A comparison of the histochemical and image-derived proteoglycan content of articular cartilage

There are several methods for determining the proteoglycan content of cartilage in biomechanics experiments. Many of these include assay-based methods and the histochemistry or spectrophotometry protocol where quantification is biochemically determined. More recently a method based on extracting data to quantify proteoglycan content has emerged using the image processing algorithms, e.g., in ImageJ, to process histological micrographs, with advantages including time saving and low cost. However, it is unknown whether or not this image analysis method produces results that are comparable to those obtained from the biochemical methodology. This paper compares the results of a well-established chemical method to those obtained using image analysis to determine the proteoglycan content of visually normal (n=33) and their progressively degraded counterparts with the protocols. The results reveal a strong linear relationship with a regression coefficient (R2) = 0.9928, leading to the conclusion that the image analysis methodology is a viable alternative to the spectrophotometry.

A light and electron microscopic study of NADPH-diaphorase-,calretinin- and parvalbumin-containing neurons in the rat nucleus accumbens

The rat nucleus accumbens contains medium-sized, spiny projection neurons and intrinsic, local circuit neurons, or interneurons. Sub-classes of interneurons, revealed by calretinin (CR) or parvalbumin (PV) immunoreactivity or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, were compared in the nucleus accumbens core, shell and rostral pole. CR, PV and NADPH-diaphorase-containing neurons are shown to form three non-co-localising populations in these three areas. No significant differences in neuronal population densities were found between the subterritories. NADPH-diaphorase-containing neurons could be further separated morphologically into three sub-groups, but CR- and PV-immunoreactive neurons form homogeneous populations. Ultrastructurally, NADPH-diaphorase-, CR- and PV-containing neurons in the nucleus accumbens all possess nuclear indentations. These are deeper and fewer in neurons immunoreactive for PV than in CR- and NADPH-diaphorase-containing neurons. CR-immunoreactive boutons form asymmetrical and symmetrical synaptic specialisations on spines, dendrites and somata, while PV-immunoreactive boutons make only symmetrical synaptic specialisations. Both CR- and PV-immunoreactive boutons form symmetrical synaptic specialisations with medium-sized spiny neurons and contact other CR- and PV-immunoreactive somata, respectively. A novel non-carcinogenic substrate for the peroxidase reaction (Vector Slate Grey, SG) was found to be characteristically electron-dense and may be distinguishable from the diaminobenzidine reaction product. We conclude that the three markers used in this study are localised in distinct populations of nucleus accumbens interneurons. Our studies of their synaptic connections contribute to an increased understanding of the intrinsic circuitry of this area.

Unmodified silver nanoparticles for rapid analysis of the organophosphorus pesticide, dipterex, often found in different waters

A novel, uncomplicated and rapid method of analysis for organophosphorus (OP) pesticides was researched and developed using the important, common OP, dipterex, as a typical example. The basis of the method involved the citrate-capped silver nanoparticles (citrate-capped AgNPs) and Acetylthiocholine (ATCh). The latter compound can be catalyzed by Acetylcholinesterase (AChE) to form thiocholine (TCh), which induces the aggregation of AgNPs. Correspondingly, the color of AgNPs in solution changes from bright yellow to pink, and the UV–vis characteristic absorption peak of AgNPs at about 400 nm decreases; simultaneously, a new absorption band appears at about 520 nm. Irreversible inhibition of AChE activity caused by dipterex, prevents the aggregation of AgNPs. Thus, a UV–vis spectrophotometric method was developed for the analysis of dipterex. The absorbance ratio A396 nm/A520 nm was found to be linearly related to the concentration of dipterex in the range of 0.25–37.5 ng mL−1 with a detection limit of 0.18 ng mL−1. This method was used successfully to analyse dipterex in spiked, different water samples.

Different correlation of Sphingosine-1-Phosphate receptor 1 with receptor activator of nuclear factor kappa B ligand and regulatory T cells in rat periapical lesions

Introduction Sphingosine-1-phosphate receptor 1 (S1P1) is crucial for regulation of immunity and bone metabolism. This study aimed to investigate the expression of S1P1 in rat periapical lesions and its relationship with receptor activator of nuclear factor kappa B ligand (RANKL) and regulatory T (Treg) cells. Methods Periapical lesions were induced by pulp exposure in the first lower molars of 55 Wistar rats. Thirty rats were killed on days 0, 7, 14, 21, 28, and 35, and their mandibles were harvested for x-ray imaging, micro–computed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. The remaining 25 rats were killed on days 0, 14, 21, 28, and 35, and mandibles were harvested for flow cytometry. Results The volume and area of the periapical lesions increased from day 0 to day 21 and then remained comparably stable after day 28. S1P1-positive cells were observed in the inflammatory periapical regions; the number of S1P1-positive cells peaked at day 14 and then decreased from day 21 to day 35. The distribution of S1P1-positive cells was positively correlated with the dynamics of RANKL-positive cells but was negatively correlated with that of Treg cells. Conclusions S1P1 expression was differentially correlated with RANKL and Treg cell infiltration in the periapical lesions and is therefore a contributing factor to the pathogenesis of such lesions.

Dracocephalum moldavica L. and Melissa officinalis L. : Chemistry and Bioactivities Relevant in Alzheimer’s Disease Therapy

Oksidatiivisen stressin eli liiallisen reaktiivisten happiyhdisteiden määrän soluissa on jo pitkään arveltu olevan tärkeä Alzheimerin taudin kehittymiseen ja etenemiseen vaikuttava tekijä. Tämän vuoksi kiinnostus erilaisten antioksidanttien (yhdisteitä, jotka neutraloivat näitä happiradikaaleja soluissa) mahdollisia terapeuttisia ominaisuuksia Alzheimerin taudin hoidossa on tutkittu laajalti. Tähän mennessä ei kuitenkaan ole vielä onnistuttu löytämään antioksidanttia, joka olisi hyödyksi Alzheimerin taudin hoidossa. Tämän vuoksi on tärkeää pyrkiä löytämään uusia antioksidanttien lähteitä sekä tutkia niistä löytyviä aktiivisia yhdisteitä. Kiinnostus luonnon antioksidantteja kohtaan on kasvanut voimakkaasti viime aikoina. Huomio on kiinnittynyt erityisesti aromaattisista sekä lääkekasveista löytyviin antioksidantteihin. Lamiaceae- perheeseen kuuluvia tuoksuampiaisyrttiä (Dracocephalum moldavica L.) ja sitruunamelissaa (Melissa officinalis L.) on käytetty Iranissa pitkään sekä ruoanlaitossa että lääkinnässä, minkä vuoksi näiden kasvien uutteiden antioksidanttisisältöä päätettiin analysoida käyttäen useaa erilaista in vitro- menetelmää. Näissä kokeissa ilmeni, että uutteilla oli useita antioksidanttisia vaikutuksia. Näistä antioksidanttisista vaikutuksista vastaavia yhdisteitä pyrittiin tunnistamaan käyttäen HPLC-PDA- tekniikkaa, minkä seurauksena niiden havaittiin sisältävän erilaisia polyfenoleita, kuten hydroksyloituneita bentsoeeni- ja cinnamamidihapon johdannaisia sekä flavonoideja. Kummankin kasvin uutteissa runsaimmin esiintynyt yhdiste oli rosmariinihappo. Sitruunamelissaa (M. officinalis) on käytetty antiikin ajoista alkaen kognitiivisten toimintojen häiriöiden hoidossa. Perustuen tietoon kasvin käytöstä perinteisessä lääkinnässä, sen tehoa Alzheimerin taudin hoidossa on tutkittu viime aikoina kliinisin kokein. Sitruunamelissan todettiinkin olevan hyödyksi lievää ja keskivaikeaa Alzheimeimerin tautia sairastavien potilaiden hoidossa. Väitöskirjan osanan olevasta kooste-artikkelista käy ilmi, että tutkimalla lääkekasvien ominaisuuksia voidaan saada arvokkaita suuntaa-antavia vihjeitä Alzheimerin taudin lääkehoidon kehittämiseen. Tämän perusteella päätettiinkin testatata myös sitruunamelissauutteen kykyä estää asetyylikoliiniesteraasin (AChE) toimintaa, koska tämän entsyymin toiminna estämisen tiedetään olevan hyödyksi Alzheimerin taudin hoidossa. Uute kykeni estämään AChE:n toimintaa, minkä vuoksi uutteen sisältämiä komponentteja päätettiin tutkia terkemmin. Uute jaettiin erilaisiin fraktioihin käyttäen HPLC-menetelmää, minkä jälkeen testattiin jokaisen fraktion kykyä inhiboida AchE. Suurin osa fraktioista kykeni inhiboimaan AChE:n toimintaa selkeästi tehokkaammin, kuin raakauute. Kaikista tehokkainta fraktiota analysoitiin tarkemmin sen aktiivisten yhdisteiden tunnistamiseksi, minkä seurauksena sen sisältämät yhdisteet tunnistettiin cis ja trans-rosmariinihapoiksi. Tässä tutkimuksessa tunnistettujen yhdisteiden hyödyllisyyttä Alzheimerin taudin hoidossa tulisi seuraavaksi tutkia erilaisissa in vivo-malleissa. Lisäksi jäljellä olevien fraktioiden kemiallinen koostumus tulisi selvittää sekä antioksidanttiaktiivisuuden ja AChE:n toiminnan inhiboinnin välistä mahdollista yhteyttä tulisi tutkia tarkemmin. Tämä tutkimus osoittaa tuoksuampiasyrtin (D. moldavica) sekä sitruunamelissan (M. officinalis) sisältävän monenlaisia aktiivisia antioksidantteja. Lisäksi sitruunamelissan sisältämät yhdisteet kykenivät estämään asetyylikoliiniesteraasin (AchE) toimintaa. Nämä tulokset tukevat osaltaan väitöskirjan osana olevan kooste-artikkelin johtopäätöksiä, joiden mukaan etnofarmakologinen kasvitutkimus voi osoittautua erittäin hyödylliseksi kehitettäessä uutta lääkehoitoa Alzheimerin tautiin. Lisäksi tässä väitöskirjassa kuvattu tutkimus osoittaakin, että perinteisesti lääkekasvina käytettyä sitruunamelissaa voidaan mahdollisesti hyödyntää uusien Alzheimerin taudin hoitoon käytettävien lääkkeiden kehityksessä.

Insecticide resistance and implications for future aphid management in Australian grains and pastures: A review

Aphids can cause substantial damage to cereals, oilseeds and legumes through direct feeding and through the transmission of plant pathogenic viruses. Aphid-resistant varieties are only available for a limited number of crops. In Australia, growers often use prophylactic sprays to control aphids, but this strategy can lead to non-target effects and the development of insecticide resistance. Insecticide resistance is a problem in one aphid pest of Australian grains in Australia, the green peach aphid (Myzus persicae). Molecular analyses of field-collected samples demonstrate that amplified E4 esterase resistance to organophosphate insecticides is widespread in Australian grains across Australia. Knockdown resistance to pyrethroids is less abundant, but has an increased frequency in areas with known frequent use of these insecticides. Modified acetylcholinesterase resistance to dimethyl carbamates, such as pirimicarb, has not been found in Australia, nor has resistance to imidacloprid. Australian grain growers should consider control options that are less likely to promote insecticide resistance, and have reduced impacts on natural enemies. Research is ongoing in Australia and overseas to provide new strategies for aphid management in the future.

Mammalian brain plasma membranes: Isolation, enzymatic, and chemical characterization

Two variants of a simplified procedure for the isolation of plasma membrane fractions from monkey and rat brains, are described. The preparations show marked enrichments in the marker enzymes, (Na+-K+) adenosine triphosphatase, acetylcholinesterase, 5′-nucleotidase and adenylate cyclase. Lipid analysis and a protein electrophoretic pattern are presented. An enzymatic check has been made to assess for contamination by other cellular organelles. The amino acid composition of brain membrane proteins show a resemblance to the reported composition of erythrocyte ghost proteins but differ from myelin proteins.

Spatiotemporal Organization of AT- and GC-rich DNA and Their Association With Transition Proteins TP1 and TP2 in Rat Condensing Spermatids

Transition protein 1 (TP1) and TP2 replace histones during midspermiogenesis (stages 12-15) and are finally replaced by protamines. TPs play a predominant role in DNA condensation and chromatin remodeling during mammalian spermiogenesis. TP2 is a zinc metalloprotein with two novel zinc finger modules that condenses DNA in vitro in a GC-preference manner. TP2 also localizes to the nucleolus in transfected HeLa and Cos-7 cells, suggesting a GC-rich preference, even in vivo. We have now studied the localization pattern of TP2 in the rat spermatid nucleus. Colocalization studies using GC-selective DNA-binding dyes chromomycin A3 and 7-amino actinomycin D and an AT-selective dye, 4',6-diamidino-2-phenylindole, indicate that TP2 is preferentially localized to GC-rich sequences. Interestingly, as spermatids mature, TP2 and GC-rich DNA moves toward the nuclear periphery, and in the late stages of spermatid maturation, TP2 is predominantly localized at the nuclear periphery. Another interesting observation is the mutually exclusive localization of GC- and AT-rich DNA in the elongating and elongated spermatids. A combined immunofluorescence experiment with anti-TP2 and anti-TP1 antibodies revealed several foci of overlapping localization, indicating that TP1 and TP2 may have concerted functional roles during chromatin remodeling in mammalian spermiogenesis.

Localized agglutinin staining in muscle capillaries from normal and very old atrophic human muscle using winged bean ( Psophocarpus tetragonolobus ) lectin

The winged bean (Psophocarpus tetragonolobus) agglutinin (total lectin) and its basic (WBA I) and acidic isoform (WBA II) were used to analyze capillaries in sections from human muscle. The microvessels were clearly labeled after incubation with the lectins in both normal muscle and in old muscles with age-related type II atrophy or muscle fiber grouping. Muscle fibers, nerves, and connective tissue remained unstained. The total lectin detected muscle capillaries from all blood group AB0 individuals. The isoform WBA I reacted only with blood vessels in blood group A and B individuals, while the blood vessels in blood group 0 individuals were demonstrated with WBA II. WBA I staining was inhibited by p-nitrophenyl α-galactopyranoside and N-acetylgalactosamine, whereas 2′-fucosyllactose and preincubation with an antibody against type-1 chain H abolished capillary staining with WBA II. The study demonstrates the usefulness of WBA as a marker of capillaries in human muscle.

Assessment of In-House Natural Product and Synthetic Compound Libraries Based on In vitro Inhibition of Cholinesterases

The first line medication for mild to moderate Alzheimer s disease (AD) is based on cholinesterase inhibitors which prolong the effect of the neurotransmitter acetylcholine in cholinergic nerve synapses which relieves the symptoms of the disease. Implications of cholinesterases involvement in disease modifying processes has increased interest in this research area. The drug discovery and development process is a long and expensive process that takes on average 13.5 years and costs approximately 0.9 billion US dollars. Drug attritions in the clinical phases are common due to several reasons, e.g., poor bioavailability of compounds leading to low efficacy or toxic effects. Thus, improvements in the early drug discovery process are needed to create highly potent non-toxic compounds with predicted drug-like properties. Nature has been a good source for the discovery of new medicines accounting for around half of the new drugs approved to market during the last three decades. These compounds are direct isolates from the nature, their synthetic derivatives or natural mimics. Synthetic chemistry is an alternative way to produce compounds for drug discovery purposes. Both sources have pros and cons. The screening of new bioactive compounds in vitro is based on assaying compound libraries against targets. Assay set-up has to be adapted and validated for each screen to produce high quality data. Depending on the size of the library, miniaturization and automation are often requirements to reduce solvent and compound amounts and fasten the process. In this contribution, natural extract, natural pure compound and synthetic compound libraries were assessed as sources for new bioactive compounds. The libraries were screened primarily for acetylcholinesterase inhibitory effect and secondarily for butyrylcholinesterase inhibitory effect. To be able to screen the libraries, two assays were evaluated as screening tools and adapted to be compatible with special features of each library. The assays were validated to create high quality data. Cholinesterase inhibitors with various potencies and selectivity were found in natural product and synthetic compound libraries which indicates that the two sources complement each other. It is acknowledged that natural compounds differ structurally from compounds in synthetic compound libraries which further support the view of complementation especially if a high diversity of structures is the criterion for selection of compounds in a library.

Acute toxicity of methyl isocyanate in rabbit: In vitro and in vivo effects on rabbit erythrocyte membrane

Methyl isocyanate (MIC) interaction with the rabbit erythrocyte membrane increased the fluidity of the membrane and decreased the osmotic fragility of erythrocytes both in vitro and in vivo in rabbits intoxicated with MIC subcutaneously. MIC inhibited both acetylcholinesterase (AChE) and adenosine triphosphatase (ATPase) activities of erythrocytes dose-dependently in vitro, while in vivo a decreased trend in ATPase activity with unaltered AChE activity was observed. MIC also caused significant decrease in plasma sodium level with corresponding increase in potassium level in rabbits. The observed effects are due to MIC, per se, as the hydrolysis products of MIC, methylamine and N,Nprime-dimethylurea did not affect the erythrocyte fluidity and enzymes activities both in vitro and in vivo while they increased the osmotic fragility of erythrocytes in vivo in rabbits administered subcutaneously in equimolar concentration to MIC dosage. Inhibition of Na+-K+-dependent ATPase with altered permeability to cations and also probably water transport of plasma membrane due to MIC interaction are envisaged.