Isolation of soybean lipoxygenase-2 by affinity chromatography

Isoenzyme lipoxygenase-2 from soybean was isolated by affinity chromatography. Gel electrophoresis showed it to be a single protein. Its pH optimum of 6.5, range of 5.0–8.0 and activity which increased when Ca²⁺ was added identified the isolated enzyme as lipoxygenase-2.     

Synchrony measures for biological neural networks

 Synchronous firing of a population of neurons has been observed in many experimental preparations; in addition, various mathematical neural network models have been shown, analytically or numerically, to contain stable synchronous solutions. In order to assess the level of synchrony of a particular network over some time interval, quantitative measures of synchrony are needed. We develop here various synchrony measures which utilize only the spike times of the neurons; these measures are applicable in both experimental situations and in computer models. Using a mathematical model of the CA3 region of the hippocampus, we evaluate these synchrony measures and compare them with pictorial representations of network activity. We illustrate how synchrony is lost and synchrony measures change as heterogeneity amongst cells increases. Theoretical expected values of the synchrony measures for different categories of network solutions are derived and compared with results of simulations. Received: 6 June 1994/Accepted in revised form: 13 January 1995     

The effects of naloxone on the changes in breathing and behaviour induced by morphine in the foetal sheep

In the foetal sheep, administration of morphine induces apnoea followed by hyperpnoea; during hyperpnoea the foetus arouses. We tested the hypothesis that naloxone, an opiate antagonist, would block these responses. In 14 foetal sheep between 123 and 140 days of gestation, we measured electrocortical activity (ECoG), eye movements (EOG), diaphragmatic activity (EMGdi), blood pressure and amniotic pressure. Morphine (1 mg/kg) was injected in the foetal jugular vein during low-voltage ECoG. Saline or naloxone (0.1, 0.5 and 2.0 mg) were given, in randomized order, before the morphine injection, shortly after morphine injection during apnoea, and during maximum hyperpnoea. Saline alone had no effect on breathing or behaviour. When saline and naloxone preceded the morphine injection the length of apnoea was 26.6 +/- 7.7 and 19.5 +/- 7.0 min (SEM, P = 0.25) while the length of sustained hyperpnoea was 104.8 +/- 11.4 and 29.6 +/- 8.4 min respectively (P = 0.001). When administered during the maximum breathing response, naloxone decreased the length of breathing from 92.2 +/- 8.4 (saline) to 8.8 +/- 2.9 min (P = 0.001). Respiratory output (fEMGdi x f) also decreased from 6545 +/- 912 arbitrary units post saline to 3841 +/- 629 arbitrary units after naloxone (P = 0.05). Arousal disappeared with the decrease in breathing response. The negligible effect of naloxone on apnoea and its strong inhibition of hyperpnoea suggest that morphine may act on two distinct central regions or on two subtypes of opioid receptors to produce apnoea, hyperpnoea and arousal.     

Genotypes and haplotypes in the insulin-like growth factors, their receptors and binding proteins in relation to plasma metabolic levels and mammographic density

Background

Chronic inflammatory diseases including inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis), psoriasis and rheumatoid arthritis (RA) afflict millions of people worldwide, but their pathogenesis is still not well understood. It is also not well known if distinct changes in gene expression characterize these diseases and if these patterns can discriminate between diseased and control patients and/or stratify the disease. The main focus of our work was the identification of novel markers that overlap among the 3 diseases or discriminate them from each other.

Methods

Diseased (n = 13, n = 15 and n = 12 in IBD, psoriasis and RA respectively) and healthy patients (n = 18) were recruited based on strict inclusion and exclusion criteria; peripheral blood samples were collected by clinicians (30 ml) in Venous Blood Vacuum Collection Tubes containing EDTA and peripheral blood mononuclear cells were separated by Ficoll gradient centrifugation. RNA was extracted using Trizol reagent. Gene expression data was obtained using TaqMan Low Density Array (TLDA) containing 96 genes that were selected by an algorithm and the statistical analyses were performed in Prism by using non-parametric Mann-Whitney U test (P-values < 0.05).

Results

Here we show that using a panel of 96 disease associated genes and measuring mRNA expression levels in peripheral blood derived mononuclear cells; we could identify disease-specific gene panels that separate each disease from healthy controls. In addition, a panel of five genes such as ADM, AQP9, CXCL2, IL10 and NAMPT discriminates between all samples from patients with chronic inflammation and healthy controls. We also found genes that stratify the diseases and separate different subtypes or different states of prognosis in each condition.

Conclusions

These findings and the identification of five universal markers of chronic inflammation suggest that these diseases have a common background in pathomechanism, but still can be separated by peripheral blood gene expression. Importantly, the identified genes can be associated with overlapping biological processes including changed inflammatory response. Gene panels based on such markers can play a major role in the development of personalized medicine, in monitoring disease progression and can lead to the identification of new potential drug targets in chronic inflammation.     

Using the method of homogenization to calculate the effective diffusivity of the stratum corneum with permeable corneocytes

The stratum corneum is the outermost layer of the skin, which acts as a barrier membrane against the penetration of molecules into and out of the body. It has a biphasic structure consisting of keratinized cells (corneocytes) that are embedded in a lipid matrix. The macroscopic transport properties of the stratum corneum are functions of its microstructure and the transport properties of the corneocytes and the lipid matrix, and are of considerable interest in the context of transdermal drug delivery and quantifying exposure to toxins, as well as for determining the relation of skin disorders to disruption of the stratum corneum barrier. Due to the complexity of the tissue and the difference in length scales involved in its microstructure, a direct analysis of the mass transport properties of the stratum corneum is not feasible. In this study, we undertake an approach where the macroscopic diffusion tensor of the stratum corneum is obtained through homogenization using the method of asymptotic expansions. The biphasic structure of the stratum corneum is fully accounted for by allowing the corneocytes to be permeable and considering the partitioning between the corneocytes and the lipid phases. By systematically exploring the effect of permeable corneocytes on the macroscopic transport properties of the stratum corneum, we show that solute properties such as lipophilicity and relative permeabilities in the two phases have large effects on its transdermal diffusion behavior.     

Methylmercuric iodide modification of lipoxygenase-1. Effects on the anaerobic reaction and pigment bleaching

The effect of methylmercuric iodide modification of sulfhydryl groups in soybean lipoxygenase-1 on linoleate oxidation, carbonyl production and beta-carotene and chlorophyll alpha bleaching were determined under aerobic and anaerobic conditions. Linoleate oxidation at pH 9.0 was strongly inhibited by modification of the enzyme. On the other hand, pigment bleaching was enhanced with the modified enzyme. Unmodified lipoxygenase-1 was not sensitive to chlorophyll inhibition, but activity of modified lipoxygenase-1 was affected. Linoleate oxidation was inhibited up to 70% when 2.2 microM chlorophyll was present in the reaction mixture. Chlorophyll inhibition was similar with affinity chromatography-purified lipoxygenase-2 and modified lipoxygenase-1. Unmodified lipoxygenase-1 exhibited high bleaching activity under anaerobic conditions and relatively low activity under aerobic (oxygen or air) conditions. Modified lipoxygenase-1 showed a significant increase in carotene and chlorophyll bleaching under both anaerobic and aerobic conditions. Under anaerobic conditions in the presence of either pigment, both modified and unmodified lipoxygenase-1 exhibited high 285 nm absorbing material production. Antioxidants (butylated hydroxyanisole, butylated hydroxytoluene, alpha-tocopherol, propyl gallate and tertiary butylated hydroxyquinone ) were powerful inhibitors of pigment bleaching by modified lipoxygenase-1. However, only tertiary butylated hydroxyquinone and propyl gallate blocked the increase in the rate of absorbance at 285 nm.     

Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis

Background

Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species.

Methodology/Principal Findings

For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay.

Conclusions/Significance

The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.