New evidence of neuroprotection by lactate after transient focal cerebral ischaemia: extended benefit after intracerebroventricular injection and efficacy of intravenous administration.

BACKGROUND: Lactate protects mice against the ischaemic damage resulting from transient middle cerebral artery occlusion (MCAO) when administered intracerebroventricularly at reperfusion, yielding smaller lesion sizes and a better neurological outcome 48 h after ischaemia. We have now tested whether the beneficial effect of lactate is long-lasting and if lactate can be administered intravenously. METHODS: Male ICR-CD1 mice were subjected to 15-min suture MCAO under xylazine + ketamine anaesthesia. Na L-lactate (2 µl of 100 mmol/l) or vehicle was administered intracerebroventricularly at reperfusion. The neurological deficit was evaluated using a composite deficit score based on the neurological score, the rotarod test and the beam walking test. Mice were sacrificed at 14 days. In a second set of experiments, Na L-lactate (1 µmol/g body weight) was administered intravenously into the tail vein at reperfusion. The neurological deficit and the lesion volume were measured at 48 h. RESULTS: Intracerebroventricularly injected lactate induced sustained neuroprotection shown by smaller neurological deficits at 7 days (median = 0, min = 0, max = 3, n = 7 vs. median = 2, min = 1, max = 4.5, n = 5, p < 0.05) and 14 days after ischaemia (median = 0, min = 0, max = 3, n = 7 vs. median = 3, min = 0.5, max = 3, n = 7, p = 0.05). Reduced tissue damage was demonstrated by attenuated hemispheric atrophy at 14 days (1.3 ± 4.0 mm(3), n = 7 vs. 12.1 ± 3.8 mm(3), n = 5, p < 0.05) in lactate-treated animals. Systemic intravenous lactate administration was also neuroprotective and attenuated the deficit (median = 1, min = 0, max = 2.5, n = 12) compared to vehicle treatment (median = 1.5, min = 1, max = 8, n = 12, p < 0.05) as well as the lesion volume at 48 h (13.7 ± 12.2 mm(3), n = 12 vs. 29.6 ± 25.4 mm(3), n = 12, p < 0.05). CONCLUSIONS: The beneficial effect of lactate is long-lasting: lactate protects the mouse brain against ischaemic damage when supplied intracerebroventricularly during reperfusion with behavioural and histological benefits persisting 2 weeks after ischaemia. Importantly, lactate also protects after systemic intravenous administration, a more suitable route of administration in a clinical emergency setting. These findings provide further steps to bring this physiological, commonly available and inexpensive neuroprotectant closer to clinical translation for stroke.

Bacterial membrane injuries induced by lactacin F and nisin

The combined action of nisin and lactacin F, two bacteriocins produced by lactic acid bacteria, is additive. In this report, the basis of this effect is examined. Channels formed by lactacin F were studied by experiments using planar lipid bilayers, and bactericidal effects were analyzed by flow cytometry. Lactacin F produced pores with a conductance of 1 ns in black lipid bilayers in 1 mM KClat 10 mV at 20°C. Pore formation was strongly dependent on voltage. Although lactacin F formed pores at very low potential (10 mV), the dependence was exponentialabov e 40 mV. The injuries induced by nisin and lactacin F in the membranes of Lactobacillus helveticus produced different flow cytometric profiles. Probably, when both bacteriocins are present, each acts separately; their cooperation may be due to an increase in the number of single membrane injuries

Establishment of an In Vitro Photoallergy Test Using NCTC2544 Cells and IL-18 Production

Differentiation between photoallergenic and phototoxic reactions induced by low molecular weight compounds represents a current problem. The use of eratinocytes as a potential tool for the detection of photoallergens as opposed to photoirritants is considered an interesting strategy for developing in vitro methods. We have previously demonstrated the possibility to use the human keratinocyte cell line NCTC2455 and the production of interleukin-18 (IL-18) to screen low molecular weight sensitizers. The purpose of this work was to explore the possibility to use the NCTC2544 assay to identify photoallergens and discriminate from phototoxic chemicals. First, we identified suitable condition of UV-irradiation (3.5 J/cm2) by investigating the effect of UVAirradiation on intracellular IL-18 on untreated or chloropromazine (a representative phototoxic compound)- treated NCTC2544 cells. Then, the effect of UVA-irradiation over NCTC2544 cells treated with increasing concentrations of 15 compounds including photoallergens (benzophenone, 4-ter-butyl-4-methoxydibenzoylmethane, 2-ethylexyl-p-methoxycinnamate, ketoprofen, 6-methylcumarin); photoirritant and photoallergen (4-aminobenzoic acid, chlorpromazine, promethazine); photoirritants (acridine, ibuprofen, 8-methoxypsoralen, retinoic acid); and negative compounds (lactic acid, SDS and p-phenilendiamine) was investigated. Twenty-four hours after exposure, cytotoxicity was evaluated by the MTT assay or LDH leakage, while ELISA was used to measure the production of IL-18. At the maximal concentration assayed with non-cytotoxic effects (CV80 under irradiated condition), all tested photoallergens induced a significant and a dose-dependent increase of intracellular IL-18 following UVA irratiation, whereas photoirritants failed. We suggest that this system may be useful for the in vitro evaluation of the photoallergic potential of chemicals.

Kalvon modifioinnin vaikutus pienten molekyylien nanosuodatukseen

Tässä työssä tutkittiin pienten molekyylien nanosuodatusta kolmella kalvolla. Lappeenrannan teknillisen korkeakoulun laboratoriomittakaavan nanosuodatuslaitteistolla suodatettiin glukoosin, maitohapon ja oktaanihapon vesiliuoksia 0,45 m/s virtausnopeudella. Lisäksi NF 45, NF 270 ja NTR 7450 –kalvoja modifioitiin UV-säteilytyksellä modifiointiaineen kanssa tai ilman. Modifiointiaineina olivat maito- ja oktaanihappo. Kalvon modifioinnilla pyrittiin parantamaan permeabiliteettia ilman retentiohäviöitä. Kirjallisuusosassa käsiteltiin nanosuodatuksen erottumisperiaatteita ja konsentraatiopolarisaation merkitystä liuenneiden aineiden erottumisessa. Lisäksi käsiteltiin kalvon modifioinnin merkitystä suodatuksen parantamiseen ja pienten orgaanisten molekyylien nanosuodatusta. Aluksi suodatettiin glukoosi- ja oktaanihappoliuoksia NF 270 –kalvolla. Glukoosin retentio oli 80% pitoisuudesta riippumatta, mutta oktaanihapon retentio, 70-100%, riippui pitoisuudesta. 100 ppm oktaanihapon pitoisuudessa retentio oli 100% ja suuremmilla pitoisuuksilla alhaisempi. Kun oktaanihappoa suodatettiin modifioimattomilla kalvoilla pH:n funktiona, niin retentiot olivat pH-riippuvaisia. Alhaisilla pH-arvoilla oktaanihapon retentiot olivat lähes nolla ja nousivat jyrkästi pH:ssa 6 siten, että korkeilla pH-arvoilla retentiot olivat yli 80%. Glukoosin suodatuksissa NF 270 –kalvolla modifiointi aina hieman paransi vuota, mutta retentiot huononivat. Oktaanihapon suodatuksissa vuo parani hieman, kun kalvoja (NF 270 ja NTR 7450) oli modifioitu 20 minuuttia UV-säteilytyksellä 100 ppm maitohappoliuoksessa. NTR 7450 –kalvon vuo moninkertaistui modifioimattoman kalvon vuohon verrattuna, kun kalvoa oli UV-säteilytetty 20 minuuttia 2000 ppm maitohappoliuoksessa. Oktaanihapposuodatuksissa retentiot modifioiduilla kalvoilla olivat suurimmat pH-alueella 7-10. Modifioitujen kalvojen permeabiliteetit nousivat jyrkästi pH:ssa 12 kaikilla malliaineilla, mikä viittaa siihen, että modifiointiaine irtosi pH:ssa 12. Toisaalta korkeilla pH-arvoilla kalvo muuttuu avoimemmaksi, joten modifiointiaineen irtoamista ei voitu näiden mittausten perusteella varmentaa. Kalvon modifiointi oli onnistunut, sillä malliainesuodatuksissa havaittiin vuo- ja retentiomuutoksia.

Métodos de preparação e atividade biológica do ácido quinolínico e derivados

In 1981 2,3-pyridine dicarboxylic acid (quinolinic acid) was discovery to be a selective agonist for the N-methyl -D-aspartic acid (NMDA) receptor. As a consequence it possesses neurotoxic activity resulting from overstimulation of the receptor. Quinolinic acid is implicated as an etiological factor in a range of neurodegenerative disease including AIDS related dementia, Huntington´s disease and Lyme disease. In the design of novel therapies to treat these diseases, some molecules have been identified as an important target. In this paper we described different methods to prepare quinolinic acid and derivatives.

CARACTERIZAÇÃO E AVALIAÇÃO IN VITRO DE NANOCOMPÓSITOS DE POLI (L-ÁCIDO LÁTICO) E NANOTUBOS DE CARBONO DE PAREDES MÚLTIPLAS PURIFICADOS

Carbon nanotubes (CNT) have been studied for biomedical applications due to their unique properties. However, pristine CNT have structural features and impurities that can cause toxicity to biological systems. In this work, we describe a method to purify multiwalled carbon nanotubes (MWCNT) by chemical modification and subsequent attachment of hydroxyl and carboxyl groups to improve dispersion and to decrease toxic effects. Nanocomposites from poly (L-lactic acid) (PLLA) and nanotubes were produced by the solvent casting method and characterized and evaluated for cytocompatibility with Vero cells. The nanocomposite interactions with Vero cells demonstrated that the cells were able to adhere and sustain proliferation and showed favorable cytocompatibility. In vitro studies also revealed an increase in fibroblast cell viability in the nanocomposites, compared with neat PLLA.

Physical-chemical characterization and wet milling yield of four maize hybrids

The influence of physical-chemical characteristics of maize grains and lactic acid concentrations on byproduct yields, generated by conventional wet milling, was studied during steeping, for four maize hybrids and two lactic acid concentrations (0.55 and 1.00%). For physical-chemical characterization, grain dimensions (length, thickness, and width) were determined, as well as mass of 100 grains, percentage of floating grains, volumetric mass, and centesimal composition. Statistical differences were found for percentage of floating grains (2.33 to 24.67%), volumetric mass (0.814 to 0.850 kg.L-1), mass of 100 grains (0.033 to 0.037 kg), water content (11.86 to 12.20%), proteins (8.21 to 9.06%), lipids (3.00 to 4.77%), and ashes (1.07 to 1.26%). There were no relationships of wet milling yields with maize appearance and physical-chemical characteristics. The addition of 1.00% lactic acid did not statistically improve byproduct yields; however, it favored separation of the grain components.

Membraanin modifiointi happo- ja alkoholikäsittelyillä

Tässä diplomityössä tutkittiin alkoholilla ja orgaanisella hapolla tehtävien esikäsittelyiden vaikutusta nanosuodatuskalvon ominaisuuksiin. Työn tarkoituksena oli parantaa nanosuodatuskalvon fraktiointiominaisuuksia sekä kasvattaa monosakkaridien suotautuvuutta. Tarkasteluissa käytetty membraani oli GE Osmonicsin valmistamaa Desal-5 DL nanosuodatuskalvoa, jota modifioitiin erilaisilla maitohappo- ja isopropanoliesikäsittelyillä. Suodatukset tehtiin kahdella erilaisella laboratoriomittakaavan levysuotimella käyttäen malliaineina väkevää sokeriliuosta sekä laimeampaa sokeri-suola-liuosta. Happo- ja alkoholiesikäsiteltyjen kalvojen vuo- ja retentioarvoja verrattiin referensseinä käytettyjen vesiliotettujen kalvojen vastaaviin arvoihin. Puhtaat esikäsitellyt kalvot analysoitiin myös tarkemmin kalvoissa tapahtuneiden muutosten ymmärtämiseksi. Suodatusten ja analyysitulosten perusteella sekä alkoholi- että happoesikäsittelyt paransivat nanosuodatuskalvon ominaisuuksia parantaen sokerivuota, heikentämättä kuitenkaan kalvon fraktiointikykyä eri moolimassan omaavien sokereiden suhteen. Hapolla saavutettiin hieman alkoholikäsittelyä suotuisammat muutokset, mutta molemmilla käsittelyillä haluttujen komponenttien retentio monesti jopa parani referenssikalvoon verrattuna. Havaitut muutokset kalvoissa olivat pääosin fysikaalisia, mutta erityisesti happokäsittely muutti kalvon rakennetta myös kemiallisesti. Molemmat käsittelyt lisäsivät myös tarkasteltavan membraanin hydrofiilisuutta.

Sawfly larval poisoning in cattle: Report on new outbreaks and brief review of the literature

Sawfly larval poisoning (SLP) is an acute hepatotoxicosis documented in livestock in Australia, Denmark and in countries of South America. It is caused by the ingestion of the larval stage of insects of the suborder Symphyta, order Hymenoptera, commonly known as "sawfly". Three species of sawfly are reportedly involved in the toxicosis. The insect involved in Australian SLP is Lophyrotoma interrupta (Pergidae), in Denmark the cause of SLP is the ingestion of the larvae Arge pullata (Argidae), and in South American countries documented outbreaks of SLP were caused by the ingestion of yet another sawfly, Perreyia flavipes (Pergidae). In all geographical areas where it occurred, SLP causes important livestock losses. In cattle, as well as in other affected species, the disease has a short clinical course and in many outbreaks affected cattle can be found dead. When observed, clinical signs include apathy, recumbence, tremors, paddling movements and death in 24-48 hours. Neurological signs such aggressiveness attributable to hepatic encephalopathy are also observed. In cases with a more protracted course icterus and photodermatitis may develop. Gross findings included ascites, petechiae and ecchymosis over serosal surfaces of thoracic and abdominal cavities, and an enlarged liver that displays accentuation of the lobular pattern and edema of the gall bladder wall. Sawfly larval body fragments and heads are consistently found in the fore stomachs and occasionally abomasum of affected cattle. Main microscopic lesions are restricted to the liver and consist of centrolobular (periacinar) to massive hepatocellular necrosis. In most lobules necrotic areas extended up to the portal triads where only a few viable hepatocytes remain. Mild to moderate lymphocyte necrosis is seen in lymphatic tissues. Cases occur in the winter months when the larval stages of the sawfly are developing. D-amino acid-containing peptides have been found to be the toxic principle in each sawfly involved in SLP. The octapeptide lophyrotomin is the major toxin in the in the larvae of Australian and Danish sawflies and is present in small amounts in the larvae of South American sawfly. The heptadecapeptide pergidin is the main toxin in the South American sawfly while small amounts of pergidin have been found in the other two species of toxic sawfly. During the winter of 2011 (July-August) four outbreaks of SLP were diagnosed in the State of Rio Grande do Sul, Brazil. The findings in those outbreaks are reported here and a brief review of the literature regarding SLP around the world is provided.

Glutamatergic transmission in the nucleus tractus solitarii: from server to peripherals in the cardiovascular information superhighway

Afferent nerves carrying signals from mechanoreceptors in the aortic arch and carotid sinus terminate predominantly in the nucleus tractus solitarii (NTS). Signal transduction and neurotransmission in the NTS are critical for central cardiovascular reflex control, but little was known about either until the late 1970's. None of the numerous neuroactive chemicals found in the NTS had met strict criteria as a neurotransmitter in the baroreflex arc until data suggested that the excitatory amino acid L-glutamate (GLU) might be released from baroreceptor afferent terminals in the NTS. In anesthetized animals microinjection into the NTS of GLU, which can be demonstrated in terminals in the NTS, produces cardiovascular responses like those seen with activation of the baroreceptor reflex. Similar responses occur in awake animals if the chemoreceptor reflex is eliminated; otherwise, in conscious animals responses mimic those of chemoreceptor reflex activation. GLU is released in the NTS upon selective activation of the baroreceptor, and possibly the chemoreceptor, reflex. Responses to selective agonists as well as baroreflex responses are eliminated by GLU antagonists microinjected into the NTS. Non-NMDA (N-methyl-D-aspartic acid) receptors seem to predominate at primary baroreceptor synapses in the NTS while NMDA receptors may be involved at later synapses. Although inhibition of soluble guanylate cyclase attenuates responses to ionotropic glutamate agonists in the NTS, nitric oxide does not seem to play a role in glutamate transmission in the NTS. GLU may also participate in transmission at cardiovascular neurons beyond the NTS. For example, a role has been suggested for GLU in the ventrolateral medulla and spinal cord. Work continues concerning GLU signal transduction and mechanisms that modulate that transduction both at the NTS and at other cardiovascular nuclei

Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

Lactic acid bacteria (LAB) are Gram-positive bacteria and are generally regarded as safe (GRAS) organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc) was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE) of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV) epitope-protein fusion (BCV-Nuc). BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

Kullan talteenotto takaisinuuttoliuoksista

Työssä tutkittiin kirjallisuuden ja laboratoriomittausten avulla vaihtoehtoja kullan pelkistämiseen ja talteenottoon kultauuton takaisinuuttoliuoksista. Tavoitteena oli löytää menetelmä, jolla saadaan puhdasta kiinteää lopputuotetta ilman kullan häviöitä. Käytettyjä pelkistimiä olivat D-(+)-glukoosi, natriumboorihydridi, L-askorbiinihappo, D-(-)-isoaskorbiinihappo ja aktiivihiili. Laboratoriokokeiden perusteella D-(-)-isoaskorbiinihappo sekä aktiivihiili olivat sopivimmat pelkistimet kokeissa käytetylle kultaliuokselle. Isoaskorbiinihapolla suoritettiin panoskokeita lasireaktorissa eri alku-pH:ssa sekä erilaisilla pelkistimen ja kullan moolisuhteilla. Tulosten perusteella havaittiin pH:n ja pelkistimen ylimäärän vaikuttavan merkittävästi lopputuotteen puhtauteen. Myös redox-potentiaalia säätämällä ja happopesulla pelkistyksen jälkeen voidaan vaikuttaa lopputuotteen puhtauteen. Aktiivihiilellä suoritettiin panoskokeita adsorptiotasapainojen (latausisotermi) ja kinetiikan tutkimiseksi. Hiileen on mahdollista saada kultaa 383 mg/g kuivaa hiiltä. Suurempi lataus voitaisiin saavuttaa käyttämällä hiiltä, jolla on pienempi partikkelikoko. Kolonnikokeita tehtiin eri virtausnopeuksilla. Kolonnikokeissa kullan dynaaminen adsorptiokapasiteetti hiileen odotetusti kasvoi virtausnopeuden laskiessa. Pienin käytetty virtausnopeus oli 2,40 BV/h, jolloin kapasiteetti oli 75,4 mg/g kuivaa hiiltä (c (Au feed) = 129 mg/L). Kullasta voidaan poistaa myös kolonnipelkistyksen jälkeen epäpuhtauksia happopesulla. Isoaskorbiinihapolla pelkistyksen kinetiikka on nopea ja sillä saatiin pelkistettyä puhdasta lopputuotetta. Sekä isoaskorbiinihappo, että aktiivihiili ovat potentiaalisia menetelmiä kullan talteenottoon.

Galactosaminoglycans from normal myometrium and leiomyoma

In many tumors, the amount of chondroitin sulfate in the extracellular matrix has been shown to be elevated when compared to the corresponding normal tissue. Nevertheless, the degree of chondroitin sulfate increase varies widely. In order to investigate a possible correlation between the amount of chondroitin sulfate and tumor size, several individual specimens of human leiomyoma, a benign uterine tumor, were analyzed. The glycosaminoglycans from eight tumors were extracted and compared with those from the respective adjacent normal myometrium. The main glycosaminoglycan found in normal myometrium was dermatan sulfate, with small amounts of chondroitin sulfate and heparan sulfate. In leiomyoma, both dermatan sulfate and chondroitin sulfate were detected and the total amounts of the two galactosaminoglycans was increased in all tumors when compared to normal tissue. In contrast, the heparan sulfate concentration decreased in the tumor. To assess the disaccharide composition of galactosaminoglycans, these compounds were incubated with bacterial chondroitinases AC and ABC. The amounts of L-iduronic acid-containing disaccharides remained constant, whereas the concentration of D-glucuronic acid-containing disaccharides increased from 2 to 10 times in the tumor, indicating that D-glucuronic acid-containing disaccharides are responsible for the elevation in galactosaminoglycan concentration. This increase is positively correlated with tumor size.

Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with ß-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed ß-D-glucuronic acid and N-acetyl ß-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

Protective mechanism of agmatine pretreatment on RGC-5 cells injured by oxidative stress

Agmatine has neuroprotective effects on retinal ganglion cells (RGCs) as well as cortical and spinal neurons. It protects RGCs from oxidative stress even when it is not present at the time of injury. As agmatine has high affinity for various cellular receptors, we assessed protective mechanisms of agmatine using transformed RGCs (RGC-5 cell line). Differentiated RGC-5 cells were pretreated with 100 μM agmatine and consecutively exposed to 1.0 mM hydrogen peroxide (H2O2). Cell viability was determined by measuring lactate dehydrogenase (LDH), and the effects of selective alpha 2-adrenergic receptor antagonist yohimbine (0-500 nM) and N-methyl-D-aspartic acid (NMDA) receptor agonist NMDA (0-100 µM) were evaluated. Agmatine’s protective effect was compared to a selective NMDA receptor antagonist MK-801. After a 16-h exposure to H2O2, the LDH assay showed cell loss greater than 50%, which was reduced to about 30% when agmatine was pretreated before injury. Yohimbine almost completely inhibited agmatine’s protective effect, but NMDA did not. In addition, MK-801 (0-100 µM) did not significantly attenuate the H2O2-induced cytotoxicity. Our results suggest that neuroprotective effects of agmatine on RGCs under oxidative stress may be mainly attributed to the alpha 2-adrenergic receptor signaling pathway.