NMR and multivariate methods: Identification of chemical markers in extracts of pedra-ume-caá and their antiglycation, antioxidant, and enzymatic inhibition activities.

INTRODUCTION: In Brazil, the plant group popularly known as "pedra-ume-caá" is used in folk medicine for the treatment of diabetes, and its raw material is commonly sold. OBJECTIVE: The aim of the study was to apply a method for chemical identification of extracts of dry pedra-ume-caá leaves using HPLC-high-resolution mass spectrometry (HRMS) and NMR and develop a multivariate model with NMR data to authenticate commercial samples. In addition, to evaluate the biological activities of the extracts. MATERIALS AND METHODS: Dry extracts of Myrcia multiflora, Myrcia amazonica, Myrcia guianensis, Myrcia sylvatica, Eugenia punicifolia leaves, and 15 commercial samples (sold in Manaus and Belém, Brazil) were prepared by infusion. All the extracts were analysed using HPLC-high-resolution mass spectrometry (HRMS), NMR, principal component analysis (PCA), and hierarchical cluster analysis (HCA). The antidiabetic effect of extracts was evaluated according to enzymatic inhibition. Their content of total phenols, cell viability, and antioxidant and antiglycation activities were also determined. RESULTS: HPLC-HRMS and NMR analysis of these extracts permitted the identification of 17 compounds. 1H NMR data combined with multivariate analyses allowed us to conclude that catechin, myricitrin, quercitrin, and gallic and quinic acids are the main chemical markers of pedra-ume-caá species. These markers were identified in 15 commercial samples of pedra-ume-caá. Additionally, only the extracts of M. multiflora and E. punicifolia inhibited α-glucosidase. All the extracts inhibited the formation of advanced glycation end products (AGEs) and showed free-radical-scavenging activity. These extracts did not present cytotoxicity. CONCLUSION: This study revealed the chemical markers of matrices, and it was possible to differentiate the materials marketed as pedra-ume-caá. Moreover, this study corroborates the potential of these species for treating diabetes.

Impulsivity as a Multifaceted Construct Related to Excessive Drinking Among UK Students.

AIMS: A substantial number of university students exceed alcohol guidelines. Impulsivity has been repeatedly implicated in heavy alcohol use, yet despite knowledge that impulsivity is multifaceted, there have previously been few studies applying multiple measures of self-report and behavioural impulsivity to examine the relationship with excessive student drinking. This results in a limited understanding of the relationship of various facets of impulsivity to student drinking. METHODS: Participants completed a comprehensive battery of impulsivity measures: the Barratt Impulsiveness Scale as a self-report index and the Stop Signal Task, Information Sampling Task and Monetary Choice Questionnaire as behavioural measures of three facets of impulsivity. Participants who exceeded UK drinking guidelines were compared to those who did not on measures of impulsivity. Hierarchical linear regression was then employed to test whether indices of impulsivity were associated with the average units consumed per week. RESULTS: Participants who exceeded UK guidelines reported increased impulsivity in facets of self-report impulsivity. They also displayed performance deficits in normal adjustment of Go responses on the Stop Signal Task. In the regression model, nonplanning impulsivity on the Barratt Impulsiveness Scale was seen to predict quantity of alcohol consumed per month. CONCLUSIONS: The study applies a comprehensive selection of behavioural and self-report measures of impulsivity and indicates that excessive drinkers are more impulsive in some but not all aspects. The results indicate that the wide range of deficits apparent in alcohol-dependent individuals are not evident in this younger, heavy drinking population, but that specific performance and self-identified deficits are already apparent.

Thrombospondin 4 deficiency in mouse impairs neuronal migration in the early postnatal and adult brain.

In the post-natal rodent brain, neuronal precursors originating from the sub-ventricular zone (SVZ) migrate over a long distance along the rostral migratory stream (RMS) to eventually integrate the olfactory bulb neuronal circuitry. In order to identify new genes specifically expressed in the RMS, we have screened the Allen Brain Atlas Database. We focused our attention on Thrombospondin 4 (Thbs4), one of the 5 members of the Thrombospondin family of large, multidomain, extracellular matrix proteins. In post-natal and adult brain Thbs4 mRNA and protein are specifically expressed in the neurogenic regions, including the SVZ and along the entire RMS. RMS cells expressing Thbs4 are GFAP (Glial Fibrillary Acidic Protein) positive astrocytes. Histological analysis in both wild-type and Thbs4 knock-out mice revealed no major abnormality in the general morphology of these neurogenic regions. Nevertheless, immunostaining for doublecortin demonstrates that in Thbs4-KO, migration of newly formed neurons along the RMS is somehow impaired, with several neurons migrating out of the RMS. This is further supported by a Bromodeoxyuridine-based in vivo approach showing a decrease in the number of newly born neuronal precursors reaching the olfactory bulb, while proliferation in the SVZ is not affected compared to wild-type, both in young animals (P15) and in adults (8 to 12 weeks of age). Corroborating this observation, the number of Parvalbumin- and Calbindin-immunoreactive interneurons in the olfactory bulb is also reduced in Thbs4-KO. Together, these observations support a role for the astrocyte-secreted protein Thbs4 in the migration of newly form neurons within the RMS to the olfactory bulb.

Gene expression analysis in the parvalbumin-immunoreactive PV1 nucleus of the mouse lateral hypothalamus.

A solitary, elongated cluster of parvalbumin-immunoreactive neurons has been previously observed in the rodent ventrolateral hypothalamus. However, the function of this so-called PV1 nucleus is unknown. In this article, we report the results of an unbiased, broad and in-depth molecular characterization of this small, compact group of neurons. The Allen Brain Atlas database of in situ hybridization was screened in order to identify genes expressed in the PV1-nucleus-containing area of the hypothalamus, and those that might be co-expressed with parvalbumin. Although GABA is the principal neurotransmitter in parvalbumin-expressing cells in various other brain areas, we found that PV1 neurons express the vesicular glutamate transporter (VGlut) VGlut2-encoding gene Slc17a6 and are negative for the glutamic acid decarboxylase 1 (GAD1) gene. These cells also express the mRNA for the neuropeptides Adcyap1 and possibly Nxph4, express several types of potassium and sodium channels, are under the control of the neurotransmitter acetylcholine, bear receptors for the glial-derived neurotrophic factor, and produce an extracellular matrix rich in osteopontin. The PV1 nucleus is thus composed of glutamatergic nerve cells, expressing some typical markers of long-axon, projecting neurons (e.g. VGlut2), but also co-expressing genes typical of short-axon GABA neurons (e.g. a variety of potassium channels). Hence, neurons of the PV1 nucleus combine physiological characteristics of interneurons with those of projection neurons.

Piper capitarianum essential oil: a promising insecticidal agent for the management of Aedes aegypti and Aedes albopictus.

Mosquitoes are responsible for serious public health problems worldwide, and as such, Aedes aegypti and Aedes albopictus are important vectors in the transmission of dengue, chikungunya, and Zika in Brazil and other countries of the world. Due to growing resistance to chemical insecticides among populations of vectors, environmentally friendly strategies for vector management are receiving ever more attention. Essential oils (EOs) extracted from plants have activities against insects with multiple mechanisms of action. These mechanisms hinder the development of resistance, and have the advantages of being less toxicity and biodegradable. Thus, the present study aimed to evaluate the chemical composition of the EOs obtained from Piper capitarianum Yunck, as well as evaluating their insecticidal potential against Aedes aegypti and A. albopictus, and their toxicity in relation to Artemia salina. The yields of the EOs extracted from the leaves, stems, and inflorescences of P. capitarianum were 1.2%, 0.9%, and 0.6%, respectively, and their main constituents were trans-caryophyllene (20.0%), α-humulene (10.2%), ß-myrcene (10.5%), α-selinene (7.2%), and linalool (6.0%). The EO from the inflorescences was the most active against A. aegypti and A. albopictus, and exhibited the respective larvicidal (LC50 = 87.6 µg/mL and 76.1 µg/mL) and adulticide activities (LC50 = 126.2 µg/mL and 124.5 µg/mL). This EO was also the most active in the inhibition of AChE, since it presented an IC50 value of 14.2 µg/mL. Its larvicidal effect was observed under optical and scanning electron microscopy. Additionally, non-toxic effects against A. salina were observed. Docking modeling of trans-caryophyllene and α-humulene on sterol carrier protein-2 (SCP-2) suggests that both molecules have affinity with the active site of the enzyme, which indicates a possible mechanism of action. Therefore, the essential oil of P. capitarianum may be used in the development of new insecticide targets for the control of A. aegypti and A. albopictus in the Amazonian environment.

Parvalbumin in most gamma-aminobutyric acid-containing neurons of the rat cerebral cortex.

gamma-Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters in the central nervous system. In the cerebral cortex, GABA-containing cells represent a subpopulation of interneurons. With semithin frozen sections, it is possible to demonstrate that most GABA neurons in the rat somatosensory cortex contain the calcium-binding protein parvalbumin and that parvalbumin is found virtually only in GABA neurons. Parvalbumin seems to influence the electrical properties and enzymatic machinery to modulate neuronal excitability and activity. The specific role of parvalbumin in GABA-containing cortical cells may be related to controlling the effectiveness of their inhibitory action.

Neurons in the Nucleus papilio contribute to the control of eye movements during REM sleep.

Rapid eye movements (REM) are characteristic of the eponymous phase of sleep, yet the underlying motor commands remain an enigma. Here, we identified a cluster of Calbindin-D28K-expressing neurons in the Nucleus papilio (NPCalb), located in the dorsal paragigantocellular nucleus, which are active during REM sleep and project to the three contralateral eye-muscle nuclei. The firing of opto-tagged NPCalb neurons is augmented prior to the onset of eye movements during REM sleep. Optogenetic activation of NPCalb neurons triggers eye movements selectively during REM sleep, while their genetic ablation or optogenetic silencing suppresses them. None of these perturbations led to a change in the duration of REM sleep episodes. Our study provides the first evidence for a brainstem premotor command contributing to the control of eye movements selectively during REM sleep in the mammalian brain.

Pedra-ume caá fruit: An Amazon cherry rich in phenolic compounds with antiglycant and antioxidant properties.

Species of Eugenia have been used as an antidiabetic natural source. Chemical, antioxidant and antiglycant screening of extracts from pedra-ume caá (Eugenia punicifolia) fruits were performed. 1H NMR assisted by non-supervised chemometric methods were employed for the evaluation of the chemical profiles which were distinguished according to the color of fruit maturation stages, as well as for pulp and seed fruit. Furthermore, 1H NMR fingerprint analysis of the crude extract allowed the identification of quercitrin and myricitrin, beside other nine compounds. The extracts of the yellow (YP) and green (GP) pulps presented higher antiglycant and antioxidant activities. Fresh juice from E. punicifolia was encapsulated in microcapsules produced with dextrose equivalent (DE) of 10, 20 or 30 as wall materials for the maintainment of their antioxidant and antiglycant properties. The more efficient retention of the bioactive compounds was found using the DE30. The Encapsulation Efficiency (EE) and the Retention Efficiency (RE) of this system was found around 89.7% and 97.6%, respectively. In addition, NMR spectra revealed the presence of flavonoids O-glycosylated (quercitrin and myricitrin) which might be related to the antiglycant and antioxidant activities. The YP presented larger content of quercitrin (117.6 ±â€¯0.4 mg per each 100 g of fresh fruit). Therefore, pedra-ume caá should be employed as an alternative nutraceutical source, as well as intherapeutic pourposes.

The role of parvalbumin and calbindin D28k in experimental scrapie.

AIMS: Prion diseases are generally characterized by pronounced neuronal loss. In particular, a subpopulation of inhibitory neurones, characterized by the expression of the calcium-binding protein parvalbumin (PV), is selectively destroyed early in the course of human and experimental prion diseases. By contrast, nerve cells expressing calbindin D28 k (CB), another calcium-binding protein, as well as PV/CB coexpressing Purkinje cells, are well preserved. METHODS: To evaluate, if PV and CB may directly contribute to neuronal vulnerability or resistance against nerve cell death, respectively, we inoculated PV- and CB-deficient mice, and corresponding controls, with 139A scrapie and compared them with regard to incubation times and histological lesion profiles. RESULTS: While survival times were slightly but significantly diminished in CB-/-, but not PV-/- mice, scrapie lesion profiles did not differ between knockout mice and controls. There was a highly significant and selective loss of isolectin B(4)-decorated perineuronal nets (which specifically demarcate the extracellular matrix surrounding the 'PV-expressing' subpopulation of cortical interneurones) in scrapie inoculated PV+/+, as well as PV-/- mice. Purkinje cell numbers were not different in CB+/+ and CB-/- mice. CONCLUSIONS: Our results suggest that PV expression is a surrogate marker for neurones highly vulnerable in prion diseases, but that the death of these neurones is unrelated to PV expression and thus based on a still unknown pathomechanism. Further studies including the inoculation of mice ectopically (over)expressing CB are necessary to determine whether the shortened survival of CB-/- mice is indeed due to a neuroprotective effect of this molecule.

Calcium-binding proteins in the nervous system.

Among the many calcium-binding proteins in the nervous system, parvalbumin, calbindin-D28K and calretinin are particularly striking in their abundance and in the specificity of their distribution. They can be found in different subsets of neurons in many brain regions. Although it is not yet known whether they play a 'triggering' role like calmodulin, or merely act as buffers to modulate cytosolic calcium transients, they are valuable markers of neuronal subpopulations for anatomical and developmental studies.

Perineuronal nets: past and present.

Golgi ranked the peripheral reticulum--which adheres intimately to nerve cell surfaces--alongside the intracellular reticulum, or Golgi apparatus,which immortalized his name. At first dismissed as an artefact of capricious staining techniques, this peripheral reticulum, or perineuronal net, is now recognized as a genuine entity in neurocytology. It represents a complex of extracellular matrix molecules interposed between the meshwork of glial processes, from which they are indistinguishable, and nerve-cell surfaces. In no other branch of neuroscience has the waxing and waning of interest in any morphological entity been so pronounced as in the case of the perineuronal net. This review traces the history of this enigmatic structure from its conception to the present time, brings to light the keen observational powers of morphologists at the turn of the century and reveals how their sagacious forethought anticipated current thinking on the role of perineuronal nets.

Deficiency in parvalbumin, but not in calbindin D-28k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells.

The Ca(2+)-binding proteins parvalbumin (PV) and calbindin D-28k (CB) are key players in the intracellular Ca(2+)-buffering in specific cells including neurons and have profound effects on spatiotemporal aspects of Ca(2+) transients. The previously observed increase in mitochondrial volume density in fast-twitch muscle of PV-/- mice is viewed as a specific compensation mechanism to maintain Ca(2+) homeostasis. Since cerebellar Purkinje cells (PC) are characterized by high expression levels of the Ca(2+) buffers PV and CB, the question was raised, whether homeostatic mechanisms are induced in PC lacking these buffers. Mitochondrial volume density, i.e. relative mitochondrial mass was increased by 40% in the soma of PV-/- PC. Upregulation of mitochondrial volume density was not homogenous throughout the soma, but was selectively restricted to a peripheral region of 1.5 microm width underneath the plasma membrane. Accompanied was a decreased surface of subplasmalemmal smooth endoplasmic reticulum (sPL-sER) in a shell of 0.5 microm thickness underneath the plasma membrane. These alterations were specific for the absence of the "slow-onset" buffer PV, since in CB-/- mice neither changes in peripheral mitochondria nor in sPL-sER were observed. This implicates that the morphological alterations are aimed to specifically substitute the function of the slow buffer PV. We propose a novel concept that homeostatic mechanisms of components involved in Ca(2+) homeostasis do not always occur at the level of similar or closely related molecules. Rather the cell attempts to restore spatiotemporal aspects of Ca(2+) signals prevailing in the undisturbed (wildtype) situation by subtly fine tuning existing components involved in the regulation of Ca(2+) fluxes.

Change of calretinin expression in the human colon adenocarcinoma cell line HT29 after differentiation.

Calretinin is a Ca(2+)-binding protein of the EF-hand family which is expressed in colon adenocarcinomas and colon-derived tumor cell lines (e.g. WiDr), but is absent from normal human enterocytes. Its function has not as yet been elucidated, but some lines of evidence lead us to postulate its involvement in cell proliferation in these cells. In order to test whether calretinin is correlated with an undifferentiated, proliferating, or with a differentiated, state of cells, its expression was studied in the human colon adenocarcinoma clonal cell line HT29-18, which can be caused to differentiate into enterocyte-like cells by replacing glucose with galactose in the culture medium (glucose starvation differentiation). Treatment of HT29-18 cells with galactose led to a drop in the calretinin mRNA level and in protein expression as evidenced by immunocytochemical staining and Western blot analysis of cytosolic cell extracts. These results suggest that calretinin is present in HT29-18 cancer cells, mostly in those which are in the undifferentiated state. The possibility that calretinin is involved in maintaining the cells in an undifferentiated (cancerous) state is discussed.

The EF-hand Ca(2+)-binding protein super-family: a genome-wide analysis of gene expression patterns in the adult mouse brain.

In mice, 249 putative members of the superfamily of EF-hand domain Ca(2+)-binding proteins, manifesting great diversity in structure, cellular localization and functions have been identified. Three members in particular, namely, calbindin-D28K, calretinin and parvalbumin, are widely used as markers for specific neuronal subpopulations in different regions of the brain. The aim of the present study was to compile a comprehensive atlas of the gene-expression profiles of the entire EF-hand gene superfamily in the murine brain. This was achieved by a meticulous examination of the in-situ hybridization images in the Allen Brain Atlas database. Topographically, our analysis focused on the olfactory bulb, cerebral cortex (barrel cortex in the primary somatosensory area), basal ganglia, hippocampus, amygdala, thalamus, hypothalamus, cerebellum, midbrain, pons and medulla, and on clearly identifiable sub-structures within each of these areas. The expression profiles of four family-members, namely hippocalcin-like 4, neurocalcin-δ, plastin 3 and tescalcin, that have not been hitherto reported, at either the mRNA (in-situ-hybridization) or the protein (immunohistochemical) levels, are now presented for the first time. The fruit of our analysis is a document in which the gene-expression profiles of all members of the EF-hand family genes are compared, and in which future possible neuronal markers for specific cells/brain areas are identified. The assembled information could afford functional clues to investigators, conducive to further experimental pursuit.

Characterization of a polyclonal antiserum against the purified human recombinant calcium binding protein calretinin.

We have purified recombinant human calretinin (CR) from Escherichia coli lysates and have produced a polyclonal antiserum against it. The antiserum recognizes determinants conserved in fish, chicken, rat, monkey and human CR. We show its use in the qualitative detection of CR by different methods of immunohistochemistry as well as in the detection of CR on immunoblots.

Monoclonal antibodies directed against the calcium binding protein parvalbumin.

We have produced twelve monoclonal antibodies (McAB) against carp-II parvalbumin. Three of them, designated 235, 239, 267 recognize determinants conserved in fish, chicken, mouse, rat, monkey and human parvalbumin. We show their use in the qualitative detection of parvalbumin (PV) by immunohistochemistry, in the quantitation of parvalbumin by radioimmunoassay and in the detection of parvalbumin on immunoblots.

The calcium-binding protein calretinin-22k, an alternative splicing product of the calretinin gene is expressed in several colon adeno carcinoma cell lines.

An alternatively spliced mRNA for the calcium-binding protein calretinin (CR) is present in the colon adenocarcinoma cell line WiDr. As a consequence of a frame shift, the resulting protein, calretinin-22k (CR-22k), consists of the first 178 amino acids of calretinin followed by a carboxy-terminal peptide of 14 amino acids that is not present in full-length calretinin. Antibodies specific for this C-terminal region have been generated by 2 different methods. A peptide corresponding to the specific C-terminal region of CR-22k was either chemically synthesized and coupled to a carrier protein or was expressed in Escherichia coli as a carboxyterminal fusion to a carrier protein applying recombinant techniques. Both antisera produced in rabbits were tested in Western blots and immuno-histochemical experiments. The antisera recognized human recombinant CR-22k overexpressed in E. coli, but not fulllength calretinin and stained fixed WiDr cells. The presence of CR-22k was also confirmed in the colon cell lines CO115/3 in which mRNA coding for CR-22k mRNA coding for CR-22k mRNA is present as well as in the lines COLO205 and LS-180, all of which also express full-length calretinin. Although the intracellular distribution of CR-22k and CR are similar as evidenced by immunohistochemical stainings, CR-22k is preferentially localized in the nucleus in the cell lines LS-180 and Co115/3 suggesting potentially different roles for the two proteins.

Monoclonal antibodies directed against the calcium binding protein Calbindin D-28k.

We have produced 25 clones secreting antibodies directed against chicken Calbindin D-28k. Two of them, 300 and 318, recognize determinants conserved in fish, chicken, mouse, rat, rabbit, monkey and human Calbindin D-28k. We demonstrate their use in the immunohistochemical localization of Calbindin D-28k, and in the detection of Calbindin D-28k on immunoblots.

Parvalbumin-immunoreactive neurons in the neocortex are resistant to degeneration in Alzheimer's disease.

Recent studies have stressed the fact that specific neuronal subtypes may display a differential sensitivity to degeneration in Alzheimer's disease. For example, large pyramidal neurons have been shown to be vulnerable, whereas smaller neurons are resistant to pathology. Using a monoclonal antibody against the calcium-binding protein parvalbumin, we investigated the possible changes in a subpopulation of interneurons in two cortical areas known to be strongly damaged in Alzheimer's disease. In the prefrontal cortex as well as in the inferior temporal cortex, we observed no differences in parvalbumin-immunoreactive cell counts or cell size in Alzheimer's disease brains as compared to control cases. Moreover, the general cellular morphology of these neurons was preserved in the Alzheimer's disease cases, in that their perikarya and dendritic arborizations were intact. These results suggest that paravalbumin-immunoreactive cells represent a neuronal subset resistant to degeneration, and further support the hypothesis that the pathological process in Alzheimer's disease involves specific neuronal subtypes with particular morphological and molecular characteristics.

Preservation of calretinin-immunoreactive neurons in the hippocampus of epilepsy patients with Ammon's horn sclerosis.

Selective neuronal vulnerability and aberrant axonal reorganization in the hippocampus may play an important role for the pathogenesis of pharmaco-resistant temporal lobe epilepsy (TLE). Interneurons containing calcium-binding proteins (CaBPs) are candidates for pathogenetically relevant neurons in the hippocampus of patients with TLE. Here we have examined the cellular localization and distribution of calretinin (CR), a recently discovered CaBP, in the hippocampus of 35 patients with TLE. There was a striking preservation of CR-immunoreactive neurons in TLE patients with Ammon's horn sclerosis (AHS). No significant differences in the distribution of CR-immunoreactive neurons were observed between patients with lesion-associated TLE and control patients without epilepsy. However, a subpopulation of CR-immunoreactive interneurons with morphological features of Cajal-Retzius-like cells, which are only transiently detectable in the normally developing hippocampus, was markedly increased in epilepsy patients with AHS. This increase did not correlate with the duration of the epileptic disorder. Another significant finding was a striking increase and reorganization of CR-immunoreactive neuropil throughout the entire molecular layer of the dentate gyrus (DG-ML) in patients with AHS as compared to patients with focal lesions and control specimens. Ultrastructural analysis identified the CR-immunoreactive axonal profiles as components of an inhibitory, intrinsic neuronal system. The presence of a CR-positive, aberrant cell population, in combination with sprouting of CR-positive axonal processes may significantly alter the gating function of the dentate gyrus and thereby increase hippocampal epileptogenicity in epilepsy patients with AHS.