Two new species of Monopisthocotyla (Dactylogyridea: Dactylogyridae) parasitizing the nasal cavities of Bryconops melanurus (Characiformes: Iguanodectidae) from coastal drainages of the Eastern Amazon, Brazil.

The present study integrates molecular and morphological data to support the proposal of new species of Telethecium Kritsky, Van Every & Boeger, 1996 and Diaphorocleidus Jogunoori, Kritsky & Venkatanarasaiah, 2004 from the nasal cavities of Bryconops melanurus (Bloch) of the coastal drainages of the Eastern Amazon. Telethecium tiquira sp. n. is characterized by possessing a male copulatory organ (MCO) with two circular sclerotized brims on the base, a coiled tubular shaft having 1 counterclockwise rings, an accessory piece with enlarged base, pincer-shaped at the distal portion; a sclerotized calyx-shaped vaginal vestibule, and hooks with proximal shank dilatation comprising 3/4 of the shank length. Also, Telethecium tiquira sp. n. can be easily distinguished from other species of the genus by the absence of a protruding bag located at the level of the copulatory complex. Diaphorocleidus forficata sp. n. is characterized by having a MCO with two counterclockwise rings, circular sclerotized tandem brim associated with the base of the MCO; accessory piece non-articulated with the MCO, bifurcate, pincer-shaped; vaginal pore sinistral-ventral with opening marginal, vaginal canal sclerotized, elongated, comprising one loop in the proximal portion before entering to the seminal receptacle; ventral anchor with shaft elongated and evenly curved on the axis; point short and slightly curved, and hooks similar in shape and size, hooks with proximal dilatation comprising approximately of the shank length. Furthermore, D. forficata sp. n. is supported by phylogenetic analysis based on sequences of the partial 28S rDNA gene, which placed D. forficata sp. n. in a well-supported clade of Diaphorocleidus spp. of characiform fishes. Thus, the two new species described here expand our knowledge about the diversity of monopisthocotylan parasites from the nasal cavities of Neotropical fishes. The findings of this study provide valuable insights into the biodiversity of the region and highlight the importance of further research in this area.

Two new species of Elachistocleis Parker, 1927 (Anura: Microhylidae: Gastrophryninae) from Colombia.

The genus Elachistocleis consists of small to medium size species of Neotropical microhylids. It is the second largest genus of New World microhylids and an important component of the anuran diversity in the Neotropical region. Herein, based on morphological, acoustical, and molecular evidence, we describe two new species and their advertisement calls. The new species have reticulated venter and inhabit the Orinoco basin of Colombia.

Visualization of stimulated nerve endings by preferential calcium accumulation of mitochondria.

Calcium was detected by X-ray microanalysis in the mitochondria of electrically stimulated nerve endings. The phenomenon described here offers a simple means for identifying the stimulated nerve endings in the electron microscope and appears to be a promising new method for following spontaneous and drug-stimulated translocation of calcium in relation to the regulation of neurotransmitter release.

Endothelial cells of the brain and other organ systems: some similarities and differences.

The cerebral endothelium represents an active interface between blood and central nervous system. The blood-brain barrier restricts the free passage of nutrients, hormones, drugs and cellular elements to the brain. Recent studies performed on freshly isolated cerebral microvessels and cultured endothelial cells of brain capillaries provided a better understanding of the properties and functions of cerebral endothelial cells. This review summarizes the main findings of the in vitro approach in the blood-brain barrier research, describes the common endothelial and unique cerebral features of the brain endothelium, and provides a short overview on how these blood-brain barrier characteristics can be induced in cerebral endothelial cells by the neighbouring cells.

Highly efficient dehydrogenation of formic acid in aqueous solution catalysed by an easily available water-soluble iridium(iii) dihydride.

The new water-soluble cis-mer-[IrH2Cl(mtppms)3] (mtppms = monosulfonated triphenylphosphine) was employed as a catalyst for selective decomposition of formic acid to H2 + CO2 in aqueous solution at T = 30-100 °C. The easily synthesized compound showed high catalytic activity (TOF up to 298 000 h(-1)) and could be reused several times with no loss of activity (total TON = 67 650). A sharp maximum in the reaction rate was observed at pH = 3.75; its coincidence with the pKa of formic acid shows that both H(+) or HCOOH and HCOO(-) play important roles in the reaction mechanism.

The activation of rat platelets increases the exposure of polyunsaturated fatty acid enriched phospholipids on the external leaflet of the plasma membrane.

Rat platelets have been hydrogenated in the presence of colloidal palladium adsorbed on the surface of the non water-soluble polymer polyvinylpolypyrrolidone. This non-permeating catalyst restricts hydrogenation of the fatty acyl double bonds of phospholipids only in the outer half of the plasma membrane. The pattern of hydrogenation of the molecular species present on the external side of the membrane is determined using desorption-chemical soft ionization-mass spectrometry (DCI-MS) before and after cell activation by the calcium ionophore A23187. The accessibility to the catalyst of the polyunsatured molecular species within each phospholipid class is compared for resting and activated cells. The abundance of polyunsaturated species of phosphatidyl-ethanolamine and -serine in the inner half of the resting biomembrane is confirmed in rat platelets. Phosphatidylcholine is especially rich in disaturated species in this membrane. The induced exposure of the polyunsaturated species of diacyl- and ether-phosphatidylethanolamine, and of phosphatidylserine on the external side of the membrane appears after activation by the calcium ionophore. A detailed quantitative analysis within a phospholipid class shows an unequal scrambling for diacyl-, alkyl-, alkenyl-phosphatidylethanolamine, and a variable involvement in the transmembrane redistribution following cell activation of the various molecular species as a function of the acyl moities.

The hydrogenation of phospholipid-bound unsaturated fatty acids by a homogeneous, water-soluble, palladium catalyst.

The hydrogenation of unsaturated phospholipids by palladium di(sodium alizarine monosulphonate) activated for 5 min under H2 proceeded rapidly at 20 degrees C and 1 atm. H2. Multibilayer liposomes of dioleoyl- and dilinolenoylphosphatidylcholine were hydrogenated at similar rates while dilinoleoyl- and 1-palmitoyl-2-oleoylphosphatidylcholine were hydrogenated at slightly slower rates. The reduction of polyunsaturated fatty acids gave rise to a variety of natural and unnatural positional cis and trans isomers which were largely reduced further to saturated fatty acids as the hydrogenation continued. Dioleoylphosphatidylethanolamine was attacked by the catalyst more slowly at 20 degrees C than was the equivalent phosphatidylcholine molecular species. Experiments conducted using mixtures of phosphatidylethanolamine and phosphatidylcholine in varying proportions also suggested that phospholipids are slightly more susceptible to catalytic hydrogenation in the bilayer phase than in the hexagonalII phase. Understanding the sequence of hydrogenation reactions involving these one and two component lipid preparations is useful in interpreting the action of the palladium catalyst on living cells under the same mild conditions.

Catalytic hydrogenation of polyunsaturated biological membranes: effects on membrane fatty acid composition and physical properties.

The relationship between phospholipid saturation and membrane physical structure in a complex, highly polyunsaturated biological membrane (trout liver microsomes) has been studied by the graded and specific hydrogenation of polyunsaturated fatty acids. The homogeneous catalyst Pd(QS)2 caused rapid and effective hydrogenation, increasing the proportion of saturated fatty acids from 20-30% up to 60%, without loss or fragmentation. Long chain, polyunsaturated fatty acids (20:5 omega 3, 22:6 omega 3) were rapidly converted to a large number of partially hydrogenated isomers, and ultimately to the fully saturated C20 or C22 fatty acids. C18 mono- and di-unsaturates showed slower rates of hydrogenation. Increased saturation was closely associated with an increased membrane physical order as determined by the fluorescence anisotropy probe, 1,6-diphenyl-1,3,5-hexatriene. However, extensive hydrogenation led to highly ordered membranes exhibiting a gel-liquid crystalline phase transition between 30 and 60 degrees C. Polyunsaturated membranes can thus be converted into partially or substantially saturated membranes with measurable phase structure without direct alteration of other membrane components. This offers a less equivocal means of assessing the influence of polyunsaturation upon membrane structure and function.

Action of a homogeneous hydrogenation catalyst on living Tetrahymena mimbres cells.

Various conditions were tested in an attempt to hydrogenate the unsaturated fatty acids of living Tetrahymena mimbres with the homogeneous catalyst palladium di-(sodium alizarine monosulfonate) without causing serious damage to the cells. Using a low (20 micrograms/ml) catalyst concentration in the external medium, hydrogenation of greater than 20% of surface membrane lipid double bonds were obtained, but hydrogenation of intracellular membranes was minimal. When exposed to H2, cells preincubated with inactive catalyst for several hours and visibly loaded with the catalyst lost viability as soon as hydrogenation exceeded trace levels. Material secreted by Tetrahymena into their medium effectively inhibited hydrogenation of added oleic acid, normally a good substrate. Mucus secreted by the cells, soluble proteins isolated from cell homogenates, bovine serum albumin, and cysteine were also inhibitory, but the inhibition could be overcome by employing higher catalyst concentrations. Although some enzymatic retroconversion of saturated lipids back to unsaturated lipids appeared to take place, the scale of the conversion was small, and further experimentation will be required to understand the mechanism involved. The selective hydrogenation of surface membranes achieved by these methods may be especially useful to those interested in fluidity effects on plasma membrane properties.

Simple and reliable conditions for routine, long-term culturing of fetal human pancreatic tissue fragments.

Fetal human pancreatic tissue fragments were isolated and cultured for 18 wk. Insulin production was almost continuous during this period. Multiplication of cells was observed at the second week, and these cells later aggregated as epithelioid cells and formed pseudoislets. The growth characteristics, insulin-like immunoreactivity, and endocrine properties of these cells were evidenced by light microscopy, immunocytochemistry, electron microscopic examination, and measurement of the total insulin content. These results indicate that long-term culture of fetal islets may be useful in clinical work and provides a possible method for increasing islet mass and reducing immunogenicity.

Platelet membrane fluidity and plasma malondialdehyde levels in Alzheimer's demented patients with and without family history of dementia.

Platelet membrane fluidity (PMF) was measured with three different fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 3-(p-phenyl-1,3,5-hexatrienyl)phenyl-propionic acid (DPH-PA), which labeled different parts of the bilayer (the hydrophobic core and the positively and negatively charged regions, respectively) in Alzheimer's disease (AD) patients with and without a family history of dementia, and in a control group. In support of earlier findings in the literature, significantly increased PMF was found by the application of DPH in both groups with AD. The use of the fluorescence probe TMA-DPH, however, revealed no differences between the groups. In contrast, significant rigidification was observed with DPH-PA, but only in the AD group with a positive family history of dementia. The plasma malondialdehyde levels appeared to be similar in each group. Our findings are discussed in light of the controversies regarding the value of PMF measurements in AD.

Lack of histamine synthesis and down-regulation of H1 and H2 receptor mRNA levels by dexamethasone in cerebral endothelial cells.

The purpose of this work was to determine whether cerebral endothelial cells have the capacity to synthesize histamine or to express mRNA of receptors that specifically respond to available free histamine. The histamine concentrations and the expression of L-histidine decarboxylase (HDC) and histamine H1 and H2 receptor mRNA, both in adult rat brain and in cultured immortalized RBE4 cerebral endothelial cells, were investigated. In this study endothelial cells were devoid of any kind of detectable histamine production, both in vivo and in the immortalized RBE4 cells in culture. Both the immunostainings for histamine and the in situ hybridizations for HDC were negative, as well as histamine determinations by HPLC, indicating that endothelial cells do not possess the capacity to produce histamine. Also, glucocorticoid (dexamethasone) treatment failed to induce histamine production in the cultured cells. Although the cerebral endothelial cells lack histamine production, a nonsaturable uptake in RBE4 cells is demonstrated. The internalized histamine is detected both in the cytoplasm and in the nucleus, which could indicate a role for histamine as an intracellular messenger. Histamine H1 and H2 receptor mRNA was expressed in RBE4 cells, and glucocorticoid treatment down-regulated the mRNA levels of both H1 and H2 receptors. This mechanism may be involved in glucocorticoid-mediated effects on cerebrovascular permeability and brain edema.

Global cerebral ischemia associated with cardiac arrest in the rat: I. Dynamics of early neuronal changes.

Light microscopic neuronal changes were studied in rats subjected to 10 min of global ischemia produced by compression of the major cardiac vessels. Observations of cresyl violet-stained sections revealed early changes involving predominantly GABAergic neurons in various locations. In rats killed 15 min after recirculation, the changes were characterized by the appearance of a clear peripheral zone with condensation of the remaining neuronal cytoplasm. After 1 h, these zones appeared to be compartmentalized into individual pearl-like vacuoles, especially prominent in the nucleus reticularis thalami. After 3 h, the cytoplasmic vacuoles disappeared and the neuronal changes, particularly in the cerebral cortex, striatum, hippocampus, and pars reticulata of the substantia nigra, consisted mainly of hyperchromasia or loss of Nissl substance. After 2 days, the cerebral cortex and thalamus contained occasional neurons with conspicuously large nucleoli. After 7 days, the hippocampus revealed an approximately 50% loss of CA1 pyramidal neurons, associated with intense microglial reactivity in the stratum radiatum, whereas the neuronal destruction was more complete in the nucleus reticularis thalami. Our observations suggest a possibility that early changes in GABAergic neurons may provide a period of neuronal disinhibition and thus contribute to an excitatory ischemic damage in regions connected by GABAergic circuitry.

Neonatal enucleation induces correlated modification in sensory responsive areas and pial angioarchitecture of the parietal and occipital cortex of albino rats.

This study was carried out to investigate whether correlations existing in normal adult rats (Ambach et al., '86) between functional characteristics of neocortical areas and their pial angioarchitecture can be correspondingly modified under pathological conditions. The right eyes of albino rats were enucleated on the 1st, 8th, 15th and 30th day after birth, respectively. At the age of 3 to 4 months, the responsiveness of the parieto-occipital cortex to sensory stimuli was studied in enucleated animals and age matched controls. After the mapping of visually and somatosensorily evoked potentials, the vascular system was filled with dye. Monocular enucleation at birth induced bilateral modifications in sensory responsiveness and corresponding changes in pial angioarchitecture, especially in the venous drainage fields. In comparison with the controls, a considerable expansion was observed in the overlapping zone between visually and somatosensorily responsive areas. In contrast, borders of the visual cortex toward the auditory and retrosplenial areas were essentially stable. Corresponding changes were found in the pial distribution patterns of cerebral veins but not of arteries. The major effect of neonatal enucleation on angioarchitecture was a change in the subdivision of the parieto-occipital veins drainage fields. This was due to a significant enlargement of the anterior accessory occipital (O3) vein, which compressed the drainage fields of the parietal and occipital veins and completely separated them from one another. The results suggest that during ontogenesis: (1) alterations in the formation of sensory input may interfere with neocortical angiogenesis, especially the structuring of veins, (2) after monocular enucleation this influence is prominent in parietal and occipital cerebral veins, and (3) these angiogenetic processes are vulnerable only during the perinatal and early postnatal period.

Distribution of GABA-immunoreactive nerve fibers and cells in the cervical and thoracic paravertebral sympathetic trunk of adult rat: evidence for an ascending feed-forward inhibition system.

Neurochemical and immunohistochemical evidence suggests that the superior cervical ganglion (SCG) contains all components of a gamma-aminobutyric acid (GABA)ergic transmission system, which includes GABAergic axons of unknown origin. The number of nerve fibers with and without GABA-like immunoreactivity was determined in interganglionic connectives at all cervical and thoracic levels of the paravertebral sympathetic trunk. In addition, the distribution of GABA-immunoreactive (IR) neurons was established within the ganglion chain and compared with the relative frequency of principal neurons richly innervated by GABA-IR axon terminals. The following results were obtained: 1) the total number of nerve fibers in cross sections did not significantly vary between the cervical levels, but it increased steadily from upper to lower thoracic segments; 2) in contrast, the number of GABA-IR fibers decreased from the cervical sympathetic trunk below the SCG (approximately 300 fibers) down to the seventh to tenth thoracic ganglion, below which no such fiber was seen; 3) GABA-IR nerve fibers originate from a subclass of GABA-IR cells; these are small, bipolar neurons with predominantly ascending, unmyelinated axon-like processes; 4) the number of principal neurons richly innervated by GABA-IR nerve fibers decreased from the SCG to the upper thoracic ganglia, and was very small below; and 5) apart from basket-like innervation, GABA-IR axons also formed diffuse networks around GABA-negative principal neurons predominantly in cervical and upper thoracic ganglia. These data suggest that the GABAergic innervation of paravertebral sympathetic ganglia is more complex than previously suspected. What appears as preganglionic afferents from several spinal segments (C8-Th7) innervate GABAergic neurons in the sympathetic trunk which have ascending axons and focus their inhibitory effects on the cervical sympathetic ganglia, predominantly the SCG. These data suggest that GABAergic small interganglionic neurons form a feed-forward inhibition system, which may be driven by multisegmental spinal input in the paravertebral sympathetic ganglion chain.

Quantitative analysis of the number and distribution of neurons richly innervated by GABA-immunoreactive axons in the rat superior cervical ganglion.

The superior cervical ganglion of rats contains a considerable number of nerve fibers with GABA-like immunoreactivity which show a nonuniform distribution within the ganglion. The topography of these fibers has been analyzed by using antibodies raised against GABA-BSA-glutaraldehyde complexes. GABA-positive axons and axon varicosities accumulated around a subpopulation of principal ganglion cells forming basketlike patterns. These neurons richly innervated by GABA-positive axons (RIG-neurons) in turn were aggregated in patches with strong immunoreactivity. The size and packing density of the patches containing RIG-neurons and GABA-positive axons approaching them had rostral-to-caudal and medial-to-lateral gradients. Similar patterns were found in right and left ganglia. In five ganglia, a quantitative analysis revealed on average 1,344 RIG-neurons per ganglion representing about 5% of the total neuron population, with small variations (standard deviation 122) despite the highly variable shape of the ganglia. The distribution of RIG-neurons resembles that of neurons sending their axons into the internal carotid nerve. To check this possible correlation, HRP was injected into the eye and applied to the transected external carotid nerve. Double staining for the retrogradely transported peroxidase and GABA immunohistochemistry revealed that RIG-neurons formed a small subpopulation of retrogradely labelled neurons in both experiments. This suggests that RIG-neurons innervate various target organs. This conclusion is in agreement with the observation that RIG-neurons also exist in other sympathetic ganglia. Data presented suggest that sympathetic ganglion cells can be classified on the basis of non-uniform innervation patterns formed by axons that use different neurotransmitters.

An immune-mediated guinea pig model for lower motor neuron disease.

Guinea pigs were immunized with motor neurons from swine spinal cords. One month after the last of five serial immunizations, the recipients showed progressive weight loss. By seven months of age, five of the six immunized animals had died. Pathological examination showed destruction of motor neurons in the spinal cords without demyelination, but with atrophy of the related skeletal muscle groups. By immunofluorescent and histochemical tests, serum from the guinea pigs was shown to react with motor neurons of swine and guinea pig cord. This experimental disorder of guinea pigs appeared to be based on the immunologically mediated destruction of motor neurons and it may serve as a model for the human motor neuron diseases.

Distinct subsets of neuropeptide Y-negative principal neurons receive basket-like innervation from enkephalinergic and gabaergic axons in the superior cervical ganglion of adult rats.

The distributions of axons immunoreactive for [Leu]- or [Met]enkephalin and GABA were studied in the superior cervical ganglion of adult rats. The antigens were visualized separately and in combination with neuropeptide Y by the immunoperoxidase technique, using reaction end-products of different colors. Similarities and differences were found in the light-microscopic innervation patterns of enkephalin- and GABA-immunoreactive nerve fibers. Both fiber systems were heterogeneously distributed within the superior cervical ganglion, forming denser networks in its rostral part than elsewhere in the ganglion. The appearance of labeled nerve fibers differed in the two systems. Enkephalin-immunoreactive axons exhibited dotted profiles due to a strong immunoreaction in the axonal varicosities as compared with that in the intervaricose segments, whereas GABA-positive fibers were evenly labeled in both parts of the axons. The most marked difference between the innervation patterns from enkephalin- and GABA-immunoreactive axons was the presence of bundles of varicose axons in conjunction with the basket-like aggregation of enkephalin-immunoreactive nerve terminals. The possibility that enkephalins and GABA are co-localized in certain axons was excluded in double-labeling studies, silver intensification being used for the first antigen and the nickel-enhanced diaminobenzidine reaction for the second antigen. Different subsets of principal neurons were richly innervated in a basket-like manner by axons immunoreactive for enkephalins and GABA. Additionally, combined staining with antisera against either enkephalin and neuropeptide Y or GABA and neuropeptide Y revealed that both subsets of principal neurons richly innervated either by enkephalin-immunoreactive or by GABA-immunoreactive axons were devoid of neuropeptide Y immunoreactivity. Thus, the enkephalinergic and GABAergic axons have different subpopulations of neuropeptide Y-negative principal neurons as targets in the superior cervical ganglion. These results provide further evidence that sympathetic ganglion cells can be classified on the basis of their receiving input from different sources.

Capsaicin differentially influences somatosensory cortical responses evoked by peripheral electrical or mechanical stimulation.

The effects of capsaicin injected intraperitoneally (200 micrograms/kg) or applied locally to the cortical surface (10(-5) M) were studied on cortical potentials evoked by peripheral electrical or mechanical stimulation. Capsaicin treatment (i.p.) differentially influenced the cortical evoked potentials depending on the type of stimulation. Just after both types of capsaicin application, the responses to both kinds of stimuli decreased in amplitude. Additionally, during this time a short fall in blood pressure was observed. Half an hour later, however, only in the case of interperitoneal application the potentials evoked by electrical stimulation were facilitated, while the potentials evoked by vibrissa deflection had recovered and stayed around the control levels thereafter. In addition, the responsive cortex area activated by electrical stimulation became enlarged after the i.p. injection of capsaicin, while that of the cortex region activated by mechanical stimulation did not change significantly. Capsaicin applied locally to the cortex resulted neither in the facilitation of evoked potentials nor in the enlargement of the responsive cortical area. The present findings are the first to demonstrate that the i.p. (but not local) administration of capsaicin, in low dosage, differentially influences the cortical responses evoked by electrical and mechanical stimulation of somatosensory afferents.

Effects of dexamethasone on brain edema induced by kainic acid seizures.

The histopathological alterations developing in the hippocampus, piriform cortex and thalamus of the rat brain, the blood-brain barrier damage, and the effects of dexamethasone pretreatment on the brain edema were investigated 4 h following intraperitoneal kainic acid administration. The most pronounced Evans Blue extravasation accompanied by increases in the water and sodium contents and a decrease in the potassium content, were observed in the thalamus. Dexamethasone, injected in a dose of 5 mg/kg 2 h before kainic acid administration, reduced considerably the vasogenic edema and neuronal damage in the thalamus, but the cytotoxic edema of the hippocampus and piriform cortex remained unaltered. Kainic acid-induced seizures lead to the development of vasogenic brain edema mainly in the thalamus, as well as to cytotoxic edema in the hippocampus and piriform cortex. The vasogenic edema seems to contribute to the cell damage in the thalamus. Dexamethasone reduces the vasogenic edema and cell damage in the thalamus, possibly by inducing the synthesis of certain protein(s) with antiphospholipase A2 activity.