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.     

Intrinsic and network rhythmogenesis in a reduced traub model for CA3 neurons

We have developed a two-compartment, eight-variable model of a CA3 pyramidal cell as a reduction of a complex 19-compartment cable model [Traub et al, 1991]. Our reduced model segregates the fast currents for sodium spiking into a proximal, soma-like, compartment and the slower calcium and calcium-mediated currents into a dendrite-like compartment. In each model periodic bursting gives way to repetitive soma spiking as somatic injected current increases. Steady dendritic stimulation can produce periodic bursting of significantly higher frequency (8–20 Hz) than can steady somatic input (<8 Hz). Bursting in our model occurs only for an intermediate range of electronic coupling conductance. It depends on the segregation of channel types and on the coupling current that flows back-and-forth between compartments. When the soma and dendrite are tightly coupled electrically, our model reduces to a single compartment and does not burst. Network simulations with our model using excitatory AMPA and NMDA synapses (without inhibition) give results similar to those obtained with the complex cable model [Traub et al, 1991; Traub et al, 1992]. Brief stimulation of a single cell in a resting network produces multiple synchronized population bursts, with fast AMPA synapses providing the dominant synchronizing mechanism. The number of bursts increases with the level of maximal NMDA conductance. For high enough maximal NMDA conductance synchronized bursting repeats indefinitely. We find that two factors can cause the cells to desynchronize when AMPA synapses are blocked: heterogeneity of properties amongst cells and intrinsically chaotic burst dynamics. But even when cells are identical, they may synchronize only approximately rather than exactly. Since our model has a limited number of parameters and variables, we have studied its cellular and network dynamics computationally with relative ease and over wide parameter ranges. Thereby, we identify some qualitative features that parallel or are distinguished from those of other neuronal systems; e.g., we discuss how bursting here differs from that in some classical models.     

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.     

Tissue-resident ecto-5' nucleotidase (CD73) regulates leukocyte trafficking in the ischemic brain

Ectoenzymes expressed on the surface of vascular cells and leukocytes modulate the ambient nucleotide milieu. CD73 is an ecto-5' nucleotidase that catalyzes the terminal phosphohydrolysis of AMP and resides in the brain on glial cells, cells of the choroid plexus, and leukocytes. Though CD73 tightens epithelial barriers, its role in the ischemic brain remains undefined. When subjected to photothrombotic arterial occlusion, CD73(-/-) mice exhibited significantly larger (49%) cerebral infarct volumes than wild-type mice, with concordant increases in local accumulation of leukocyte subsets (neutrophils, T lymphocytes, macrophages, and microglia). CD73(-/-) mice were rescued from ischemic neurologic injury by soluble 5'-nucleotidase. In situ, CD73(-/-) macrophages upregulated expression of costimulatory molecules far more than wild-type macrophages, with a sharp increase of the CD80/CD86 ratio. To define the CD73-bearing cells responsible for ischemic cerebroprotection, mice were subjected to irradiative myeloablation, marrow reconstitution, and then stroke following engraftment. Chimeric mice lacking CD73 in tissue had larger cerebral infarct volumes and more tissue leukosequestration than did mice lacking CD73 on circulating cells. These data show a cardinal role for CD73 in suppressing ischemic tissue leukosequestration. This underscores a critical role for CD73 as a modulator of brain inflammation and immune function.     

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.