Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21 parts per thousand salinity), as well as potential routes of Na(+) uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase alpha-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell inviginations, respectively. These findings ire discussed regarding the putative movement of Na(+) across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations. (C) 2008 Elsevier Inc. All rights reserved.

Low prenatal vitamin D alters morphology, cell density and gene expression in adult rat brain: Correlations with schizophrenia

Statement of the study: Based on data from ecological and analytic epidemiological studies, we have proposed that low prenatal vitamin D is a candidate risk-modifying factor for schizophrenia. Previously, we demonstrated that low prenatal vitamin D adversely affected brain development in neonatal rats (Eyles et al, 2003). Here we examine the impact of both prenatal and early life hypovitaminosis D on various outcomes in the adult rat brain. Methods: Female Sprague-Dawley rats were made vitamin D deficient via the use of a special diet (Dyets CA) and lighting conditions that excluded UVB radiation. Animals were kept under these conditions for 6 weeks then mated with males kept under normal conditions. Vitamin deplete dams were kept under these conditions during pregnancy. Offspring from two test groups were examined. Offspring were either reared with dams repleted with vitamin D at birth or remained under deplete conditions till weaning. Both test groups were weaned under normal vitamin D conditions and remained so till testing at adulthood. We compared the brains of adult offspring kept under both test conditions with animals from control environments. Summary of results: We found a significant persistent dose-related increase in lateral ventricle volume and alterations in anterior cingulate and prefrontal cortical cell densities (consistent with the known prodifferentiation properties of this steroid). In both test groups we observed a reduced expression of NGF as well as a down-regulation of transcripts coding for GABAA alpha 4 receptor and two neuronal structural elements; MAP2 and Neurofilament L. Conclusion: These findings provide further evidence that vitamin D is involved in brain development. An increase in prefrontal cortical cell density, a reduction neuronal structural elements and persistent ventriculomegaly are all common anatomical findings in the brains of patients with schizophrenia. The specific reduction in transcripts for neuronal structural proteins but not GFAP is also in accordance with the proposal that frontal cortical architecture in schizophrenia reflects a reduction in connectivity rather than a reduction in glial processes(Goldman-Rakic and Selemon, 1997). These findings confirm the biological plausibility of early life hypovitaminosis D as a risk factor for schizophrenia.

Process development for a recombinant Chinese Hamster Ovary (CHO) cell line utilizing a metal induced and amplified metallothionein expression system

The suspension Chinese Hamster Ovary cell line, 13-10-302, utilizing the metallothionein (MT) expression system producing recombinant human growth hormone (hGH) was studied in a serum-free and cadmium-free medium at different fermentation scales and modes of operation. Initial experiments were carried out to optimize the concentration of metal addition to induce the MT promoter. Subsequently, the cultivation of the 13-10-302 cell line was scaled up from spinner flasks into bioreactors, and the cultivation duration was extended with fed-batch and perfusion strategies utilizing 180 muM zinc to induce the promoter controlling expression of recombinant hGH. It was shown that a fed-batch process could increase the maximum cell numbers twofold, from 3.3 to 6.3 x 10(6) cell/mL, over those obtained in normal batch fermentations, and this coupled with extended fermentation times resulted in a fourfold increase in final hGH titer, from 135 +/- 15 to 670 +/- 70 mg/L at a specific productivity q(hGH) value of 12 pg cell(-1)d(-1). The addition of sodium butyrate increased the specific productivity of hGH in cells to a value of approximately 48 pg cell(-1)d(-1), resulting in a final hGH titer of over a gram per liter during fed-batch runs. A BioSep acoustic cell recycler was used to retain the cells in the bioreactor during perfusion operation. It was necessary to maintain the specific feeding rates (SFR) above a value of 0.2 vvd/(10(6) cell/mL) to maintain the viability and productivity of the 13-10-302 cells; under these conditions the viable cell number increased to over 107 cell/mL and resulted in a volumetric productivity of over 120 mg(hGH) L(-1)d(-1). Process development described in this work demonstrates cultivation at various scales and sustained high levels of productivity under cadmium free condition in a CHO cell line utilizing an inducible metallothionein expression system. (C) 2004 Wiley Periodicals, Inc.

Synthesis of homoallylic oxygenated alpha-methylene-gamma-butyrolactones: a model for preparing biologically active natural lactones

In this Letter we describe a 12% overall yield synthesis of a model for homoallylic oxygenated alpha-methylene-gamma-butyrolactones with relative stereochemistry defined by selective hydrogenation with Rh/Al(2)O(3). The synthesis was realized in 9 steps involving simple reactions. (C) 2008 Elsevier Ltd. All rights reserved.

Virtual models of the HLA class I antigen processing pathway

Antigen recognition by cytotoxic CD8 T cells is dependent upon a number of critical steps in MHC class I antigen processing including proteosomal cleavage, TAP transport into the endoplasmic reticulum, and MHC class 1 binding. Based on extensive experimental data relating to each of these steps there is now the capacity to model individual antigen processing steps with a high degree of accuracy. This paper demonstrates the potential to bring together models of individual antigen processing steps, for example proteosome cleavage, TAP transport, and MHC binding, to build highly informative models of functional pathways. In particular, we demonstrate how an artificial neural network model of TAP transport was used to mine a HLA-binding database so as to identify H LA-binding peptides transported by TAP. This integrated model of antigen processing provided the unique insight that HLA class I alleles apparently constitute two separate classes: those that are TAP-efficient for peptide loading (HLA-B27, -A3, and -A24) and those that are TAP-inefficient (HLA-A2, -B7, and -B8). Hence, using this integrated model we were able to generate novel hypotheses regarding antigen processing, and these hypotheses are now capable of being tested experimentally. This model confirms the feasibility of constructing a virtual immune system, whereby each additional step in antigen processing is incorporated into a single modular model. Accurate models of antigen processing have implications for the study of basic immunology as well as for the design of peptide-based vaccines and other immunotherapies. (C) 2004 Elsevier Inc. All rights reserved.

Biochemical and structural characterization of a beta-1,3-1,4-glucanase from Bacillus subtilis 168

beta-1,3-1,4-Glucanases (E.C. 3.2.1.73) hydrolyze linked beta-D-glucans, such as lichenan and barley beta-glucan. Recombinant beta-1,3-1,4-glucanase from Bacillus subtilis expressed in Escherichia coil and purified by Ni-NTA chromatography exhibited optimum activity at 50 degrees C and pH 6.0. The catalytic half-life at 60 degrees C decreased from 90 to 5 min when the enzyme was incubated in the presence and absence of Ca(2+) respectively. The kinetic parameters of lichenan hydrolysis were 2695, 3.1 and 1220 for V(max) (mu mol/min/mg), K(m) (mg mL(-1)) and K(cat) (s(-1)), respectively. Analysis by DLS, AUC and SAXS demonstrated the enzyme is monomeric in solution. Chemical denaturation monitored by ITFE and far-UV CD yielded Delta G(H2O) values of 9.6 and 9.1 kcal/mol, respectively, showing that the enzyme has intermediate stability when compared with other Bacillus beta-1,3-1,4-glucanases. The crystal structure shows the anti-parallel jelly-roll beta-sheet conserved in all GH16 beta-1,3-1,4-glucanases, with the amino acid differences between Bacillus sp. enzymes that are likely determinants of stability being distributed throughout the protein. (C) 2011 Elsevier Ltd. All rights reserved.

Differential adjustment in gill Na(+)/K(+)- and V-ATPase activities and transporter mRNA expression during osmoregulatory acclimation in the cinnamon shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

We evaluate osmotic and chloride (Cl(-)) regulatory capability in the diadromous shrimp Macrobrachium amazonicum, and the accompanying alterations in hemolymph osmolality and [Cl(-)], gill Na(+)/K(+)-ATPase activity, and expression of gill Na(+)/K(+)-ATPase alpha-subunit and V-ATPase B subunit mRNA during salinity (S) acclimation. We also characterize V-ATPase kinetics and the organization of transport-related membrane systems in the gill epithelium. Macrobrachium amazonicum strongly hyper-regulates hemolymph osmolality and [Cl(-)] in freshwater and in salinities up to 25 parts per thousand S. During a 10-day acclimation period to 25 parts per thousand S, hemolymph became isosmotic and hypo-chloremic after 5 days, [Cl(-)] alone remaining hyporegulated thereafter. Gill Na(+)/K(+)-ATPase alpha-subunit mRNA expression increased 6.5 times initial values after 1 h, then decreased to 3 to 4 times initial values by 24 h and to 1.5 times initial values after 10 days at 25 parts per thousand S. This increased expression was accompanied by a sharp decrease at 5 h then recovery of initial Na(+)/K(+)-ATPase activity within 24 h, declining again after 5 days, which suggests transient Cl(-) secretion. V-ATPase B-subunit mRNA expression increased 1.5-fold within 1 h, then reduced sharply to 0.3 times initial values by 5 h, and remained unchanged for the remainder of the 10-day period. V-ATPase activity dropped sharply and was negligible after a 10-day acclimation period to 21 parts per thousand S, revealing a marked downregulation of ion uptake mechanisms. The gill epithelium consists of thick, apical pillar cell flanges, the perikarya of which are coupled to an intralamellar septum. These two cell types respectively exhibit extensive apical evaginations and deep membrane invaginations, both of which are associated with numerous mitochondria, characterizing an ion transporting epithelium. These changes in Na(+)/K(+)- and V-ATPase activities and in mRNA expression during salinity acclimation appear to underpin ion uptake and Cl(-) secretion by the palaemonid shrimp gill.

Association of haplotypes in the IL8 gene with susceptibility to chronic periodontitis in a Brazilian population

Background: Interleukin 8 (IL-8) is a chemokine related to the initiation and amplification of acute and chronic inflammatory processes. Polymorphisms in the IL8 gene have been associated with inflammatory diseases. We investigated whether the - 845(T/C) and - 738(T/A) single nucleotide polymorphisms (SNPs) in the IL8 gene, as well as the haplotypes they form together with the previously investigated -353(A/T), are associated with susceptibility to chronic periodontitis. Methods: DNA was extracted from buccal epithelial cells of 400 Brazilian individuals (control n =182, periodontitis n=218). SNPs were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Disease associations were analyzed by the chi(2) test, Exact Fisher test and Clump program. Haplotypes were reconstructed using the expectation-maximization algorithm and differences in haplotype distribution between the groups were analyzed to estimate genetic susceptibility for chronic periodontitis development. Results: When analyzed individually, no SNPs showed different distributions between the control and chronic periodontitis groups. Although, nonsmokers carrying the TTA/CAT (OR = 2.35, 95% CI = 1.03-5.36) and TAT/CTA (OR= 6.05, 95% CI = 1.32-27.7) haplotypes were genetically susceptible to chronic periodontitis. The ITT/TAA haplotype was associated with protection against the development of periodontitis (for nonsmokers OR= 0.22, 95% CI = 0.10-0.46). Conclusion: Although none of the investigated SNPs in the IL8 gene was individually associated with periodontitis, some haplotypes showed significant association with susceptibility to, or protection against, chronic periodontitis in a Brazilian population. (C) 2010 Elsevier B.V. All rights reserved.

Refolding and purification of the human secreted group IID phospholipase A(2) expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A(2) (sPLA(2)s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A(2) (hsPLA(2)-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA(2)-IID in Escherichia coli, the DNA-coding sequence for hsPLA(2)-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA(2)-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A(2). The refolded recombinant hsPLA(2)-IID demonstrated Ca(2+)-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA(2)-IID which will advance our understanding of the structure-function relationship and biological effects of the protein. (C) 2009 Elsevier Inc. All rights reserved.

Na(+), K(+)-ATPase activity in gill microsomes from the blue crab, Callinectes danae, acclimated to low salinity: Novel perspectives on ammonia excretion

This investigation provides an extensive characterization of the modulation by ATP, Mg(2+), Na(+), K(+) and NH(4)(+) of a gill microsomal (Na(+),K(+))-ATPase from Callinectes danae acclimated to 15 parts per thousand salinity. Novel findings are the lack of high-affinity ATP-binding sites and a 10-fold increase in enzyme affinity for K(+) modulated by NH4+, discussed regarding NH4+ excretion in benthic marine crabs. The (Na(+),K(+))-ATPase hydrolyzed ATP at a maximum rate of 298.7 +/- 16.7 nmol Pi min(-1) mg(-1) and K(0.5) = 174.2 +/- 9.8 mmol L(-1) obeying cooperative kinetics (n(H) = 1.2). Stimulation by sodium (V = 308.9 +/- 15.7 nmol Pi min(-1) mg(-1), K(0.5) = 7.8 +/- 0.4 mmol L(-1)), magnesium (299.2 +/- 14.1 nmol Pi min(-1) mg(-1), K(0.5) = 767.3 +/- 36.1 mmol L(-1)), potassium (300.6 +/- 153 nmol Pi min(-1) mg(-1), K(0.5) = 1.6 +/- 0.08 mmol L(-1)) and ammonium (V = 345.1 +/- 19.0 nmol Pi min(-1) mg(-1), K(0.5) = 6.0 +/- 0.3 mmol L(-1)) ions showed site-site interactions. Ouabain inhibited (Na(+),K(+))-ATPase activity with K(1) = 45.1 +/- 2.5 mu mol L(-1), although affinity for the inhibitor increased (K(1) = 22.7 +/- 1.1 mu mol L(-1)) in 50 mmol L(-1) NH(4)(+). Inhibition assays using ouabain plus oligomycin or ethacrynic acid suggest mitochondrial F(0)F(1)- and K(+)-ATPase activities, respectively. Ammonium and potassium ions synergistically stimulated specific activity up to 72%, inferring that these ions bind to different sites on the enzyme molecule, each modulating stimulation by the other. (C) 2009 Elsevier Inc. All rights reserved.

Effects of reversible inactivation of the medial septum on rat exploratory behavior in the elevated plus-maze using a test-retest paradigm

The effect of intraseptal injections of lidocaine before a first or a second session in the elevated plus-maze, in a test-retest paradigm, was investigated. In addition to gross session analyses, a minute-by-minute analysis of the sessions was used to evaluate both anxiety and memory. Lidocaine injections before the test session produced increases in the frequency of entries, time spent and distance run in the open arms without affecting activity occurring in the closed arms. During the retest session, saline- and lidocaine-treated rats exhibited increased indices of anxiety and lidocaine-treated rats exhibited decreased closed-arm entries. The minute-by-minute analysis showed a faster decrease in anxiety-related behaviors during the test session by saline- than by lidocaine-treated rats and a significant decrease in closed-arm exploration by saline-treated rats, but not by lidocaine-treated ones. Lidocaine injection before the retest session produced increases in the frequency of entries, time spent and distance run in the open arms in the second session when compared with saline-treated rats. Minute-by-minute analysis showed an increase in the time spent in the open arms by lidocaine animals at the beginning of the retest session in comparison to saline animals and a significant decrease in closed-arm exploration by both groups. These results suggest that inactivation of the medial septum by lidocaine affects the expression of unconditioned and conditioned forms of anxiety in the elevated plus-maze and, in a lesser way, the acquisition and retention of spatial information. (C) 2010 Elsevier B.V. All rights reserved.

L-Allylglycine dissociates the neural substrates of fear in the periaqueductal gray of rats

The dorsal (dPAG) and ventral (vPAG) regions of the periaqueductal gray are well known to contain the neural substrates of fear and anxiety. Chemical or electrical stimulation of the dPAG induces freezing, followed by a robust behavioral reaction that has been considered an animal model of panic attack. In contrast, the vPAG is part of a neural system, in which immobility is the usual response to its stimulation. The defense reaction induced by the stimulation of either region is accompanied by anti nociception. Although GABAergic mechanisms are known to exert tonic inhibitory control on the neural substrates of fear in the dPAG, the role of these mechanisms in the vPAG is still unclear. The present study examined defensive behaviors and antinociception induced by microinjections of an inhibitor of gamma-aminobutyric acid synthesis, L-allylglycine (L-AG; 1, 3, and 5 mu g/0.2 mu l), into either the dPAG or vPAG of rats subjected to the open field and tail-flick tests. Passive or tense immobility was the predominant behavior after L-AG (1 or 3 mu g) microinjection into the vPAG and dPAG, respectively, which was replaced with intense hyperactivity, including jumps or rearings, after injections of a higher dose (5 mu g/0.2 mu l) into the dPAG or vPAG. Moreover, whereas intra-dPAG injection of 3 mu g L-AG produced intense antinociception, only weak antinociception was induced by intra-vPAG injections of 5 mu g L-AG. These findings suggest that GABA mechanisms are involved in the mediation of antinociception and behavioral inhibition to aversive stimulation of the vPAG and exert powerful control over the neural substrates of fear in the dPAG to prevent a full-blown defense reaction possibly associated with panic disorder. (C) 2009 Elsevier Inc. All rights reserved.

THE ANTERIOR CINGULATE CORTEX IS A TARGET STRUCTURE FOR THE ANXIOLYTIC-LIKE EFFECTS OF BENZODIAZEPINES ASSESSED BY REPEATED EXPOSURE TO THE ELEVATED PLUS MAZE AND FOS IMMUNOREACTIVITY

Prior experience with the elevated plus maze (EPM) increases the avoidance of rodents to the open arms and impairs the anxiolytic-like effects of benzodiazepines on the traditional behaviors evaluated upon re-exposure to the maze, a phenomenon known as one-trial tolerance. Risk assessment behaviors are also sensitive to benzodiazepines. During re-exposure to the maze, these behaviors reinstate the information-processing initiated during the first experience, and the detection of danger generates stronger open-arm avoidance. The present study investigated whether the benzodiazepine midazolam alters risk assessment behaviors and Fos protein distribution associated with test and retest sessions in the EPM. Naive or maze-experienced Wistar rats received either saline or midazolam (0.5 mg/kg i.p.) and were subjected to the EPM. Midazolam caused the usual effects on exploratory behavior, increasing exploratory activity of naive rats in the open arms and producing no effects on these conventional measures in rats re-exposed to the maze. Risk assessment behaviors, however, were sensitive to the benzodiazepine during both sessions, indicating anxiolytic-like effects of the drug in both conditions. Fos immunohistochemistry showed that midazolam injections were associated with a distinct pattern of action when administered before the test or retest session, and the anterior cingulate cortex, area 1 (Cg1), was the only structure targeted by the benzodiazepine in both situations. Bilateral infusions of midazolam into the Cg1 replicated the behavioral effects of the drug injected systemically, suggesting that this area is critically involved in the anxiolytic-like effects of benzodiazepines, although the behavioral strategy adopted by the animals appears to depend on the previous knowledge of the threatening environment. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Anxiety-like symptoms induced by morphine withdrawal may be due to the sensitization of the dorsal periaqueductal grey

Withdrawal from morphine leads to the appearance of extreme anxiety accompanied of several physical disturbances, most of them linked to the activation of brainstem regions such as the locus coeruleus, ventral tegmental area, hypothalamic nuclei and periaqueductal grey (PAG). As anxiety remains one of the main components of morphine withdrawal the present study aimed to evaluating the influence of the dorsal aspects of the PAG on the production of this state, since this structure is well-known to be involved in defensive behaviour elicited by anxiety-evoking stimuli. Different groups of animals were submitted to 10 days of i.p. morphine injections, challenged 2 h after with an i.p. injection of naloxone (0.1 mg/kg), and submitted to the plus-maze, open-field and light-dark transition tests. The effects of morphine withdrawal on anxiety-induced Fos immunolabelling were evaluated in four animals that passed by the light-dark transition test randomly chosen for Fos-protein analysis. Besides the PAG, Fos neural expression was conducted in other brain regions involved in the expression of anxiety-related behaviours. Our results showed that morphine withdrawn rats presented enhanced anxiety accompanied of few somatic symptoms. Increased Fos immunolabelling was noted in brain regions well-known to modulate these states as the prelimbic cortex, nucleus accumbens, amygdala and paraventricular hypothalamus. Increased Fos labelling was also observed in the ventral and dorsal aspects of the PAG, a region involved in anxiety-related processes suggesting that this region could be a common neural substrate enlisted during anxiety evoked by dangerous stimuli as well as those elicited by opiate withdrawal. (c) 2008 Elsevier Inc. All rights reserved,

Activity of the medial prefrontal cortex and amygdala underlies one-trial tolerance of rats in the elevated plus-maze

The anxiolytic effects of benzodiazepines are reduced after a single exposure of rats to elevated plus-maze test (EPM). Midazolam showed an anxioselective profile in animals submitted to one session (T1) but did not change the usual exploratory behavior of rats exposed twice (T2) to the EPM. In this study we examined further the one-trial tolerance by performing a factor analysis of the exploratory behavior of rats injected with saline before both trials as well as an immunohistochemistry study for quantification of Fos expression in encephalic structures after these sessions. Factor analysis of all behavioral categories revealed that factor I consisted of anxiety-related categories in T1 whereas these same behavioral categories loaded on factor 2 in T2. Risk assessment was also dissociated as it loaded stronger on T2 (factor 3) than on T1 (factor 4). Locomotor activity in T1 loaded on factor 5. Immunohistochemistry analyses showed that Fos expression predominated in limbic structures in T1 group. The medial prefrontal cortex and amygdala were the main areas activated in T2 group. These data suggest that anxiety and risk assessment behaviors change their valence across the EPM sessions. T2 is characterized by the emergence of a fear factor, more powerful risk assessment and medial prefrontal cortex activation. The amygdala functions as a switch between the anxiety-like patterns of T1 to the cognitive control of fear prevalent in T2. The EPM retest session is proposed as a tool for assessing the cognitive activity of rodents in the control of fear. (c) 2007 Elsevier B.V. All rights reserved.