Interferon-γ receptors are expressed at synapses in the rat superficial dorsal horn and lateral spinal nucleus

Summary Interferon-γ can facilitate the spinal nociceptive flexor reflex and elicit neuropathic pain-related behavior in rats and mice. Immunoreactivity for the interferon-γ receptor (IFN-γR) occurs in the superficial layers of the dorsal horn and the lateral spinal nucleus in the rat and mouse spinal cord, as well as in subsets of neurons in the dorsal root ganglia. The aim of the present study was to examine the cellular localization and origin of the IFN-γR in the spinal cord. As viewed by confocal microscopy, the immunopositivity for the IFN-γR was co-localized with that of the presynaptic marker synaptophysin and with neuronal nitric oxide synthase in the lateral spinal nucleus, whereas only a minor overlap with these molecules was observed in laminae I and II of the dorsal horn. There was no co-localization of the IFN-γR with markers for astrocytes and microglial cells. Ultrastructurally, the IFN-γR was found predominantly in axon terminals in the lateral spinal nucleus but also at postsynaptic sites in dendrites in laminae I and II. The IFN-γR expressed in neurons in dorsal root ganglia was transported in axons both centrally and peripherally. Hemisection of the spinal cord caused no reduction in immunolabelling of the IFN-γR in the dorsal horn or the lateral spinal nucleus. Since rhizotomy does not effect the immunolabelling in the lateral spinal nucleus, our observation indicates that the presynaptic receptors in this nucleus are derived from intrinsic neurons. The localization of the IFN-γR in the spinal cord differed from that of the AMPA glutamate receptor subunits 2 and 3 and the substance P receptor (NK1). Our results, showing localization of IFN-γR to pre- and postsynaptic sites in the dorsal horn and lateral spinal nucleus indicate that IFN-γ can modulate nociception at the spinal cord level.     

Layer-dependent role of collagen recruitment during loading of the rat bladder wall

In this work, we re-evaluated long-standing conjectures as to the source of the exceptionally large compliance of the bladder wall. Whereas these conjectures were based on indirect measures of loading mechanisms, in this work we take advantage of advances in bioimaging to directly assess collagen fibers and wall architecture during biaxial loading. A custom biaxial mechanical testing system compatible with multiphoton microscopy was used to directly measure the layer-dependent collagen fiber recruitment in bladder tissue from 9 male Fischer rats (4 adult and 5 aged). As for other soft tissues, the bladder loading curve was exponential in shape and could be divided into toe, transition and high stress regimes. The relationship between collagen recruitment and loading curves was evaluated in the context of the inner (lamina propria) and outer (detrusor smooth muscle) layers. The large extensibility of the bladder was found to be possible due to folds in the wall (rugae) that provide a mechanism for low resistance flattening without any discernible recruitment of collagen fibers throughout the toe regime. For more extensible bladders, as the loading extended into the transition regime, a gradual coordinated recruitment of collagen fibers between the lamina propria layer and detrusor smooth muscle layer was found. A second important finding was that wall extensibility could be lost by premature recruitment of collagen in the outer wall that cut short the toe region. This change was correlated with age. This work provides, for the first time, a mechanistic understanding of the role of collagen recruitment in determining bladder extensibility and capacitance.     

Reductive inactivation of digitoxin by Eubacterium lentum cultures

The obligate anaerobe Eubacterium lentum inactivated the cardiac glycoside digitoxin by reducing the double bond in the lactone ring. This conversion was quantitative when the substrate was incubated at a concentration of 10 micrograms/ml. The reduction reaction coincided with the growth phase of the bacterium. The stereochemical configuration at C-20 of the reduction product dihydrodigitoxin was found to be R. Incubation of digitoxigenin and its mono- and bisdigitoxosides individually with E. lentum led to the formation of their respective dihydro derivatives. The configuration at C-20 of these reduced metabolites was also found to be R.     

Biological activity and metabolic clearance of recombinant human follicle stimulating hormone produced in Sp2/0 myeloma cells

Human follicle stimulating hormone is a pituitary glycoprotein that is essential for the maintenance of ovarian follicle development and testicular spermatogenesis. Like other members of the glycoprotein hormone family, it contains a common a subunit and a hormone specific subunit. Each subunit contains two glycosylation sites. The specific structures of the oligosaccharides of human follicle stimulating hormone have been shown to influence both thein vitro andin vivo bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the isoform profiles,in vitro bioactivity and metabolic clearance of a preparation of purified recombinant human follicle stimulating hormone derived from a stable, transfected Sp2/0 myeloma cell line, and pituitary human follicle stimulating hormone. Isoelectric focussing and chromatofocussing studies of human follicle stimulating hormone preparations both showed a more basic isoform profile for the recombinant human follicle stimulating hormone compared to that of pituitary human follicle stimulating hormone. The recombinant human follicle stimulating hormone had a significantly higher radioreceptor activity compared to that of pituitary human follicle stimulating hormone, consistent with a greaterin vitro potency. Pharmacokinetic studies in rats indicated a similar terminal half life (124 min) to that of the pituitary human follicle stimulating hormone (119 min). Preliminary carbohydrate analysis showed recombinant human follicle stimulating hormone to contain high mannose and/or hybrid type, in addition to complex type carbohydrate chains, terminating with both2,3 and2,6 linked sialic acids. These results demonstrate that recombinant human follicle stimulating hormone made in the Sp2/0 myeloma cells is sialylated, has a more basic isoform profile, and has a greaterin vitro biological potency compared to those of the pituitary human follicle stimulating hormone.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Depletion of brainstem epinephrine stores by alpha-methyldopa: possible relation to attenuated sympathetic outflow

The antihypertensive effect of alpha-methyldopa (MD) is believed to be critically dependent on its ability to deplete endogenous catecholamines or cause the synthesis of false neurotransmitters. We used liquid chromatography with electrochemical detection (LCEC) and negative chemical ionization gas chromatography-mass spectrometry (GC-MS) for quantitation of catecholamines and MD metabolites in rat. MD intraperitoneally (100 mg/kg q12 hr X 12 days), significantly increased alpha-methylnorepinephrine (MNE) in brain (1.02 +/- 0.33 micrograms/g), heart (1.67 +/- 0.57 micrograms/g) and adrenal glands (114.93 +/- 50.47 micrograms/g) Endogenous norepinephrine (NE), epinephrine (E) and dopamine (DA) were reduced. ME levels were 2.19 +/- 0.44 micrograms/g (n = 6) in the adrenal gland but only 99 +/- 26 pg/g (n = 3) in the brainstem. MD-induced endogenous brainstem NE depletion was more than compensated by MNE production, but brainstem E depletion was not compensated for by a stoichiometric production of brainstem ME. We conclude (1) although ME is a metabolite of MD, it is present in extremely low concentrations in brainstem and (2) central epinephrine-containing neurons are depleted of neurotransmitter by MD therapy. If this selective epinephrine depletion occurs in the bulbospinal tract neurons responsible for maintaining sympathetic tone, then this effect could contribute to the antihypertensive effect of MD.     

Chorusing Behaviour,a Density-dependent Alternative Mating Strategy in Male Common Toads (Bufo bufo)

Male chorusing behaviour was studied in a population of common toads (Bufo bufo) on the island of Öland south Sweden, and the functional role of male advertisement calling in this species was experimentally examined. Calling males were larger and heavier than non-calling males (t = 2.41, p < 0.025 and t = 2.36, p < 0.025, respectively). However, small males were also found to call. This indicates that large males persisted in calling for longer and/or called more often. The proportion of calling males decreased as population size increased during the breeding season, indicating that calling is a low density strategy. Females responded more readily to calls than males. There were insufficient data to determine if the dominant frequencies of advertisement calls were inversely correlated with male body size, however, this relationship was found for the similar release calls. Females were found not to discriminate between high and low frequency calls, but when given a choice between two calls of different sound pressure levels (SPL), females were attracted to the louder calls. Thus, the function of chorusing is to advertise the position of males to mate-seeking females when the population density is low.     

Survival of Cryptosporidium parvum oocysts under various environmental pressures.

The survival of various isolates of Cryptosporidium parvum oocysts under a range of environmental pressures including freezing, desiccation, and water treatment processes and in physical environments commonly associated with oocysts such as feces and various water types was monitored. Oocyst viability was assessed by in vitro excystation and by a viability assay based on the exclusion or inclusion of two fluorogenic vital dyes. Although desiccation was found to be lethal, a small proportion of oocysts were able to withstand exposure to temperatures as low as -22 degrees C. The water treatment processes investigated did not affect the survival of oocysts when pH was corrected. However, contact with lime, ferric sulfate, or alum had a significant impact on oocyst survival if the pH was not corrected. Oocysts demonstrated longevity in all water types investigated, including seawater, and when in contact with feces were considered to develop an enhanced impermeability to small molecules which might increase the robustness of the oocysts when exposed to environmental pressures.     

Phylogeography of the pantropical sea urchin Tripneustes: contrasting patterns of population structure between oceans

To understand how allopatric speciation proceeds, we need information on barriers to gene flow, their antiquity, and their efficacy. For marine organisms with planktonic larvae, much of this information can only be obtained through the determination of divergence between populations. We evaluated the importance of ocean barriers by studying the mitochondrial DNA phylogeography of Tripneustes, a pantropical genus of shallow water sea urchin. A region of cytochrome oxidase I (COI) was sequenced in 187 individuals from locations around the globe. The COI phylogeny agreed with a previously published phylogeny of bindin that barriers important to the evolution of Tripneustes are: (1) the cold water upwelling close to the tip of South Africa, (2) the Isthmus of Panama, (3) the long stretch of deep water separating the eastern from the western Atlantic, and (4) the freshwater plume of the Orinoco and the Amazon rivers between the Caribbean and the coast of Brazil. These barriers have previously been shown to be important in at least a subset of the shallow water marine organisms in which phylogeography has been studied. In contrast, the Eastern Pacific Barrier, 5000 km of deep water between the central and the eastern Pacific that has caused the deepest splits in other genera of sea urchins, is remarkably unimportant as a cause of genetic subdivision in Tripneustes. There is also no discernible subdivision between the Pacific and Indian Ocean populations of this genus. The most common COI haplotype is found in the eastern, central, and western Pacific as well as the Indian Ocean. Morphology, COI, and bindin data agree that T. depressus from the eastern Pacific and T. gratilla from the western Pacific are, in fact, the same species. The distribution of haplotype differences in the Indo-Pacific exhibits characteristics expected from a sea urchin genus with ephemeral local populations, but with high fecundity, dispersal, and growth: there is little phylogenetic structure, and mismatch distributions conform to models of recent population expansion on a nearly global scale. Yet, comparisons between local populations produce large and significant F(ST) values, indicating nonrandom haplotype distribution. This apparent local differentiation is only weakly reflected in regional divergence, and there is no evidence of isolation by distance in correlations between F(ST) values and either geographical or current distance. Thus, Tripneustes in the Indo-Pacific (but not in the Atlantic) seems to be one large metapopulation spanning two oceans and containing chaotic, nonequilibrium local variation, produced by the haphazard arrival of larvae or by unpredictable local extinction.