Immunohistochemical, Histochemical and Radioassay Analysis of Nitric Oxide Synthase Immunoreactivity in the Lumbar and Sacral Dorsal Root Ganglia of the Dog

Summary In this study, immunohistochemistry for neuronal nitric oxide synthase (bNOS-IR), nicotinamide adenine dinucleotide phosphate diaphorase histochemistry (NADPHd) and nitric oxide synthase radioassay were used to study the occurrence, number and distribution pattern of nitric oxide synthesizing neurons in the lumbar (L1–L7) and sacral (S1–S3) dorsal root ganglia of the dog. Nitric oxide synthase immunolabelling was present in a large number of small- (area <1000 μm²) and medium-sized (area 1000–2000 μm²) as well as in a limited number of large-sized (area >2000 μm²) neurons. Although neuronal nitric oxide synthase immunolabelling and histochemical staining provided intense staining of multiple small- and medium-sized neurons in all lumbar and sacral dorsal root ganglia, immunolabelled or histochemically stained somata exhibited little topographic distribution in individual dorsal root ganglia. Great heterogeneity was noticed in the immunolabelling of medium-sized nitric oxide synthase immunopositive neurons ranging from lightly immunolabelled somata to heavily immunoreactive ones with completely obscured nuclei. Both staining procedures proved to be highly effective in visualizing intraganglionic fibers of various diameters. In general, the largest fibers revealed at the peripheral end of lumbar and sacral dorsal root ganglia were larger, 6.49–9.35 μm in diameter, while those running centrally and proceeding into the dorsal roots were about 30% reduced, ranging between 5.32 and 8.67 μm in diameter. Peripherally, the occurrence of nitric oxide synthase detected in axonal profiles, and confirmed histochemically, in the specimens of the femoral and sciatic nerves, is the first indication of the presence of nitric oxide synthase in the peripheral processes of somata located in L4–S2 dorsal root ganglia. Large and thin central nitric oxide synthase immunoreactive processes of L1–S3 dorsal root ganglion neurons segregate shortly before entering the spinal cord, the former making a massive medial bundle in the dorsal root accompanied by a slim lateral bundle penetrating Lissauer's tract. Quantitative assessment of the distribution of bNOS-IR and/or NADPHd-stained neurons showed a peculiar pattern in relation to spinal levels. Apparent incongruity was found in the total number of NADPHd-stained versus bNOS-IR neurons, demonstrating a clear prevalence of small bNOS-IR somata in all lumbar ganglia, while medium-sized NADPHd-stained somata clearly prevailed all along the rostrocaudal axis with a peak in L5 ganglion. While the number of small bNOS-IR neurons clearly outnumbered NADPHd-stained and NADPHd-unstained somata in S1–S3 ganglia, an inverse relation appeared comparing the total number of medium-sized NADPHd-stained and NADPHd-unstained somata compared with the number of moderate and intense bNOS-IR neurons. Densitometry of bNOS-IR and NADPHd-stained neurons in lumbar and sacral ganglia revealed two distinct subsets of densitometric profiles, one relating to more often found medium-sized bNOS immunolabelled and the other, characteristic for moderately bNOS immunoreactive somata of the same cell size. Considerable differences in catalytic nitric oxide synthase activity, determined by conversion of [³H]arginine to [³H]citrulline were obtained in lumbosacral dorsal root ganglia all along the lumbosacral intumescence, the lowest (0.898± 0.2 dpm/min/μg protein) being in the L4 dorsal root ganglion and the highest (4.194± 0.2 dpm/min/μg protein) in the S2 dorsal root ganglion.     

Molecular dynamics study of major urinary protein-pheromone interactions: a structural model for ligand-induced flexibility increase

Recently, two independent (15)N NMR relaxation studies indicated that in contrast to the decreased flexibility expected for induced-fit interactions, the backbone flexibility of major urinary protein isoform I (MUP-I) slightly increased upon complex formation with its natural pheromone 2-sec-butyl-4,5-dihydrothiazol. We have investigated the subtle details of molecular interactions by molecular dynamics simulations in explicit solvent. The calculated order parameters S(2) for a free- and ligand-bound protein supply evidence that mobility in various regions of MUP-I can be directly related to small conformational changes of the free- and complexed protein resulting from modifications of the hydrogen bonding network.     

Effect of fatty acids on growth of conjugated-linoleic-acids-producing bacteria in rumen

Microorganisms with high activity of linoleic acid delta12-cis,delta11-trans-isomerase were isolated from the digestive tract of ruminants and characterized. The isolate with the highest isomerase activity was identified as Pseudobutyrivibrio ruminis. The susceptibility of this strain to 3 fatty acids added to the grow medium was determined. A significant inhibition of bacterial growth (during a 3-d period) by linoleic acid (0.1 %) and oleic acid (5 ppm) was observed; no inhibition was found in the presence of stearic acid.     

Isolation and characterization of a novel semi-lethal Arabidopsis thaliana mutant of gene for pentatricopeptide (PPR) repeat-containing protein

A novel Arabidopsis thaliana mutant of one member of the pentatricopeptide repeat (PPR) gene family has been identified among T-DNA insertion lines. Tagging of the At1g53330 gene caused the appearance of a semi-lethal mutation with a complex phenotypic expression from embryo lethality associated with the abnormal pattern of cell division during globular to heart transition to fertile plants with just subtle phenotypic changes. The PPR protein At1g53330.1 was predicted to be targeted to mitochondria by TargetP and MitoProt programs. Complementation analysis confirmed that the phenotype is a result of a single T-DNA integration. A thorough functional analysis of this mutant aimed at finding a particular organelle target of At1g53330.1 protein will follow.     

MOESIN crosslinks actin and cell membrane in Drosophila oocytes and is required for OSKAR anchoring

In Drosophila, development of the embryonic germ cells depends on posterior transport and site-specific translation of oskar (osk) mRNA and on interdependent anchoring of the osk mRNA and protein within the posterior subcortical region of the oocyte. Transport of the osk mRNA is mediated by microtubules, while anchoring of the osk gene products at the posterior pole of the oocyte is suggested to be microfilament dependent. To date, only a single actin binding protein (TropomyosinII) has been identified with a putative role in osk mRNA and protein anchoring. This communication demonstrates that mutations in the Drosophila moesin (Dmoe) gene that encodes another actin binding protein result in delocalization of osk mRNA and protein from the posterior subcortical region and, as a consequence, in failure of embryonic germ cell development. In Dmoe mutant oocytes, the subcortical actin network is detached from the cell membrane, while the polarized microtubule cytoskeleton is unaffected. In line with the earlier observations, colocalization of ectopic actin and OSK protein in Dmoe mutants suggests that the actin cytoskeleton anchors OSK protein to the subcortical cytoplasmic area of the Drosophila oocyte.     

Towards characterization of DNA structure under physiological conditions in vivo at the single-molecule level using single-pair FRET

Fluorescence resonance energy transfer (FRET) under in vivo conditions is a well-established technique for the evaluation of populations of protein bound/unbound nucleic acid (NA) molecules or NA hybridization kinetics. However, in vivo FRET has not been applied to in vivo quantitative conformational analysis of NA thus far. Here we explored parameters critical for characterization of NA structure using single-pair (sp)FRET in the complex cellular environment of a living Escherichia coli cell. Our measurements showed that the fluorophore properties in the cellular environment differed from those acquired under in vitro conditions. The precision for the interprobe distance determination from FRET efficiency values acquired in vivo was found lower (～31%) compared to that acquired in diluted buffers (13%). Our numerical simulations suggest that despite its low precision, the in-cell FRET measurements can be successfully applied to discriminate among various structural models. The main advantage of the in-cell spFRET setup presented here over other established techniques allowing conformational analysis in vivo is that it allows investigation of NA structure in various cell types and in a native cellular environment, which is not disturbed by either introduced bulk NA or by the use of chemical transfectants.     

Conservation of chromosomes syntenic with avian autosomes in squamate reptiles revealed by comparative chromosome painting

In contrast to mammals, birds exhibit a slow rate of chromosomal evolution. It is not clear whether high chromosome conservation is an evolutionary novelty of birds or was inherited from an earlier avian ancestor. The evolutionary conservatism of macrochromosomes between birds and turtles supports the latter possibility; however, the rate of chromosomal evolution is largely unknown in other sauropsids. In squamates, we previously reported strong conservatism of the chromosomes syntenic with the avian Z, which could reflect a peculiarity of this part of the genome. The chromosome 1 of iguanians and snakes is largely syntenic with chromosomes 3, 5 and 7 of the avian ancestral karyotype. In this project, we used comparative chromosome painting to determine how widely this synteny is conserved across nine families covering most of the main lineages of Squamata. The results suggest that the association of the avian ancestral chromosomes 3, 5 and 7 can be dated back to at least the early Jurassic and could be an ancestral characteristic for Unidentata (Serpentes, Iguania, Anguimorpha, Laterata and Scinciformata). In Squamata chromosome conservatism therefore also holds for the parts of the genome which are homologous to bird autosomes, and following on from this, a slow rate of chromosomal evolution could be a common characteristic of all sauropsids. The large evolutionary stasis in chromosome organization in birds therefore seems to be inherited from their ancestors, and it is particularly striking in comparison with mammals, probably the only major tetrapod lineage with an increased rate of chromosomal rearrangements as a whole.     

Hepatocyte-specific deletion of Janus kinase 2 (JAK2) protects against diet-induced steatohepatitis and glucose intolerance

Non-alcoholic fatty liver disease (NAFLD) is becoming the leading cause of chronic liver disease and is now considered to be the hepatic manifestation of the metabolic syndrome. However, the role of steatosis per se and the precise factors required in the progression to steatohepatitis or insulin resistance remain elusive. The JAK-STAT pathway is critical in mediating signaling of a wide variety of cytokines and growth factors. Mice with hepatocyte-specific deletion of Janus kinase 2 (L-JAK2 KO mice) develop spontaneous steatosis as early as 2 weeks of age. In this study, we investigated the metabolic consequences of jak2 deletion in response to diet-induced metabolic stress. To our surprise, despite the profound hepatosteatosis, deletion of hepatic jak2 did not sensitize the liver to accelerated inflammatory injury on a prolonged high fat diet (HFD). This was accompanied by complete protection against HFD-induced whole-body insulin resistance and glucose intolerance. Improved glucose-stimulated insulin secretion and an increase in β-cell mass were also present in these mice. Moreover, L-JAK2 KO mice had progressively reduced adiposity in association with blunted hepatic growth hormone signaling. These mice also exhibited increased resting energy expenditure on both chow and high fat diet. In conclusion, our findings indicate a key role of hepatic JAK2 in metabolism such that its absence completely arrests steatohepatitis development and confers protection against diet-induced systemic insulin resistance and glucose intolerance.