Hetero-concatemeric KIR6.X4/SUR14 channels display distinct conductivities but uniform ATP inhibition.

K(IR)6.1 and K(IR)6.2 are the pore-forming subunits of K(NDP)(,) the nucleotide-diphosphate-activated K(ATP) channels, and classical K(ATP) channels, respectively. "Hybrid" channels, in which the structure is predetermined by concatemerizing K(IR)6.1 and K(IR)6.2, exhibit distinct conductivities specified by subunit number and position. Inclusion of one K(IR)6.2 is sufficient to open K(IR)6. X-X-X-X/SUR1(4) in the absence of nucleotide stimulation through sulfonylurea receptor-1 (SUR1). ATP inhibited the spontaneous bursting of hybrid channels with an IC(50(ATP)) approximately 10(-)(5) m, similar to that of K(IR)6.2(4)-containing channels. These findings and a transient increase in K(NDP) channel activity following rapid wash-out of MgATP suggested that K(IR)6.1 is not ATP-insensitive as previously believed. We propose that SUR-dependent, inhibitory ATP-enhanced interactions of the cytoplasmic domains of both K(IR)6.1 and K(IR)6.2 stabilize a closed form of the M2 bundle in the gating apparatus.

Discrimination between apo and iron-loaded forms of transferrin by transferrin binding protein B and its N-terminal subfragment.

Many pathogens of the Pasteurellaceae and Neisseriaceae possess a surface receptor that binds transferrin (Tf) as an initial step in an iron acquisition process. This receptor is comprised of two proteins, transferrin binding protein A (TbpA) and transferrin binding protein B (TbpB). Since the ability to recognize the iron-loaded form of Tf preferentially would be a useful attribute of these receptors, we examined this property in a number of bacterial species. In solid-phase binding assays with isolated membranes, only the receptor from Moraxella catarrhalis was capable of preferentially binding iron-loaded Tf. In a competitive affinity isolation assay which enabled us to resolve TbpA and TbpB, TbpA from all tested species was shown to bind both apo and iron-loaded Tf. Under these assay conditions TbpB from M. catarrhalis, Haemophilus somnus and Pasteurella haemolytica discriminated between apo and holo Tf, whereas TbpB from Neisseria meningitidis showed no discrimination. The ability of TbpB from N. meningitidis to bind iron-saturated hTf preferentially became evident in a TbpA- background or by using recombinant TbpB. In binding assays with recombinant fusion proteins, both intact TbpB and the N-terminal half of TbpB from all the tested species preferentially bound Fe-loaded Tf, indicating that this may be a conserved mechanism by which these organisms optimize their ability to acquire iron.

Hypothalamic and extrahypothalamic sauvagine-like immunoreactivity in the bullfrog (Rana catesbeiana) central nervous system.

In the present study, immunocytochemistry and radioimmunoassay were used to investigate the presence of sauvagine in both hypothalamic and extrahypothalamic areas of the central nervous system (CNS) of the bullfrog (Rana catesbeiana) using a specific antiserum raised against synthetic non-conjugated sauvagine (SVG), a frog (Phyllomedusa sauvagei) skin peptide of the corticotropin-releasing factor (CRF) family. Sauvagine-immunoreactive (SVG-ir) bipolar neurons were found in the nucleus of the fasciculus longitudinalis medialis located in the rostral mesencephalic tegmentum. In the tectal mesencephalon, beaded SVG-ir fibres were present in the optic tectum, and in the torus semicircularis. Abundant SVG-ir varicose fibres were seen in the granulosa layer of the cerebellum, the nucleus isthmi, and the obex of the spinal cord. SVG-ir fibres were also seen by the alar plate of the rombencephalon. In the diencephalon, the antiserum stained parvocellular neurons of the preoptic nucleus (PON) which extended their dendrites into the cerebro-spinal fluid (CSF) of the third ventricle and projected their ependymofugal fibres to the zona externa (ZE) of the median eminence. Immunopositive fibres were also present in the medial forebrain bundle at the chiasmatic field, the posterior thalamus, the pretectal gray, and the ventrocaudal hypothalamus. In the telencephalon (forebrain), SVG-ir fibres were seen in the medial septum, the lateral septum, and the amygdala. The SVG immunoreactivity could not be detected after using the SVG antiserum previously immunoabsorbed with synthetic SVG (0.1 microM), but immunoblock of the antiserum with sucker (Catostomus commersoni) urotensin I (sUI), sole (Hippoglossoides elassodon) urotensin I, sucker CRF, rat/human CRF, or ovine CRF (0.1-10 microM) did not eliminate visualization of the immunoreactivity. In radioimmunoassay, the SVG antiserum did not crossreact with sUI, or the SVG fragments SVG1-16, SVG16-27, and SVG26-34, but it recognized the C-terminal fragment SVG35-40. Crossreaction with mammalian ovine CRF and rat/human CRF was negligible. Both hypothalamic and mesencephalic extracts gave parallel displacement curves to SVG. The results suggest the presence in the bullfrog brain of a SVG-like neuropeptide, i.e., a peptide of the CRF family, that either is SVG or shares high homology with the C-terminus of that peptide. The function of this neuropeptide in amphibians is not known at this time, but based on its anatomical distribution to the ZE it could affect the release of adrenocorticotropin (ACTH) or other substances from the amphibian pars distalis. Involvement of the SVG-like peptide in behavioural (forebrain), visual (thalamus-tegmentum mesencephali-pretectal gray-optic tectum), motor coordination (cerebellum), and autonomic (spinal) functions, as well as an undefined interaction with the CSF in the bullfrog, seems likely.

Presence of sauvagine-like epitopes in the interrenal gland of the bullfrog Rana catesbeiana.

Immunocytochemistry was used to investigate the presence of corticotropin-releasing factor-like peptides in the interrenal (adrenal) glands of the bullfrog Rana catesbeiana by using specific antisera raised against synthetic nonconjugated rat/human corticotropin-releasing factor, urotensin I, and sauvagine. From these three antisera, covering a broad range of corticotropin-releasing factor-like immunoreactivities, only the sauvagine antiserum gave positive immunoreactivity. Sauvagine immunoreactivity was found in cortical cells grouped into cords in the renal zone of the interrenal gland. The central and subcapsular cords were less stained. Tyrosine hydroxylase-positive chromaffin cells were not sauvagine-immunoreactive. The immunoreactivity was abolished, in all cases, by previous immunoabsorption of the sauvagine antiserum with synthetic sauvagine (0.1 microM), but it was not eliminated by sucker (Catostomus commersoni) urotensin I, sole (Hippoglossoides elassodon) urotensin I, sucker corticotropin-releasing factor, rat/human corticotropin-releasing factor, or ovine corticotropin-releasing factor (0.1-10 microM). In a sauvagine radioimmunoassay, interrenal extracts displaced 125I-sauvagine from antiserum only partially, and not in parallel with the sauvagine standard curve. The results suggest that the sauvagine immunoreactivity in the R. catesbeiana interrenal gland may represent a novel sauvagine-like peptide.

Comparative localization of corticotropin and corticotropin releasing factor-like peptides in the brain and hypophysis of a primitive vertebrate, the sturgeon Acipenser ruthenus L.

The sturgeon is a primitive actinopterigian fish that, unlike modern teleosts, possess a portal vascular system that connects a true median eminence with the anterior pituitary as in mammals. The occurrence and localization of corticotropin and corticotropin releasing factor-like immunoreactivies were examined in the brain of the sturgeon (Acipenser ruthenus L.) by immunocytochemistry with antisera raised against synthetic non-conjugated human corticotropin, and rat/human corticotropin releasing factor. In the hypothalamus, corticotropin-immunoreactive parvicellular perikarya were found in the infundibular nucleus and in dendritic projections to the infundibular recess. In addition, ependymofugal corticotropin-immunoreactive fibres were found to terminate in the ventral hypothalamus. Corticotropin releasing factor-immunoreactive neurons were found in the rostral portion of the ventral hypothalamus (tuberal nucleus), and in the vicinity of the rostral aspect of the lateral recess. These cells projected to the dorsal hypothalamus, the ventral hypothalamus, the median eminence, the anterior and posterior telencephalon, the tegmentum mesencephali, and the pars nervosa of the pituitary. An affinity-purified UI antiserum failed to stain the sturgeon hypothalamus. Corticotrophs in the rostral pars distalis of the pituitary were also corticotropin-immunoreactive. In the neurointermediate lobe, only about 50% of cells of the pars intermedia appeared to be corticotropin-positive, the rest appeared unstained. These results suggest that the presence of corticotropin-like and corticotropin releasing factor-like peptides in the brain is a relatively early event in vertebrate evolution, already occurring in Chondrostean/Actinopterigian fishes, as exemplified by A. ruthenus. The close spatial relationship between corticotropin releasing factor immunoreactivity and corticotropin immunoreactivity in the ventral hypothalamus of A. ruthenus supports a possible interaction between the two systems in that area of the sturgeon brain. The pars intermedia might be an important site for corticotropin synthesis, even though the possibility cannot be excluded that the antiserum was recognizing the proopiomelanocortin molecule. The occurrence of corticotropin releasing factor immunoreactivity in the region of median eminence/pars intermedia of the sturgeon suggests that the sturgeon corticotropin releasing factor might regulate the adenohypophyseal release of proopiomelanocortin products in the same manner as in other vertebrates. The presence of extrahypothalamic corticotropin releasing factor-immunoreactive projections suggests further neuromodulatory functions for this peptide in A. ruthenus.

Comparison of the abilities of different protein sources of iron to enhance Neisseria meningitidis infection in mice.

This study was done primarily to determine whether the previously observed specificity of the meningococcal transferrin and lactoferrin receptors for human proteins was maintained in vivo during meningococcal infection in mice. Preliminary experiments evaluating the choice of host strain, the age and sex of mice, and the growth conditions of the meningococci indicated that 45-day-old female Swiss Webster mice challenged with meningococci grown on low-pH, low-iron Mueller-Hinton agar plates were appropriate for this study. The comparison of transferrins and lactoferrins from different species demonstrated that only the human forms of these proteins were utilized by meningococci; there was significantly greater mortality among mice treated with iron-saturated human transferrin or lactoferrin (93 and 100%, respectively) than among those not treated or treated with iron-saturated bovine transferrin or bovine lactoferrin (0%). Provision of exogenous hemoglobin also resulted in increased mortality, although not as great as that observed with amounts of transferrin with equivalent iron content, which parallels the more effective utilization of transferrin and lactoferrin in in vitro growth experiments. In addition, unlike with transferrin and lactoferrin, there was no difference in utilization of human and bovine hemoglobin in vitro or in vivo.

Sauvagine-like and corticotropin-releasing factor-like immunoreactivity in the brain of the bullfrog (Rana catesbeiana).

Immunocytochemical methods were used to investigate the occurrence and distribution of sauvagine, corticotropin-releasing factor-, or urotensin I-like immunoreactivities (SVG-ir, CRF-ir, UI-ir, respectively) in the bullfrog (Rana catesbeiana) brain, using specific antisera raised against non-conjugated SVG, ovine CRF, rat/human CRF, and UI. In the hypothalamus, SVG-ir was found in the magnocellular perikarya, in the dorsal and ventral regions of the preoptic nucleus, and in the hypothalamo-hypophyseal projections to the external zone as well as the internal zone of the median eminence, to pars nervosa, and in fibres running from the pars nervosa to the pars intermedia of the pituitary. In contrast, CRF-ir was found only in parvocellular perikarya, mainly localized in the rostro-ventral part of the preoptic nucleus, with fine processes protruding through the ependyma of the third ventricle, fibre projections terminating in the anterior preoptic area and in the neuropil of the periventricular gray, and a caudal projection to the external zone of the median eminence. No CRF-ir staining was seen in the pars nervosa and pars intermedia. The use of UI-specific antisera failed to give a positive response in the frog brain. It is concluded that, in the frog brain, two anatomically different CRF-like (or SVG-like) systems co-exist, comparable to the reported co-existence of UI-ir and CRF-ir neuronal systems in fish brain.