is proportional to analyte concentration (8, 9). Initial reports that used LC-MS for the analysis of the urinary proteome provided proof of principle of the use of this technique for the analysis of urinary polypeptides (10–12), and recently, using new generation LC-MS/MS instrumentation, more than 1500 proteins have been detected in urine (13). Nevertheless despite these advances in our understanding of the qualitative composition of the urinary proteome, precise and comprehensive quantification of urinary polypeptides to discover potential biomarkers remains a challenge.The ideal, and more widely used, strategies to derive quantitative information from LC-MS experiments are based on differential stable isotope labeling of proteins or peptides, which are then mixed and quantified relative to each other in single multidimensional LC-LC-MS experiments (14). This technique, however, is not ideal for biomarker discovery because of problems associated with protein derivatization in a clinical setting, because of its limited throughput, and because, although not impossible, isotope labeling techniques make it difficult to compare a large number of specimens; at present labeling reagents can be used for simultaneous comparison of up to eight protein samples (15).Novel analytical strategies for quantitative proteomics that do not require isotope labeling have been reported (4, 5, 16). These techniques can quantify polypeptides with precisions and accuracies comparable to those based on isotope labeling (17). In addition, such label-free quantitative LC-MS approaches can compare an unlimited number of samples, and it is therefore ideal for biomarker discovery as experimental designs normally involve comparing a large number of specimens to statistically validate the results. Thus, label-free quantitative LC-MS would clearly assist in analyzing the full potential of urine clinical samples as a source of disease biomarkers. The aim of the study presented herein was to prove this concept taking chronic allograft dysfunction (CAD)¹ as a paradigm.During the last years, the incidence and prevalence of end stage renal disease has increased worldwide (18). Successful renal transplantation improves the patients'' quality of life and increases survival as compared with long term dialysis treatment (19). However, despite these improvements, a substantial portion of grafts develop progressive dysfunction and fail within a decade even with the use of appropriate doses of immunosuppressive drugs to prevent acute rejection (20). CAD is responsible for more than 50% of graft losses and remains a central clinical challenge. Although patients can return to dialysis after transplant failure, loss of a functioning graft is associated with a 3-fold increase in the risk of death, a substantial decrease in quality of life for those who survive, and a 4-fold increase in healthcare costs (21).CAD is mediated by a combination of immune, ischemic, and inflammatory stimuli, and multiple pathways and mediators lead to cumulative structural damage to all compartments of the transplanted kidney. Sclerosing changes associated with tubulointerstitial injury are mediated by the processes of active fibrogenesis, resulting in epithelial loss and the phenotype of tubular atrophy and chronic interstitial fibrosis (22). Available diagnostic methods include clinical presentation, biochemical parameters, and biopsies. Currently the only non-invasive biomarker of CAD is serum creatinine and glomerular filtration rate (GFR), but neither is particularly sensitive or specific and may not reflect early alterations (20, 22). At present, biopsy allograft is regarded as the gold standard for the diagnosis of CAD allowing its early detection; however, this is a costly procedure that is associated with clinical complications (23).Clinicians are hence faced with a dilemma. On the one hand, protocol biopsies may detect rejection at an earlier subclinical stage and allow prompt initiation of treatment, which may translate into improved long term graft survival (24). On the other hand, this also implies that patients with preserved graft function, i.e. without CAD, undergo this invasive procedure unnecessarily. Therefore, identification of non-invasive biomarkers for the early diagnosis of CAD would be invaluable for alleviating the major health and economic burden that this condition causes to western countries (25).The aim of the present study was to evaluate whether the urinary peptidome, as analyzed by a novel analytical strategy based on label-free quantification of urinary polypeptides by LC-MS, would differentiate between patients with CAD, those showing stable renal transplant (SRT), and a group of living donors. To our knowledge, this represents the first study reporting urine polypeptide signatures and individual biomarkers that group patients according to their underlying renal phenotype and hence represent potential candidates for non-invasive diagnosis of CAD.