D4 Dopamine and Metabotropic Glutamate Receptors in Cerebral Cortex and Striatum in Rat Brain

The characterization of the functional interactions between the metabotropic glutamate receptors (mGluR) and the dopaminergic (DR) receptors in the corticostriatal projections may provide a possible interpretation of synaptic events in the basal ganglia. It has been suggested that presynaptic D2-type receptor located on glutamatergic corticostriatal neurons regulates the release of glutamate. In a first approach we have studied the cellular distribution of the D4R and the mGluRs in cerebral cortex and striatum employing immunocytochemistry. D4R positive neurons were particularly numerous in medial prefrontal cortex mainly occupying layers II and III. An even distribution was found on small round-shaped neurons in the striatum. Group I mGluR1-like immunoreactivity (mGluR1-LI) was found in medial and deep layers of the cerebral cortex while group III mGluR4a labeled more superficial layers; group II mGluR2/3 signal was intense on fine fibers with a punctate appearance. In the striatum, mGluR1 and mGluR2/3 stained mainly fibers while mGluR4a labeled round shaped cell bodies. After lateral ventricular injection of colchicine, an axonal transport and firing activity blocker, D4R labeling significantly increased in cerebral cortex and decreased in the striatum. mGluR1 and mGluR4a signal decreased in cerebral cortex and only mGluR4a signal decreased in the striatum. These results support previous reports indicating a presynaptic localization of D4R in the striatum. In contrast, striatal mGluR1 appears to be a postsynaptic receptor probably synthesized in situ. Our results do not support the hypothesis of a colocalization of D4 receptor and one or more of the metabotropic glutamatergic receptors studied here.     

Hyaluronan receptors in the human ocular surface: a descriptive and comparative study of RHAMM and CD44 in tissues, cell lines and freshly collected samples

The purpose of this study was to demonstrate the presence of the receptor for hyaluronan-mediated motility (RHAMM) in human conjunctival epithelium and in two widely used cell lines from human corneal (HCE) and conjunctival (IOBA-NHC) epithelia. We compared the distribution of RHAMM proteins and mRNAs in human ocular surface tissues (corneal, limbal and conjunctival), HCE and IOBA-NHC cell lines, and corneal and conjunctival epithelia primary samples from healthy donors with the previously identified hyaluronan receptor CD44. We also aimed to determine if soluble CD44 (sCD44) was present in human tears, as it could have a role in the interaction of the tear fluid with hyaluronan. Protein expression was evaluated by Western blots and immunofluorescence microscopy. mRNA expression was evaluated by RT-PCR and Q-PCR. sCD44 was analyzed by ELISA in culture supernatants and in human tears. We describe the expression of RHAMM in human healthy conjunctiva and in HCE and IOBA-NHC cells at both protein and mRNA levels, and the presence of sCD44 in human tears. Furthermore, we detected CD44 and sCD44 expression variations in in vitro inflammatory conditions. This study also focused on the necessary caution with which the conclusions extracted from cell lines should be made, and in the great value of using primary samples as often as possible.     

Stereological study of the early ultrastructural differentiation of chick embryo neuroepithelial cells during neurulation

The neuroectodermal cells of chick embryos have been analyzed during neurulation by stereological and morphometrical ultrastructural methods in an attempt to describe their cytometric evolution. A profound change of cellular form coefficient was observed which is related to the typical process of columnarization of these cells. At stages 7 and 8, the nucleus appeared round in shape, probably due to a loss of pressure of the vitelline inclusions. In this sense, the volume density of these inclusions falls during this period. There was also a significant increase of the nuclear surface density, the significance of which is discussed on the basis of the nucleo-cytoplasmic interchanges and the differentiation process. At the same time, an increase in the number of mitochondria was observed, which is related to the neural folding process. Simultaneously, the amount of rough endoplasmic reticulum increases, presumably related to the remarkable changes of the embryonic extracellular matrix.     

Selective activity against Mycobacterium tuberculosis of new quinoxaline 1,4-di-N-oxides

New series of 3-phenylquinoxaline 1,4-di-N-oxide with selective activity against Mycobacterium tuberculosis have been prepared and evaluated. Thirty-four of the seventy tested compounds showed an MIC value less than 0.2 μg/mL, a value on the order of the MIC of rifampicin. Furthermore, 45% of the evaluated derivatives showed a good in vitro activity/toxicity ratio. The most active and selective compounds carry a fluorine atom in the quinoxaline 7-position or in the phenyl substituent para-position. In conclusion, the potency, low cytotoxicity and selectivity of these compounds make them valid lead compounds for synthesizing new analogues, particularly compound 7-methyl-3-(4’-fluoro)phenylquinoxaline-2-carbonitrile 1,4-di-N-oxide (MIC <0.2 μg/mL and SI >500).     

Outcome of Arthroscopy in Patients with Advanced Osteoarthritis of the Hip

Hip arthroscopy has continued to expand its horizons in treating many conditions other than femoroacetabular impingement (FAI). However, the results of hip arthroscopy are known to be poor if the degree of articular cartilage damage is significant. We wanted to assess, whether the procedure might have a role in the management of young and active patients with advanced osteoarthritis (OA) and whether it should be offered as a treatment modality. 77 consecutive patients with Tönnis grade 2 and 3 osteoarthritis of the hip who had undergone hip arthroscopy were included in the study. Patients'' medical notes, plain radiographs and outcome scores (modified Harris hip score (mHHS), non-arthritic hip score (NAHS)) preoperatively and postoperatively at six weeks, six months, one year and annually thereafter, were analysed. 77 patients consisted of 63 men and 14 women with mean follow-up of 2.8 years (2.2 to 4.2) and mean age at surgery of 43 years (19 to 64). The mean preoperative mHHS and NAHS scores were 58 (28 to 87) and 64 (27 to 93) respectively. The mean improvements in both the mHHS and NAHS scores were significant (p = 0.003 and p = 0.0001 for mHHS at one and two years, p = 0.002 and p = 0.0003 for NAHS at one and two years, respectively). There were 34 patients (44%) who required a total hip replacement at mean of 18 months (6 to 48) after hip arthroscopy. We conclude that hip arthroscopy improves outcome scores in 56% of patients with severe OA of the hip (Tönnis grade 2 and 3) for at least two years after surgery. We thus consider the procedure to be a reasonable option for patients with hip OA, although success of the procedure will be less than if undertaken for certain other conditions.     

Protein-Based Classifier to Predict Conversion from Clinically Isolated Syndrome to Multiple Sclerosis

Multiple sclerosis is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system. In most patients, the disease initiates with an episode of neurological disturbance referred to as clinically isolated syndrome, but not all patients with this syndrome develop multiple sclerosis over time, and currently, there is no clinical test that can conclusively establish whether a patient with a clinically isolated syndrome will eventually develop clinically defined multiple sclerosis. Here, we took advantage of the capabilities of targeted mass spectrometry to establish a diagnostic molecular classifier with high sensitivity and specificity able to differentiate between clinically isolated syndrome patients with a high and a low risk of developing multiple sclerosis. Based on the combination of abundances of proteins chitinase 3-like 1 and ala-β-his-dipeptidase in cerebrospinal fluid, we built a statistical model able to assign to each patient a precise probability of conversion to clinically defined multiple sclerosis. Our results are of special relevance for patients affected by multiple sclerosis as early treatment can prevent brain damage and slow down the disease progression.Multiple sclerosis is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system, and although the etiology of the disease is not fully understood, it is probably caused by the interaction of a complex genetic architecture and environmental factors. Multiple sclerosis affects over 2 million people worldwide, and it is typically diagnosed between ages 20 and 40, thus making a significant impact on public health and its economy (1).In most patients, the disease initiates with an episode of neurological disturbance referred to as clinically isolated syndrome. However, not all patients with this syndrome develop multiple sclerosis over time (2), and currently, the magnetic resonance imaging (MRI) abnormalities and the presence of IgG oligoclonal bands in cerebrospinal fluid (CSF) are used as predictors for later conversion to clinically definite multiple sclerosis (CDMS)¹ (3–5). Although such abnormalities are considered important factors that influence the likelihood of developing CDMS, there is currently no clinical test that can conclusively establish whether a patient with a clinically isolated syndrome will eventually develop CDMS.The lack of diagnostic and prognostic biomarkers is a common problem for many diseases lacking a complete etiology, which is the case for most neurological disorders related to the central nervous system such as Parkinson''s and Alzheimer''s diseases, schizophrenia, and multiple sclerosis. In the particular case of multiple sclerosis, early treatment of patients with a clinically isolated syndrome can prevent brain damage and slow down the disease progression (6). Therefore, the availability of a diagnostic test in the initial stages of the disease is not only desirable but also of extreme relevance to attenuate the degenerative effects of the disease.Biomarker validation has traditionally been dominated by enzyme linked immuno-sorbent assays (ELISA), but recent advances in proteomics techniques have enabled the measurement of a subset of selected proteins over a large dynamic concentration range in multiple samples. Targeted mass spectrometry has thus become the method of choice when quantifying simultaneously a panel of proteins across many different biological samples (7–9). In particular, selected reaction monitoring (SRM) is the gold standard targeted mass spectrometry method for protein quantification due to its high precision, reliability, and throughput (10–13). This targeted mass spectrometry method is performed on triple quadrupole instruments, in which a predefined peptide precursor ion is first isolated, and then selected fragment ions arising from its collisional dissociation are measured over time. Each pair of precursor and fragment ion is called a transition, and multiple transitions can be coordinately measured and used to conclusively identify and quantify a peptide in a clinical complex sample.In a previous study, we used a screening mass spectrometric approach to discover potential markers for multiple sclerosis conversion in patients that initially presented a clinical isolated syndrome (14). In that discovery phase, quantitative mass spectrometry with iTRAQ labeling was used to measure protein abundances in pooled CSF samples from patients presenting a clinical isolated syndrome that either remained normal (CIS) or had eventually converted to clinically definite multiple sclerosis (CDMS) (n = 60). In the initial screening, several proteins exhibited significant differences in abundance when comparing these two groups of patients. The abundance change in one of the altered proteins, chitinase 3-like 1 (CH3L1), was confirmed by ELISA in CSF of individual patients, whereas for others, such as semaphorin 7A (SEM7A) and ala-β-his-dipeptidase (CNDP1), their abundance changes were confirmed by targeted mass spectrometry in follow-up studies with independent cohorts (15). Moreover, the levels of CH3L1 were associated with brain MRI abnormalities and disability progression during the follow-up period, as well as with shorter time to conversion to clinically definite multiple sclerosis (14).We now set out to establish a diagnostic protein classifier with high sensitivity and specificity able to differentiate between patients with a clinically isolated syndrome that have either a high or a low risk of developing clinically definite multiple sclerosis over time. For this purpose, CSF samples from an independent patient cohort from the one used in the discovery study were collected, and a set of preselected protein biomarker candidates were systematically quantified by targeted mass spectrometry (SRM) and evaluated for their classification power. Out of this study, we established a protein classifier based on the combination of abundances of proteins chitinase 3-like 1 and ala-β-his-dipeptidase, which is able to differentiate with high sensitivity and specificity between patients with a clinically isolated syndrome that have either a high or low risk of developing clinically definite multiple sclerosis. Moreover, the statistical model built around this protein classifier enables clinicians to easily assign to each patient a precise probability of conversion to clinically definite multiple sclerosis (Fig. 1).Open in a separate windowFig. 1.General workflow used in the present study. Initially, protein candidates identified in our previous discovery studies—together with several proteins described by other groups—were selected and quantified by targeted mass spectrometry (SRM) in a relatively large cohort individual patients. Protein quantities were then evaluated by their capability of classifying patients with clinical isolated syndrome, and thus, the best prognostic protein combination was identified.     

Synthesis and anti-inflammatory/antioxidant activities of some new ring substituted 3-phenyl-1-(1,4-di-N-oxide quinoxalin-2-yl)-2-propen-1-one derivatives and of their 4,5-dihydro-(1H)-pyrazole analogues

We report the synthesis, anti-inflammatory and antioxidant activities of novel ring substituted 3-phenyl-1-(1,4-di-N-oxide quinoxalin-2-yl)-2-propen-1-one derivatives and of their 4,5-dihydro-(1H)-pyrazole analogues. The tested compounds inhibit the carrageenin-induced rat paw edema (4.5-56.1%) and present important scavenging activities. Compound 2a is the most potent (56.1%) in the in vivo experiment and exhibits promising in vitro inhibition of soybean lipoxygenase (IC(50)<1microM).     

Short-term endothelial progenitor cell colonies are composed of monocytes and do not acquire endothelial markers

BACKGROUND: The aim of this study was to identify circulating endothelial progenitor cells (EPC) with colony-forming capacity and compare them with the monocytic-macrophage lineage. METHODS: Forty-two healthy donors were analyzed. EPC were cultured with VEGF and b-FGF. Sequential studies were performed on days +7 (colonies) +21 and +35. Monocytic cells were cultured using the same conditions as EPC until day +21 or alternatively by adding IGF. RESULTS: The number of EPC colonies was higher in BM than in mobilized or steady-state PB. Using EPC medium, monocytic cells formed cord-like structures but no colonies. However, colonies grew when IGF was added to the medium. By immunocytochemistry, colonies showed CD45, CD31 and lysozyme but no vWF. Colonies were CD4+, CD13+dim, CD14+, CD15++, CD16-/+dim, CD31+dim, CD33+dim, CD45+, CD105-/+dim, lysozyme+ and VE-cadherin+, and constantly negative for CD34, CD133 and KDR, when flow cytometry was used. The immunophenotype of pre-cultured and cultured monocytes was similar to that described for EPC. DISCUSSION: Our results suggest that the so-called 'EPC' obtained at 7 days of culture belong to the monocyte-macrophage lineage, as they share immunophenotypic and molecular features.     

Effects of nutrients and warming on Planktothrix dynamics and diversity: a palaeolimnological view based on sedimentary DNA and RNA

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