Plant fiber intrusive growth characterized by NMR method

Intrusively growing plant cells insert themselves between surrounding cells, thus increasing the number of membranes on the tissue cross-section. This parameter can be assessed by spin echo NMR method with a magnetic field pulse gradient. Diffusion echo decay was measured for stem regions of long-fiber flax (Linum usitatissimum L.) differing in the stages of primary fiber development, which elongate thousand-fold during intrusive growth. Additionally, the number of fibers on stem cross-sections was counted under microscope. An increase in the slow component of the echo diffusion decay was correlated with an increase in the number of fibers on the stem cross-section in the zone of intrusive growth, while other stem-structure characteristics remained unchanged. Thus, NMR method can be used for characterization of intrusive fiber growth in situ.     

Presence and co-localization of vasoactive intestinal polypeptide with neuronal nitric oxide synthase in cells and nerve fibers within guinea pig intrinsic cardiac ganglia and cardiac tissue

The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VIP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia. This work was supported by NIH grant HL65481 (R.L.P.) and HL54633 (D.B.H.). Use of the DeltaVision Restoration microscope was provided through the Imaging/Physiology Core supported by NIH Grant P20 RR16435 from the COBRE program of the National Center for Research Resources     

GABAergic system of the pineal organ of an elasmobranch (Scyliorhinus canicula): a developmental immunocytochemical study

The present immunocytochemical study provides evidence of a previously unrecognized, rich, γ-aminobutyric acid (GABA)-ergic innervation of the pineal organ in the dogfish (Scyliorhinus canicula). In this elasmobranch, the pineal primordium is initially detected at embryonic stage 24 and grows to form a long pineal tube by stage 28. Glutamic acid decarboxylase (GAD)-immunoreactive (-ir) fibers were first observed at stage 26, and by stage 28, thin GAD-ir fibers were detectable at the base of the pineal neuroepithelium. In pre-hatchling embryos, most fibers gave rise to GAD-ir boutons that were localized in the basal region of the neuroepithelium, although a smaller number of labeled terminals ascended to the pineal lumen. A few pale GAD-ir perikarya were observed within the pineal organ of stage 29 embryos, but GAD-ir perikarya were not observed at other developing stages or in adults. In contrast, GABA immunocytochemistry revealed the presence of GABAergic perikarya and fibers in the pineal organ of late stage embryos and adults. Although high densities of GABAergic cells were observed in the paracommissural pretectum, posterior tubercle, and tegmentum of dogfish embryos (regions previously demonstrated to contain pinealopetal cells), the presence of GABA-ir perikarya in the pineal organ strongly suggests that the rich GABAergic innervation of the elasmobranch pineal organ is intrinsic. This contrasts with the central origin of GABAergic fibers in the pineal gland of some mammals. This work was supported by the Spanish Education and Science Ministry and FEDER (BXX2000-0453-C02 and BFU2004-03313/BF1), the Xunta de Galicia (PGIDT99BIO20002), and NIH/NIDCD awards R01 DC01705 and P01 DC01837 (to G.R.H.).     

Activation of latent efferent sympathetic fibers in a viscero-visceral reflex circle under conditions of visceral pain in rats

Activation of “silent” efferent fibers due to stimulation of the mesenteric nerve within a definite frequency range is described; the effect is supposed to result from sensitization in reflex circles related to visceral pain. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 368–369, July–August, 2006.     

Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers

An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro.     

Caldesmon restricts the movement of both C- and N-termini of tropomyosin on F-actin in ghost fibers during the actomyosin ATPase cycle

New data on the movements of tropomyosin singly labeled at alpha- or beta-chain during the ATP hydrolysis cycle in reconstituted ghost fibers have been obtained by using the polarized fluorescence technique which allowed us following the azimuthal movements of tropomyosin on actin filaments. Pronounced structural changes in tropomyosin evoked by myosin heads suggested the "rolling" of the tropomyosin molecule on F-actin surface during the ATP hydrolysis cycle. The movements of actin-bound tropomyosin correlated to the strength of S1 to actin binding. Weak binding of myosin to actin led to an increase in the affinity of the tropomyosin N-terminus to actin with simultaneous decrease in the affinity of the C-terminus. On the contrary, strong binding of myosin to actin resulted in the opposite changes of the affinity to actin of both ends of the tropomyosin molecule. Caldesmon inhibited the "rolling" of tropomyosin on the surface of the thin filament during the ATP hydrolysis cycle, drastically decreased the affinity of the whole tropomyosin molecule to actin, and "freezed" tropomyosin in the position characteristic of the weak binding of myosin to actin.     

Severe hepatocellular disease in mice lacking one or both CaaX prenyltransferases

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) add 15- or 20-carbon lipids, respectively, to proteins that terminate with a CaaX motif. These posttranslational modifications of proteins with lipids promote protein interactions with membrane surfaces in cells, but the in vivo importance of the CaaX prenyltransferases and the protein lipidation reactions they catalyze remain incompletely defined. One study concluded that a deficiency of FTase was inconsequential in adult mice and led to little or no tissue pathology. To assess the physiologic importance of the CaaX prenyltransferases, we used conditional knockout alleles and an albumin-Cre transgene to produce mice lacking FTase, GGTase-I, or both enzymes in hepatocytes. The hepatocyte-specific FTase knockout mice survived but exhibited hepatocellular disease and elevated transaminases. Mice lacking GGTase-I not only had elevated transaminases but also had dilated bile cannaliculi, hyperbilirubinemia, hepatosplenomegaly, and reduced survival. Of note, GGTase-I-deficient hepatocytes had a rounded shape and markedly reduced numbers of actin stress fibers. Hepatocyte-specific FTase/GGTase-I double-knockout mice closely resembled mice lacking GGTase-I alone, but the disease was slightly more severe. Our studies refute the notion that FTase is dispensable and demonstrate that GGTase-I is crucial for the vitality of hepatocytes.     

GHRP-6 mimics ghrelin-induced stimulation of food intake and suppression of locomotor activity in goldfish

Ghrelin was first identified and characterized from rat stomach as an endogenous ligand for the growth hormone secretagogue (GHS) receptor (GHS-R). Ghrelin also acts as an orexigenic factor and regulates energy balance in rodents. In goldfish, native ghrelin consists of 11 molecular variants, the major form being a 17-residue peptide with n-octanoic acid modification (n-octanoyl ghrelin17), and intraperitoneal (IP) administration of n-octanoyl ghrelin17 induces central actions such as stimulation of food intake and suppression of locomotor activity through capsaicin-sensitive afferents. Four types of GHS-Rs (1a-1, 1a-2, 2a-1 and 2a-2) have been identified in goldfish, and one GHS, GHRP-6, can activate only GHS-R2a-1 in vitro. However, there is no information about the effect of GHRP-6 on food intake and locomotor activity in goldfish in vivo. Therefore, in the present study, we examined whether IP-administered GHRP-6 would mimic the orexigenic action of n-octanoyl ghrelin17 and its suppression of locomotor activity. IP administration of GHRP-6 at 1pmol/g body weight (BW) stimulated food intake, and was equipotent to the orexigenic action of n-octanoyl ghrelin17 at 10 pmol/g BW. IP-injected GHRP-6 at 1 pmol/g BW also induced a significant decrease of locomotor activity, as was the case for IP-injected n-octanoyl ghrelin17 at 10 pmol/g BW. The action of GHRP-6 was blocked by IP-preinjected capsaicin at 160 nmol/g BW. These results suggest that the central action of GHRP-6 might be mediated via the GHS-R2a-1-signaling pathway, and subsequently through capsaicin-sensitive afferents in goldfish.     

Relevance of mast cell-nerve interactions in intestinal nociception

Cross-talk between the immune- and nervous-system is considered an important biological process in health and disease. Because mast cells are often strategically placed between nerves and surrounding (immune)-cells they may function as important intermediate cells. This review summarizes the current knowledge on bidirectional interaction between mast cells and nerves and its possible relevance in (inflammation-induced) increased nociception. Our main focus is on mast cell mediators involved in sensitization of TRP channels, thereby contributing to nociception, as well as neuron-released neuropeptides and their effects on mast cell activation. Furthermore we discuss mechanisms involved in physical mast cell-nerve interactions. This article is part of a Special Issue entitled: Mast cells in inflammation.