Biomedical subjects

Leonid L Moroz

Publications and source records attributed to Leonid L Moroz.

25 records · Page 2Linked to original sources

PubMed2004

Nitric oxide regulates swimming in the jellyfish Aglantha digitale.

The cnidarian nervous system is considered by many to represent neuronal organization in its earliest and simplest form. Here we demonstrate, for the first time in the Cnidaria, the neuronal localization of nitric oxide synthase (NOS) in the hydromedusa Aglantha digitale (Trachylina). Expression of specific, fixative-resistant NADPH-diaphorase (NADPH-d) activity, characteristic of NOS, was observed in neurites running in the outer nerve ring at the base of the animal and in putative sensory cells in the ectoderm covering its tentacles. At both sites, diphenyleneiodonium (10(-4) M) abolished staining. Capillary electrophoresis confirmed that the NO breakdown products NO2- and NO3- were present at high levels in the tentacles, but were not detectable in NADPH-d-negative areas. The NADPH-d-reactive neurons in the tentacles send processes to regions adjacent to the inner nerve ring where swimming pacemaker cells are located. Free-moving animals and semi-intact preparations were used to test whether NO is involved in regulating the swimming program. NO (30-50 nM) and its precursor L-arginine (1 mM) stimulated swimming, and the effect was mimicked by 8-Br-cGMP (50-100 microM). The NO scavenger PTIO (10-100 microM) and a competitive inhibitor of NOS, L-nitroarginine methyl ester (L-NAME, 200 microM), significantly decreased the swimming frequency in free-moving animals, while its less-active stereoisomer D-nitroarginine methyl ester (D-NAME, 200 microM) had no such effect. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 5-20 microM), a selective inhibitor of soluble guanylyl cyclase, suppressed spontaneous swimming and prevented NO-induced activation of the swimming program. We suggest that an NO/cGMP signaling pathway modulates the rhythmic swimming associated with feeding in Aglantha, possibly by means of putative nitrergic sensory neurons in its tentacles.

Animals↗

PubMed2004

Simple cDNA normalization using kamchatka crab duplex-specific nuclease.

We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturation-reassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA-RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first-strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique.

Animals↗

PubMed2004

RFa-related peptides are algogenic: evidence in vitro and in vivo.

RFamide (RFa)-related peptides modulate pain processing in the mammalian CNS. The effects of these peptides are generally considered as 'anti-opioid'. They also decrease the rate of desensitization of acid-sensing ionic channels (ASICs), putative nociceptors in dorsal root ganglia neurons [C. Askwith et al. (2000) Neuron, 26, 133-141]. We have tested the role of mollusc-derived peptide, FMRFa (Phe-Met-Arg-Phe-amide) and its synthetic analogues in peripheral nociception. Here we demonstrate that RFa-related peptides powerfully excite the majority of C-fibres in the skin-nerve preparation of rat: 76% of 55 tested fibres with the conduction velocity below 2 m/s responded with long-lasting discharges to the application of peptides (20 microm). When injected subcutaneously in vivo (mice), they initiate nociceptive behaviour. We confirm the data on humans [S. Ugawa et al. (2002) J. Clin. Invest., 110, 1185-1190]: the activation of C-fibres by acid is inhibited by channel blocker of ASICs, amiloride. However, there is no correlation in the sensitivity of C-fibres to RFa peptides, protons and amiloride: 74% of tested RFa-sensitive C-fibres were insensitive to protons and in 67% of cases the response to peptides was insensitive to amiloride. Thus, powerful excitatory/algogenic action of RFa-related peptides cannot be interpreted solely in terms of their interaction with ASICs. The peptides do not activate any conductance in the somatic membrane of dorsal root ganglion neurons of rats and probably affect still unidentified molecular target(s) responsible for nociceptive signalling.

Animals↗

PubMed2002

Ascorbic acid assays of individual neurons and neuronal tissues using capillary electrophoresis with laser-induced fluorescence detection.

Ascorbic acid is an important cellular metabolite involved in many biochemical pathways. A method to quantitate ascorbic acid and dehydroascorbic acid in individual neurons and neuronal tissues is described with detection limits of 320 pM (430 zmol). The method uses microvial sampling, derivatization with 4,5-dimethyl-1,2-phenylenediamine, capillary electrophoresis separation, and laser-induced fluorescence detection and quantifies the ascorbic acid and dehydroascorbic acid levels with less than a 15-min total analysis time including sample preparation and derivatization. Ascorbic acid and dehydroascorbic acid levels are measured using functionally characterized and identified neurons of Aplysia californica, Pleurobranchaea californica, and Lymnaea stagnalis -three well-recognized models in cellular and system neuroscience. Multiple assays of a particular identified neuron (e.g., metacerebral cells from Aplysia) show a high level of reproducibility, while endogenous intracellular concentrations of ascorbate are neuron-specific. Ascorbic acid concentrations in the neurons studied range from 0.19 to 6.2 mM for Aplysia and 0.12 to 0.22 mM for Lymnaea. In contrast, concentrations of ascorbic acid observed in heterogeneous tissues such as ganglia (with connective tissues, glia, blood vessels, neuropile, and areas with intercellular spaces), 4-190 microM, are significantly lower than the single-cell values.

Animals↗

PubMed2002

Interfering with nitric oxide measurements. 4,5-diaminofluorescein reacts with dehydroascorbic acid and ascorbic acid.

4,5-Diaminofluorescein (DAF-2) is widely used for detection and imaging of NO based on its sensitivity, noncytotoxicity, and specificity. In the presence of oxygen, NO and NO-related reactive nitrogen species nitrosate 4,5-diaminofluorescein to yield the highly fluorescent DAF-2 triazole (DAF-2T). However, as reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO because it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles similar to that of DAF-2T. When DHA is present, the formation of DAF-2T is attenuated because the DHA competes for DAF-2, whereas AA decreases the nitrosation of DAF-2 to a larger extent, possibly because of additional reducing activity that affects the amount of available N(2)O(3) from the NO. The reaction products of DAF-2 with DHA and AA have been characterized using capillary electrophoresis with laser-induced fluorescence detection and electrospray mass spectrometry. The reactions of DAF-2 with DHA and AA are particularly significant because DHA and AA often colocalize with nitric-oxide synthase in the central nervous, cardiovascular, and immune systems, indicating the importance of understanding this chemistry.

Animals↗

PubMed2002

High-resolution microanalysis of nitrite and nitrate in neuronal tissues by capillary electrophoresis with conductivity detection.

Nitrites and nitrates are widely used reporters of endogenous activity of nitric oxide synthases (NOS), an important group of enzymes producing the gaseous signal molecule nitric oxide (NO). However, due to the great chemical heterogeneity of neuronal tissues, standard analytical protocols for evaluation of neuronal nitrite/nitrate concentrations are inefficient. We optimized a high-performance capillary zone electrophoresis (CZE) technique to analyze nitrite/nitrate concentrations in submicroliter samples from mammalian neuronal tissues. The measurements were made using a PrinCE 476 computerized capillary electrophoresis system with a Crystal 1000 contact conductivity detector. Isotachophoretic stacking injection of 1000- to 10000-fold diluted samples, which had been pretreated with a custom-designed solid-phase microextraction (SPME) cartridge, was employed to assay micromolar and nanomolar nitrite and nitrate levels in the presence of the high millimolar chloride concentrations characteristic of many biological samples. In the presented technique, a 10-microl volume of diluted ganglionic sample was used for chloride removal and sample cleanup. The method yields high analytical performance, including good reproducibility, resolution, and accuracy. The limits of detection relative to undiluted sample matrix were 8.9 nM (0.41 ppb) and 3.54 nM (0.22 ppb) for nitrite and nitrate, respectively. In addition, this technique resolves other anions that are present in neuronal tissues at sub-nanomolar concentrations and can be broadly applied for high-throughput anionic profiling. In rat dorsal root ganglia, endogenous levels of nitrate (231+/-29 microM; n=6) and nitrite (24-96 microM) were found. These concentrations exceeded those previously found in neuronal tissue homogenates using different techniques.

Electrophoresis, Capillary↗

PubMedDate not supplied

Endogenous thyroid hormone synthesis in facultative planktotrophic larvae of the sand dollar Clypeaster rosaceus: implications for the evolutionary loss of larval feeding.

Critical roles of hormones in metamorphic life history transitions are well documented in amphibians, lampreys, insects, and many plant species. Recent evidence suggests that thyroid hormones (TH) or TH-like compounds can regulate development to metamorphosis in echinoids (sea urchins, sand dollars, and their relatives). Moreover, previous research has provided evidence for endogenous hormone synthesis in both feeding and nonfeeding echinoderm larvae. However, the mechanisms for endogenous synthesis remain largely unknown. Here, we show that facultatively planktotrophic larvae (larvae that reach metamorphosis in the absence of food but have the ability to feed) from the subtropical sea biscuit Clypeaster rosaceus can synthesize thyroxine endogenously from incorporated iodine (I(125)). When treated with the goitrogen thiourea (a peroxidase inhibitor), iodine incorporation, thyroxine synthesis, and metamorphosis are all blocked in a dose-dependent manner. The inhibitory effect on metamorphosis can be rescued by administration of exogenous thyroxine. Finally, we demonstrate that thiourea induces morphological changes in feeding structures comparable to the phenotypic plastic response of larval structures to low food conditions, further supporting a signaling role of thyroxine in regulating larval morphogenesis and phenotypic plasticity. We conclude that upregulation of endogenous hormone synthesis might have been associated with the evolution of nonfeeding development, subsequently leading to morphological changes characteristic of nonfeeding development.

Animals↗