Concanavalin A binding by the hippocampal neurons following brief cerebral ischemia in gerbils

Immunoglobulin (Ig) fractions from the plasma of a group of newly diagnosed insulin-dependent diabetes mellitus (type 1) patients and set of control subjects were assessed for their effects on isolated mouse islet function. It was found that Igs from type 1 patients caused a significant inhibitory effect on insulin secretion when incubated with mouse islets as compared with controls (25.6 +/- 2.9 pg islet-1 h-1 vs 44.7 +/- 7.7 pg islet-1 h-1, P < 0.05). The plasma samples from which the Igs were obtained were then tested for the presence of antibodies to the mouse islet cell surface (ICSA). Four of the nine patients were positive for ICSA, and plasma samples from eight control subjects were all negative. ICSA-positive samples appeared to have the greatest inhibitory effect on insulin secretion when compared with their respective controls (53.3 +/- 7.0 pg insulin islet -1 min-1 vs 30.9 +/- 3.7 pg insulin islet -1 min-1, (P < 0.05). In contrast, it was also found that ICSA-positive Ig fractions had no significant effect on glucose oxidation when co-incubated with mouse islets as compared with the controls (11.3 +/- 2.3 pmol islet-1 h-1 vs 11.2 +/- 2.9 pmol islet-1 h-1). These studies suggest that Igs from newly diagnosed type 1 patients containing ICSA may impair insulin secretion from isolated mouse islets by mechanisms which do not involve the inhibition of B-cell glucose metabolism.     

Clostridium difficile toxin A binding to human intestinal epithelial cells

Clostridium difficile radiolabelled toxin A ([3H]-toxin A) bound to human duodenal and colonic epithelial cells isolated from endoscopic biopsies. Binding was greater at 4 degrees C than 37 degrees C, consistent with the thermal binding characteristic of toxin A to a carbohydrate moiety. At 37 degrees C colonic cells bound significantly more [3H]-toxin A than duodenal cells. The amount of [3H]-toxin A binding varied considerably between individuals. [3H]-toxin A was displaced by unlabelled toxin A by 50% for duodenal cells and 70% for colonic cells with 94.3 nM unlabelled toxin A. Low non-displacable binding was observed in some samples at 4 degrees C and 37 degrees C, suggesting that these cells came from individuals incapable of specifically binding toxin. Pre-treating cells with alpha- or beta-galactosidases to cleave terminal alpha- and beta-galactose residues reduced [3H]-toxin A binding. There was also a reduction in [3H]-toxin A binding after heat treating cells, which is suggestive of protein binding. The reduction in binding varied between individuals. The reduction of [3H]-toxin A binding, after the removal of beta-linked galactose units, implicates these as components of the receptor and adds credence to the idea that the Lewis X, Y and I antigens may be involved in toxin A binding to human intestinal epithelial cells. However, because the Lewis antigens do not possess terminal alpha-galactose units, the reduction in binding after alpha-galactosidase treatment suggests that other receptors may be involved in toxin A binding to some human intestinal cells. These data are the first demonstration of direct toxin A binding to human intestinal epithelial cells.     

A confocal microscopic evaluation of the effects of insulin imprinting on the binding of Concanavalin A by Tetrahymena Pyriformis

With confocal microscopy it is possible to study the Concanavalin A (Con A) binding characteristics of the surface and interior of a single cell by viewing optical sections. It was observed in Tetrahymena pyriformis that Con A bound both to the plasma membrane and to intracellular structures. Incubation of cells with a competing sugar a-methylmannopyranoside, decreased binding. Hormonal imprinting with insulin resulted in an increase in binding of Con A to the cell surface and a decrease in intracellular binding. It is possible that the intracellular binding sites may migrate to the plasma membrane.     

Neuropeptide FF receptors of mouse olfactory bulb: binding properties and stimulation of adenylate cyclase activity

Neuropeptide FF (NPFF) receptors have been characterized in mouse olfactory bulb membranes by using [125I][1DMe]Y8Fa. The specific binding of this NPFF analogue was time and concentration dependent, reversible, saturable, and of high affinity (Kd = 0.022 nM, Bmax = 56.4 fmol/mg protein). In olfactory bulb membranes, NaCl increased the affinity of [125I][1DMe]Y8Fa by decreasing the dissociation rate constant (k-1). In contrast, the nonhydrolyzable analogue of GTP, Gpp[NH]p, decreased the maximal number of binding sites suggesting a coupling of NPFF receptors to a G-protein. In mouse olfactory bulb and spinal cord membranes, NPFF analogues stimulated adenylate cyclase activity in a time- and dose-dependent manner, whereas in the cerebellum, which does not possess NPFF receptors, low cAMP production was stimulated by NPFF. Our data are consistent with guanine nucleotide binding protein regulation of NPFF receptors positively coupled to adenylate cyclase.     

Calretinin immunoreactivity in the developing olfactory system of the rainbow trout

OBJECTIVE: The objective of this study was to calculate and compare the effective dose and to estimate risk from the use of intraoral position-indicating devices of differing geometries. STUDY DESIGN: Thermoluminescent dosimeters were placed at selected sites in the upper portion of a tissue-equivalent human phantom to record the equivalent dose to weighted tissues and organs. The phantom was exposed to simulated complete mouth surveys with either a long (29.8 cm) or short (19.6 cm) round open-end position-indicating device, a long (35.3 cm) or short (23.3 cm) rectangular open-end position-indicating device, or a pointed (29.6 cm) closed-end position-indicating device. RESULTS: The effective dose was calculated as the sum of the equivalent doses to each organ or tissue multiplied by that organ or tissue's weighting factor. The salivary glands were included as part of the remainder. The effective dose ranged from 362 micro Sv for the pointed position-indicating device, to 63 micro Sv for both the long and the short rectangular position-indicating devices. CONCLUSIONS: These effective doses were calculated to represent a probability for stochastic effects that range in magnitude from 26 x 10(-6) to 4.6 x 10(-6).     

Compartmental organization of Purkinje cells in the mature and developing mouse cerebellum as revealed by an olfactory marker protein-lacZ transgene

In a line of transgenic mice (HpY-1), the pattern of expression of an olfactory marker protein (OMP)-lacZ fusion gene was analyzed in the cerebellum, where, in adult mice, OMP-lacZ was expressed primarily in Purkinje cells (PCs) of the posterior lobe. The transgene-expressing PCs were organized in parasagittal bands, with a boundary of expression roughly corresponding to the primary fissure that separates the cerebellum into anterior and posterior compartments. The regional expression of the lacZ gene was also analyzed during embryonic and postnatal development of the cerebellum. Within the cerebellum-isthmus region, transgene expression first was detected at embryonic day 13.5 (E13.5) in a cluster of postmitotic cells. By E14.5, lacZ was also expressed by a subpopulation of migrating PCs in the postisthmal and lateral cerebellar primordium, and, by E16.5, transgene-positive PCs formed caudally four sagittal bands symmetric to the medial embryonic fissure. The caudal pattern was retained in postnatal cerebella, where, by postnatal day 0 (P0), transgene-positive PCs in vermal lobules VIII and IX appeared to be organized in two prominent parasagittal compartments on either side of a negative midline band. In early postnatal animals, the transgene was expressed transiently in the anterior lobe vermis. Hence, from P5 onward, transgene expression appeared mostly restricted to the posterior lobe, where it followed a caudal-to-rostral gradient. In the paraflocculus, transgene-expressing PCs were confined to the rostrodorsal portion. The results indicate that the anterior and posterior cerebellar lobes are regulated by distinct ontogenetic programs, and PCs of functionally distinct cerebellar regions express the transgene differentially. Furthermore, the data suggest that ectopic expression of OMP-lacZ in the cerebellum is under the control of regulatory elements that provide positional information for the regional specification of PC subsets.     

Gonadotropin-releasing hormone neuroblasts from one olfactory placode can be present in both hemispheres in the clawed toad Xenopus laevis

Ontogenetic differentiation of the GnRH-immunoreactive (GnRHir) neuron system was studied in the clawed toad Xenopus laevis by immunocytochemistry employing polyclonal antibodies against mammalian GnRH and chicken type II GnRH, and monoclonal antibodies against GnRH exhibiting wide cross-reactivity over animal classes. Toads at different stages of differentiation as well as postmetamorphic toads subjected to uni- or bilateral ablation of the olfactory placode (OPX) between developmental stages 25 and 30 were studied. GnRHir neurons and nerve fibers could not be detected before metamorphosis. Following metamorphosis, at stage 65-66, hemi-OPX toads did not exhibit any side differences in the number and overall distribution of the GnRHir neuronal structures; however, the total number of GnRHir neurons was approximately 50% of that counted in intact controls at the same developmental stages. These findings indicate that GnRHir neuroblasts differentiating on one side in the olfactory placode can appear on both sides of the brain in the course of their migration.     

Expression of galectin-1 in the mouse olfactory system

Mast cells hold a key position in the defensive mechanisms against exogenous intruders. In this study, we investigated whether human mast cells express functional major histocompatibility complex (MHC) class II molecules that can transduce endogenous signals and present staphylococcal enterotoxin A (SEA) to T cells. Similar to HMC-1 human mast cell line, umbilical cord blood-derived mast cells express HLA-DR, -DP and -DQ molecules on their surface. MHC class II molecules expressed on HMC-1 cells bind significantly the SEA (a natural MHC class II ligand), and their ligation with specific mAbs or with SEA, leads ultrastructural changes, suggesting their degranulation. Recognition of SEA-bound MHC class II molecules on HMC-1 mast cells by the T cell receptor of K25 cells, an SEA-specific murine T cell hybridoma, triggers significant IL-2 secretion by these T cell hybridomas. Hence, our data point out the expression of functional MHC class II molecules on human mast cells, reinforcing the implication of these cells in the defense mechanisms of acquired immunity.     

A transient CD15 immunoreactive sling in the developing mouse cerebellum

The distribution of the 3-fucosyl-N-acetyl-lactosamine (FAL, CD15) epitope in the developing mouse cerebellum was examined with the aid of immunohistochemistry of paraffin sections. CD15 immunoreactivity first appeared at E15 as a discrete bundle of processes lying beneath, and slightly within, the deeper layers of the external granular layer. By E17, when the cerebellar anlagen had completed their midline fusion, these processes could be traced from the germinal trigone at the lateral limits of the cerebellar anlage around the posterior cerebellar midline to the opposite germinal trigone. By P2, this sling was no longer apparent and CD15 immunoreactivity was confined to astrocytes in the cerebellar white matter, surrounding the deep cerebellar nuclei. The CD15 immunoreactive processes pursue an unusual trajectory through the developing cerebellum, unlike any other previously described axonal or glial process bundle in the cerebellum. From its trajectory and association with the ventricular surface it seems that this structure, which we have named the transverse cerebellar sling, is composed of glial processes, although it was not immunoreactive for S-100 or glial fibrillary acidic protein. The transient appearance of this sling encircling the posterior cerebellum is suggestive of a role in prenatal cerebellar morphogenesis.     

Extracellular mutations of non-obese diabetic mouse FcgammaRI modify surface expression and ligand binding

The non-obese diabetic mouse (NOD) expresses a unique form of the high affinity receptor for IgG (FcgammaRI), containing multiple mutations that result in substitutions and insertions of amino acids and a truncated cytoplasmic tail. As a result of these major changes, receptor affinity for IgG increases 10-fold over that of wild-type FcgammaRI from BALB/c mice, while the specificity for ligand is retained. Kinetic analysis revealed that while the association rate of IgG with FcgammaRI from NOD mice (FcgammaRI-NOD) and FcgammaRI from BALB/c mice (FcgammaRI-BALB) is similar, IgG bound much more tightly to FcgammaRI-NOD as revealed by a profoundly diminished dissociation rate. Transfection studies demonstrated that FcgammaRI-NOD was expressed at one-tenth of the level of FcgammaRI-BALB. Although mouse FcgammaRI was previously not known to associate with the FcepsilonRI gamma-subunit, transfection of COS-7 cells demonstrates that like human FcgammaRI, mouse FcgammaRI is also able to associate with this signaling subunit. Furthermore, expression levels of FcgammaRI-NOD were not restored by the presence of the FcepsilonRI gamma-subunit. The difference in the levels of expression was mapped to mutations in the extracellular region of FcgammaRI-NOD as replacement of the extracellular domains with those of human FcgammaRI or FcgammaRI-BALB restored expression to that of human FcgammaRI or FcgammaRI-BALB.     

On the nature of rat hepatic and mouse olfactory sulfotransferases

Seasonal differences in the particle size fractions and mass loadings of household dust deposited on indoor surfaces were examined in four New Jersey homes. Housedust was collected during a 30-day period on non-electrostatic polyethylene sample plates on which a glass slide had been placed. In each home two samples were collected at a height of 1.5 m and two were collected at a height of 0.3 m above the floor. Dust samples were obtained from each home during a summer and winter collection period. Particle size measurement was completed using an adaptation of a Meridian ACAS 570 Interactive Laser Cytometer. Results indicated that the dust mass deposited on household surfaces during the summer was greater than during the winter. The arithmetic mean mass deposition rate for all houses was 0.37 +/- 0.13 microgram/cm2/day during the summer and 0.22 +/- 0.13 microgram/cm2/day during the winter. The total number of particles deposited, however, was greater during the winter than during the summer. The increase in winter time particle number was caused by greater numbers of particles with an equivalent spherical diameter < 2.5 microns. The most probable source of these particles was winter time combustion emissions within the residences and the subsequent particle deposition on household surfaces. The greater mass loadings measured on the low sampling plates during the summer were associated with a greater number of particles with an equivalent spherical diameter > 5 microns. In the winter, however, the particle mass and number loadings were similar at both heights. These results suggested that ventilation of the house during the summer allowed resuspended particles to enter which led to the higher levels of settled dust. Measurement of contaminant levels in housedust for exposure estimation therefore, should account for the seasonal and height differences in dust mass, and collect representative fractions of housedust that are available for human contact. Furthermore, since over 99% of the particles on indoor surfaces were < 50 microns any indirect sampling technique for dermal exposure estimation should have collection efficiencies similar to the hand of particles < 50 microns.     

Effects of unilateral olfactory deprivation in the developing opossum, Monodelphis domestica

Unilateral naris closure in young rodents leads to striking alterations in the development of the ipsilateral olfactory system. One of the most pronounced effects is a 25% reduction in the size of the experimental olfactory bulb, a change that stems in part from decreased cell survival. Since naris occlusion in rodents alters the system more during development than in adulthood, we investigated the consequences of olfactory deprivation in a species that is born in a very immature state, Monodelphis domestica. In this pouchless marsupial, offspring are born after a short 14-day gestation. In the present study, the thymidine analogue bromodeoxyuridine was used to examine early postnatal neurogenesis in the olfactory bulb. Unlike rats and mice, neurogenesis of the main output neurons (the mitral cells) continues into postnatal life. Unilateral naris closure was begun on postnatal day 4 (P4) or P5 in Monodelphis and continued for 30 or 60 days. Laminar volume measurements revealed a significant reduction in the size of the experimental bulb following 60, but not 30, days of early olfactory deprivation. Mitral cell number estimates indicated a significant reduction after both 30 and 60 days of naris closure. The immaturity of Monodelphis offspring may render the population of mitral cells susceptible to the effects of olfactory deprivation. These findings suggest that afferent activity plays a role in the survival of all bulb neurons, irrespective of cell class.     

The amino acids of Escherichia coli enterotoxin B subunit involved in binding to Bio-Gel A-5m or to the glycoprotein from mouse intestinal epithelial cells

We determined whether Arg13, Met31, and Ser95 of the heat-labile enterotoxin B subunit (LT-B) might be involved in Lt-B binding to oligosaccharides, which did not bind to the B subunit of the cholera toxin (CT-B). Three LT-B mutants, R13H, M31L, and S95A were prepared by substituting three amino acid residues that differ in CT-B. These mutants formed a pentamer and exhibited the same binding ability to the GM1 ganglioside as native LT-B. Although these mutants did not bind to Bio-Gel A-5m, they did bind to the glycoprotein from mouse intestinal cells in the order R13H > M31L > S95A. These data suggest that Ser95, Met31, and Arg13 are important for LT-B binding to Bio-Gel A-5m, and that although Ser95 is also partially responsible for LT-B binding to the glycoprotein, Arg13 has no significant involvement in it.     

Transplantation of the fetal olfactory placode restores reproductive cycles in female rhesus monkeys (Mucaca mulatta) bearing lesions in the medial basal hypothalamus

The purpose of this study was to determine whether loss of the reproductive cycle after lesions of the medial basal hypothalamus can be reversed by transplantation of the embryonic olfactory placode (OP) into female rhesus monkeys. Seven adult female rhesus monkeys with regular menstrual cycles received bilateral radiofrequency lesions in the arcuate nucleus and the median eminence. After confirmation of anovulation in these monkeys, four monkeys were stereotaxically implanted with the OP obtained from monkey fetuses on embryonic days 35-36. The remaining three monkeys were similarly implanted with embryonic cerebellum (CB) as a control. Fetuses were delivered by cesarean section, and the OP and CB were immediately dissected out using a stereomicroscope. Fetal tissue was then cut into small pieces (< 1 mm3), mixed with artificial cerebrospinal fluid containing small pieces of Gelfoam, and stereotaxically injected into the infundibular recess of the third ventricle. The recovery of ovulatory cycles in recipient monkeys was observed for at least 6 months; sex skin color changes and menstrual records were obtained daily, and serum samples for LH, estrogen, and progesterone were obtained twice a week. Three of four OP-transplanted monkeys resumed their ovulatory cycles within 2 months, whereas the fourth monkey, an elderly female, failed to recover her cycle. In contrast, none of the three CB-transplanted monkeys resumed ovulatory cycles. Histological examination indicated that 1) lesion scars were present in the median eminence-stalk region as well as the medial basal portion of the arcuate nucleus of all seven brains; and 2) cartilage was present in the third ventricles of the OP-implanted brains. Moreover, immunocytochemical staining revealed that in all OP monkeys, small, round, and immature LHRH-positive cells with fine short processes were found in the third ventricle and/or median eminence-stalk region, whereas no similar LHRH cells were found in CB-transplanted monkeys. It is concluded, therefore, that implantation of LHRH neurons derived from the fetal OP can result in resumption of the ovulatory cycle in female monkeys whose own LHRH pulse-generating mechanisms were impaired. Moreover, the results suggest that LHRH neurons derived from embryonic OP possess the physiological functions necessary for the stimulation of gonadotropin secretion.     

Anosmia and mouse killing by rats: A nonolfactory role for the olfactory bulbs.

Assigned 18 nonmuricidal Holtzman rats to 3 groups: (a) unoperated control, (b) bilateral deafferentation, and (c) bilateral olfactory bulbectomy. Ss were then presented with a Swiss albino male mouse. Pure anosmia, produced by deafferentation, did not facilitate mouse killing. Anosmia produced by olfactory bulbectomy did facilitate an irritable form of mouse killing in 80% of the Ss. Results indicate that it is not anosmia which is responsible for bulbectomy-facilitated muricide, but some nonolfactory function of the olfactory bulbs. (16 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Sulfatide from the pig jejunum brush border epithelial cell surface is involved in binding of Escherichia coli enterotoxin b

Using a quantitative dot blot overlay assay of polyvinylidene difluoride membranes, we investigated the ability of Escherichia coli heat-stable enterotoxin b (STb) to bind to various glycolipids of defined structure. STb bound strongly to acidic glycosphingolipids, including sulfatide (or 3'-sulfogalactosylceramide) and several gangliosides, but not significantly to their derivatives, galactosylceramide and asialogangliosides, respectively. STb exhibited the highest binding affinity for sulfatide. STb bound to pure sulfatide in a dose-dependent and saturable manner, with a detection level of a few nanograms. The binding was not inhibited by tetramethylurea, which is a strong disrupter of hydrophobic interactions, or by the anionic sulfated polymer of glucose, dextran sulfate, indicating that the binding is not due solely to either hydrophobic or ionic interactions via the sulfate group of the sulfatide. The specificity of the binding was confirmed by the finding that a 500-fold molar excess of sulfatide inhibited STb binding by approximately 45%, whereas no competition was obtained with galactosylceramide under the same conditions. Taken together, our data indicated that a galactose residue linked to a sulfate group is required for the binding specificity of STb. Then, total lipids extracted either from the mucous layer or from the epithelial cells of the pig jejunum brush border, the natural target of STb, were analyzed by thin-layer chromatography (TLC). Both extracts contained a lipidic molecule with a relative mobility on a TLC plate similar to that of the sulfatide standard. The migrated lipid extracted directly from a preparative TLC plate was confirmed to be sulfatide, as it was recognized by laminin, a sulfated glycolipid binding protein, and by a monoclonal antibody directed against sulfatide. In an overlay assay on PVDF membranes, STb bound to the sulfatide prepared from porcine jejunum as well as to the sulfatide standard. Thus, these findings suggest that the terminal oligosaccharide sequence Gal(3SO4)beta1- on sulfatide could mediate binding of STb to its target cells and, in support of a recent report (E. Rousset, J. Harel, and J. D. Dubreuil, Microb. Pathog. 24:277-288, 1998), probably terminal sialic acid residue on another glycosphingolipid. Moreover, pretreatment in the ligated intestinal loop assay with laminin or sulfatase altered the biological activity of STb. In summary, we present data indicating that sulfatide represents a functional receptor for the STb toxin.     

The epithelial surface of the monkey gastrointestinal tract. A scanning electron-microscopic study

A scanning electron-microscopic investigation of the luminal surface of the primate gastrointestinal tract has been performed using tissue prepared by critical point drying. This report defines criteria for identifying different segments of the normal primate gastrointestinal tract on the pasis of scanning electron-microscopic observations of the epithelial surface.     

New techniques for biopsy and culture of human olfactory epithelial neurons

OBJECTIVE: To improve the success of culturing olfactory neurons from human nasal mucosa by investigating the intranasal distribution of the olfactory epithelium and devising new techniques for growing human olfactory epithelium in vitro. DESIGN: Ninety-seven biopsy specimens were obtained from 33 individuals, aged 21 to 74 years, collected from 6 regions of the nasal cavity. Each biopsy specimen was bisected, and 1 piece was processed for immunohistochemistry or electron microscopy while the other piece was dissected further for explant culture. Four culture techniques were performed, including whole explants and explanted biopsy slices. Five days after plating, neuronal differentiation was induced by means of a medium that contained basic fibroblast growth factor. After another 5 days, cultures were processed for immunocytochemical analysis. RESULTS: The probability of finding olfactory epithelium in a biopsy specimen ranged from 30% to 76%, depending on its location. The dorsoposterior regions of the nasal septum and the superior turbinate provided the highest probability, but, surprisingly, olfactory epithelium was also found anteriorly and ventrally on both septum and turbinates. A new method of culturing the olfactory epithelium was devised. This slice culture technique improved the success rate for generating olfactory neurons from 10% to 90%. CONCLUSIONS: This study explains and overcomes most of the variability in the success in observing neurogenesis in cultures of adult human olfactory epithelium. The techniques presented here make the human olfactory epithelium a useful model for clinical research into certain olfactory dysfunctions and a model for the causes of neurodevelopmental and neurodegenerative diseases.