Spontaneous Eyeblink Activity

Spontaneous blinking is essential for maintaining a healthy ocular surface and clarity of vision. The spontaneous blink rate (SBR) is believed to reflect a complex interaction between peripheral influences mediated by the eye surface and the central dopaminergic activity. The SBR is thus extremely variable and dependent on a variety of psychological and medical conditions. Many different methods have been employed to measure the SBR and the upper eyelid kinematics during a blink movement. Each has its own merits and drawbacks, and the choice of a specific method should be tailored to the specific needs of the investigation. Although the sequence of muscle events that leads to a blink has been fully described, knowledge about the neural control of spontaneous blinking activity is not complete. The tear film is dynamically modified between blinks, and abnormalities of the blink rate have an obvious influence on the ocular surface.

Protease activity and the ultrastructure of broiler chicken PSE (pale, soft, exudative) meat

The biological cause of broiler PSE meat seems to be an excessive release of Ca(2+), promoted by a genetic mutation of ryanodine receptors located in the sarcoplasmic reticulum of skeletal muscle cells. Excessive Ca(2+), associated with protein denaturation in meat, enhances protease activity and influences the functional properties of PSE meat. Twenty-four-hour post-mortem Pectoralis major m. samples exhibited lower values for pH, water-holding capacity, and shear force than did control samples, in contrast to colour (L*) and cooking loss values. Protease activity, measured as myofibril fragmentation index, presented higher values in PSE meat than in control samples. Ultrastructural examination revealed shrinking and depolymerisation of myofilaments and Z-lines disorganisation within the sarcomere in PSE meat. Intense calpain activity was also observed, indicating that the process may initiate at the filaments, because of protein denaturation, and spread through Z-lines, resulting in the collapse of the sarcomere structure. (C) 2009 Elsevier Ltd. All rights reserved.

A quantitative analysis of the nerve fibres of the acetabular periosteum of dogs

There are many techniques for the treatment of hip dysplasia, and novel research is currently being undertaken in the hope of obtaining more efficient and less traumatic techniques. The denervation of the hip joint capsule is a simple and effective technique that allows recovery of the functional activity of the affected limbs in significantly less time than other techniques. This surgical procedure consists of removing the acetabular periosteum, thus eliminating the nerve fibres with consequent analgesia. The aim of this investigation was to quantify the number of nerve fibres present in different regions of the acetabular periosteum. The knowledge of regional differences is potentially valuable for the refining of the denervation technique of the hip joint capsule. Thirty canine acetabular fragments were used to compare the nerve fibre density of the periosteum. The results showed a significant difference between the mean density of nerve fibres at the cranial and dorsal-lateral portion (approximately 75 fibres/mm(2)) and caudal lateral portion (approximately 60 fibres/mm(2)) of the acetabulum. Those fibres at the pedosteum are almost positioned in a sagittal plane, pointing towards the joint capsule, suggesting the some density in the latter region. These results indicate a new approach to the articular denervation technique, thus obtanining even better results for the treatment of hip dysplasia in dogs.

Long-Term Maternal Separation Differentially Alters Serum Corticosterone Levels and Blood Neutrophil Activity in A/J and C57BL/6 Mouse Offspring

Objectives: In this work, we searched for maternal separation effects on serum corticosterone levels and blood neutrophil activity in adult male A/J and C57BL/6 mouse offspring. Methods: 40 male A/J mice and 40 male C57BL/6 mice were divided within each strain into two groups. Mice in the maternal separation group were separated from their mothers (1 h/day) on postnatal days 0-13. Mice in the control group were left undisturbed. On postnatal day 45, blood was drawn from all mice and used to assess neutrophil activity by flow cytometry and serum corticosterone levels by radioimmunoassay. Results: The results showed that each mouse strain responded differently to maternal separation, but in both cases, serum corticosterone levels were affected. In both strains, adult mice that experienced maternal separation showed lower serum corticosterone levels than control mice. In relation to control mice kept together with their mothers, the levels of serum corticosterone were 72.7 and 36.36% lower in A/J and C57BL/6 mice submitted to maternal separation, respectively. The current findings showed that maternal separation increased neutrophil activity in mice after reaching adulthood. The observed effects, although in the same direction, differed between A/J and C57BL/6 mice. Maternal separation increased both the percentage and intensity of phagocytosis in C57BL/6 mice, but had no effects on A/J mice. Furthermore, maternal separation increased basal and propidium iodide-labeled Staphylococcus aureus-induced oxidative burst in A/J mice but did not affect oxidative burst in C57BL/6 mice. Finally, phorbol myristate acetate-induced oxidative burst increased in both strains. Conclusion: These results indicate that early maternal separation increases innate immunity, most likely by modifying hypothalamus-pituitary-adrenal axis activity. This suggests that maternal separation is a good model for stress which produces long-term neuroimmune changes whatever the animal species and strain used. Copyright (C) 2011 S. Karger AG, Basel

Prenatal lipopolysaccharide reduces motor activity after an immune challenge in adult male offspring

Prenatal lipopolysaccharide (LPS) exposure causes reproductive, behavioral and neurochemical injuries in both the mother and pups. Previous investigations by our group showed that prenatal LPS administration (100 mu g/kg, i.p.) on gestational day 9.5 impaired the male offspring`s social behavior in infancy and adulthood. In the present study, we investigated whether these social behavioral changes were associated with motor activity impairment. Male rat pups treated prenatally with LPS or not were tested for reflexological development and open field general activity during infancy. In adulthood, animals were tested for open field general activity, haloperidol-induced catalepsy and apomorphine-induced stereotypy; striatal dopamine levels and turnover were also measured. Moreover, LPS-treated or untreated control pups were challenged with LPS in adulthood and observed for general activity in the open field. In relation to the control group, the motor behavior of prenatally treated male pups was unaffected at basal levels, both in infancy and in adulthood, but decreased general activity was observed in adulthood after an immune challenge. Also, striatal dopamine and metabolite levels were decreased in adulthood. In conclusion, prenatal LPS exposure disrupted the dopaminergic system involved with motor function, but this neurochemical effect was not accompanied by behavioral impairment, probably due to adaptive plasticity processes. Notwithstanding, behavioral impairment was revealed when animals were challenged with LPS, resulting in enhanced sickness behavior. (C) 2010 Elsevier B.V. All rights reserved.

Lercanidipine decreases vascular matrix metalloproteinase-2 activity and protects against vascular dysfunction in diabetic rats

Abnormal matrix metalloproteinases (MMPs) activity causes cardiovascular diseases. Because hyperglycemia increase MMPs activities through increased oxidative stress. we hypothesized that antioxidant effects produced by lercanidipine could attenuate the increases in MMP-2 expression/activity in diabetic rats. Control and diabetic (alloxan-induced diabetes) rats received lercanidipine 2.5 mg/kg/day (or tap water) starting three weeks after alloxan (or vehicle) injections. Blood pressure was monitored weekly. After six weeks of treatment, vascular reactivity and structural changes were assessed in aortic rings. MMP-2 levels were determined by gelatin zymography, and MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by quantitative real time RT-PCR. Plasma thiobarbituric acid reactive substances concentrations were determined by fluorimetry. Lercanidipine produced antihypertensive effects (201 +/- 5 vs. 163 +/- 7 mm Hg in diabetic rats untreated and treated with lercaniclipine, respectively; P < 0.01) and reversed the impairment in endothelium-dependent vasorelaxation in diabetic rats. Increased MMP-2 and Pro-MMP-2 levels were found in the aortas of diabetic rats (both P < 0.001). Lercandipine attenuated the increases in oxidative stress and in MMP-2 (both P < 0.05). While diabetes induced no major structural changes, it caused a 16-fold increase in the ratio of MMP-2/TIMP-2 mRNA expression, which was completely reversed by lercanidipine (both P < 0.001). These results show that antioxidant and beneficial vascular effects produced by lercanidipine in diabetic rats are associated with reversion of the imbalance in vascular MMP-2MMP-2 expression. (C) 2008 Published by Elsevier B.V.

Regulation of yeast acetohydroxyacid synthase by valine and ATP

The first step in the common pathway for the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (AHAS; EC 4.1.3.18). The enzyme is found in plants, fungi and bacteria, and is regulated by controls on transcription and translation, and by allosteric modulation of catalytic activity. It has long been known that the bacterial enzyme is composed of two types of subunit, and a similar arrangement has been found recently for the yeast and plant enzymes. One type of subunit contains the catalytic machinery, whereas the other has a regulatory function. Previously, we have shown [Pang and Duggleby (1999) Biochemistry 38, 5222-5231] that yeast AHAS can be reconstituted from its separately purified subunits. The, reconstituted enzyme is inhibited by valine, and ATP reverses this inhibition. In the present work, we further characterize the structure and the regulatory properties of reconstituted yeast AHAS. High phosphate concentrations are required for reconstitution and it is shown that these conditions are necessary for physical association between the catalytic and regulatory subunits. It is demonstrated by CD spectral changes that ATP binds to the regulatory subunit alone, most probably as MgATP. Neither valine nor MgATP causes dissociation of the regulatory subunit from the catalytic subunit. The specificity of valine inhibition and MgATP activation are examined and it is found that the only effective analogue of either regulator of those tested is the non-hydrolysable ATP mimic, adenosine 5 '-[beta,gamma -imido]triphosphate. The kinetics of regulation are studied in detail and it is shown that the activation by MgATP depends on the valine concentration in a complex manner that is consistent with a proposed quantitative model.

Genomic organization of the CC chemokine MIP-3 alpha/CCL20/LARC/EXODUS/SCYA20, showing gene structure, splice variants, and chromosome localization

We describe the genomic organization of a recently identified CC chemokine, MIP3 alpha /CCL20 (HGMW-approved symbol SCYA20). The MIP-3 alpha /CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISK analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3 alpha /CCL20, Ala MLP-3 alpha /CCL20 and Ser MIP-3 alpha /CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ata MIP-3 alpha /CCL20 or Ser MIP-3 alpha /CCL20. Both forms of MIP-3cr/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-a-activated CD4(+) and CD8(+) T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3 alpha /CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3 alpha /CCL20 and Ala MIP-3 alpha /CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known. (C) 2001 Academic Press.

Discovery and structure of a potent and highly specific blockerof insect calcium channels

We have isolated a novel family of insect-selective neurotoxins that appear to be the most potent blockers of insect voltage-gated calcium channels reported to date. These toxins display exceptional phylogenetic specificity, with at least a 10,000-fold preference for insect versus vertebrate calcium channels. The structure of one of the toxins reveals a highly structured, disulfide-rich core and a structurally disordered C-terminal extension that is essential for channel blocking activity. Weak structural/functional homology with omega -agatoxin-IVA/B, the prototypic inhibitor of vertebrate P-type calcium channels, suggests that these two toxin families might share a similar mechanism of action despite their vastly different phylogenetic specificities.

Noninvasive tests of vascular function and structure: Why and how to perform them

Background Early atherosclerosis involves the endothelium of many arteries. Information about peripheral arterial anatomy and function derived from vascular imaging studies such as brachial artery reactivity (BAR) and carotid intima media thickness (IMT) may be pertinent to the coronary circulation. The prevention and early treatment of atherosclerosis is gaining more attention, and these tests might be used as indications or perhaps guides to the effectiveness of therapy, but their application in clinical practice has been limited. This review seeks to define the anatomy and pathophysiology underlying these investigations, their methodology, the significance of their Findings, and the issues that must be resolved before their application. Methods The literature on BAR and IMT is extensively reviewed, especially in relation to clinical use. Results Abnormal flow-mediated dilation is present in atherosclerotic vessels, is associated with cardiovascular risk factors, and may be a marker of preclinical disease. Treatment of known atherosclerotic risk Factors has been shown to improve flow-mediated dilation, and some data suggest that vascular responsiveness is related to outcome. Carotid IMT is associated with cardiovascular risk factors, and increased levels can predict myocardial infarction and stroke. Aggressive risk factor management can decrease IMT. Conclusions BAR and IMT ate functional and structural markers of the atherosclerotic process. The clinical use of BAR has been limited by varying reproducibility and the influence by exogenous factors, but IMT exhibits less variability. A desirable next step in the development of BAR and IMT as useful clinical tools would be to show an association of improvement in response to treatment with improvement in prognosis.

Three-dimensional structure of RTD-1, a cyclic antimicrobial defensin from rhesus macaque leukocytes

Most mammalian defensins are cationic peptides of 29-42 amino acids long, stabilized by three disulfide bonds. However, recently Tang et al. (1999, Science 286, 498-502) reported the isolation of a new defensin type found in the leukocytes of rhesus macaques. In contrast to all the other defensins found so far, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue [Tang et al. (1999) Science 286, 498-502]. To elucidate the three-dimensional structure of RTD-1 and its open chain analogue, both peptides were synthesized using solid-phase peptide synthesis and tert-butyloxycarbonyl chemistry. The structures of both peptides in aqueous solution were determined from two-dimensional H-1 NMR data recorded at 500 and 750 MHz. Structural constraints consisting of interproton distances and dihedral angles were used as input for simulated-annealing calculations and water refinement with the program CNS. RTD-1 and its open chain analogue oRTD-1 adopt very similar structures in water. Both comprise an extended beta -hairpin structure with turns at one or both ends. The turns are well defined within themselves and seem to be flexible with respect to the extended regions of the molecules. Although the two strands of the beta -sheet are connected by three disulfide bonds, this region displays a degree of flexibility. The structural similarity of RTD-1 and its open chain analogue oRTD-1, as well as their comparable degree of flexibility, support the theory that the additional charges at the termini of the open chain analogue rather than overall differences in structure or flexibility are the cause for oRTD-1's lower antimicrobial activity. In contrast to numerous other antimicrobial peptides, RTD-1 does not display any amphiphilic character, even though surface models of RTD-1 exhibit a certain clustering of positive charges. Some amide protons of RTD-1 that should be solvent-exposed in monomeric beta -sheet structures show low-temperature coefficients, suggesting the possible presence of weak intermolecular hydrogen bonds.

Human pigmentation genes: identification, structure and consequences of polymorphic variation

The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones a-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation. (C) 2001 Elsevier Science B.V. All rights reserved.

A simulation model for analysing patient activity in dermatology

We developed a general model to assess patient activity within the primary and secondary health-care sectors following a dermatology outpatient consultation. Based on observed variables from the UK teledermatology trial, the model showed that up to 11 doctor-patient interactions occurred before a patient was ultimately discharged from care. In a cohort of 1000 patients, the average number of health-care visits was 2.4 (range 1-11). Simulation analysis suggested that the most important parameter affecting the total number of doctor-patient Interactions is patient discharge from care following the initial consultation. This implies that resources should be concentrated in this area. The introduction of teledermatology (either realtime or store and forward) changes the values of the model parameters. The model provides a quantitative tool for planning the future provision of dermatology health-care.

Characterization of pore structure and coordination of titanium in TiO2 and SiO2-TiO2 sol-pillared clays

Titania sol-pillared clay (TiO2 PILC) and silica-titania sol-pillared clay (SiO2-TiO2 PILC) were synthesized by the sol-gel method. Supercritical drying (SCD) and treatment with quaternary ammonium surfactants were used to tailor the pore structure of the resulting clay. It was found that SCD approach increased the external surface area of the PILCs dramatically and that treatment with surfactants could be used to tailor pore size because the mesopore formation in the galleries between the clay layers follows the templating mechanism as observed in the synthesis of MCM-41 materials. Highly mesoporous solids were thus obtained. In calcined TiO2 PILC, ultrafine crystallites in anatase phase, which are active for photocatalytic oxidation of organics, were observed. In SiO2-TiO2 PILCs and their derivatives, titanium was highly dispersed in the matrix of silica and no crystal phase was observed. The highly dispersed titanium sites are good catalytic centers for selective oxidation of organic compounds. (C) 2001 Academic Press.

Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.