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PubMed1979

Ultrastructural study of degeneration and regeneration in the amphibian tectum.

The processes of degeneration and reinnervation in the optic tectum of Xenopus laevis have been detailed by quantitative ultrastructural analysis. The tecta were denervated either permanently, by removing the contralateral eye, or temporarily, by cutting the optic tract. After denervation the total number of synapses decreases rapidly within 4 days of a stable level of about 40% of the original number. Vacated postsynaptic sites are subsequently removed by phagocytosis. When regenerating axons arrive in the tectum they make synaptic contact by inducing new postsynaptic membrane specializations. The ultrastructural sequence of reinnervation by optic axons resembles initial synaptogenesis.

Animals↗
PubMed1981

Regeneration of vomeronasal nerves into the main olfactory bulb in the mouse.

After surgical section of the vomeronasal nerves the neurosensory cells in the vomeronasal epithelium die. Electron microscopy has been used to demonstrate that their axons, and synaptic terminals in the accessory olfactory bulb degenerate and are removed by phagocytic astroglia. The vacated postsynaptic sites in the accessory bulb persist, and are not re-innervated, either by vomeronasal or olfactory axons, as long as 150 days post-operatively. However, new neurosensory cells which are produced in the vomeronasal epithelium after vomeronasal nerve section do form axons. Light and electron microscope autoradiography of axonally transported material has been used to show that some of these axons grow back into the cranial cavity and form glomeruli in the main olfactory bulb, in regions where it is damaged or de-afferented. The regenerated vomeronasal glomeruli contain synapses between vomeronasal nerve terminals and dendrites of main bulb neurons.

Animals↗
PubMed1994

The development of glial fibrillary acidic protein-positive cells and the appearance of laminin-like immunoreactivity in fetal olfactory bulb transplants.

Embryonic rat olfactory bulbs were transplanted into the site vacated by aspiration of an olfactory bulb from a neonatal rat. This paper presents our findings related to the development of glial fibrillary acidic protein (GFAP) positive (+ve) glial cells and the appearance of laminin-like immunoreactivity in these transplants. The GFAP+ve glial cells formed perivascular end-feet on the invading vasculature and formed a glia limitans along the pial surface of the transplant. This reconstituted glia limitans was continuous with that of the host brain, there being no glia limitans at the donor-host interface. Thus, the donor tissue was well-integrated with that of the host brain.

Animals↗
PubMed1992

Age-related development of olfactory bulb transplants in rats.

We are using the rat olfactory system to study developmental aspects of neurotransplantation (TX). Age-related TX maturation and subsequent establishment of connections are of special concern. Previous studies of deafferentation by olfactory bulb (OB) removal suggested "critical" periods of plasticity in the system. We present here preliminary attempts at relating age of host receiving TX to maturation of the TX and its connections. This investigation used hosts of postnatal age (PN) 13-14 days with fetal donors at Embryonic Day 15; the former having one OB ablated and receiving a fetal donor OB TX immediately placed in the vacated space. The fetal tissue was labeled previously in utero with tritiated thymidine. After 2 months a small coagulation lesion was placed in the OB TX and 2 days later the tissue was taken, serially sectioned, and processed for [3H] autoradiography, degeneration, and olfactory marker protein (OMP). Extensively 3H-labeled OB TXs with localized small lesions were studied. The cellular architecture of the TX is less well organized than in normals but substantial OMP reactivity occurs throughout. Degeneration occurs mainly near the lesion and little if any degeneration is seen beyond the 3H-labeled TX tissue. The results show that OB TX survive and develop in the PN 13-14 age group as they do in the younger animals and that primary olfactory neurons likewise reinnervate the TX but that PN 13-14 TX efferent projections are far more limited than those of younger hosts.

Aging↗
PubMed1992

Development of olfactory marker protein and tyrosine hydroxylase immunoreactivity in the transplanted rat olfactory bulb.

Evidence suggests that tyrosine hydroxylase (TH) expression by juxtaglomerular (JG) neurons of the olfactory bulb (OB) is dependent upon input from primary olfactory neurons (ONs), which are identifiable using immunocytochemical localization (ICC-L) methods for olfactory marker protein (OMP). When the input from the continuously regenerating ONs is temporarily removed (either surgically or chemically), JG cells cease TH production until ON contact is reestablished. We are studying this transneuronal regulation using the rat OB in a transplantation (TX) model. Fetal OBs, labeled in utero with tritiated thymidine, were transplanted (TX) into a site vacated by removal of a neonatal host OB. Host animals were sacrificed at varying periods after TX. Alternate sets of frozen sections were then processed for autoradiography or using ICC-L for TH and OMP. As early as 1 week post-TX, OMP-positive fibers and glomerulus-like structures were seen throughout the TX OB. Despite this extensive and rapid OMP reinnervation, TH expression returned very slowly and the number of TH expressing cells never approached control levels. The reduced TH activity in TXs may be due to failure of JG cells to survive or to develop the correct phenotype under TX conditions. Alternatively, input from ON fibers may only be necessary, but not sufficient, for the expression of TH.

Animals↗
PubMed1992

Plasma progesterone levels in the pregnant female rat-kangaroo (Bettongia gaimardi).

Plasma progesterone levels were measured in female bettongs (small macropodid marsupials) under two natural regimes: (1) during "delayed" gestation (initiated by removal of pouch young, RPY) and (2) during the pregnancy prior to pouch vacation when a young still occupies the pouch (i.e., during lactation). Basal levels of progesterone were 0.15-0.5 ng/ml. There was a transient peak of progesterone (0.7 ng/ml) early in gestation at Day 4 RPY. After Day 6 RPY, progesterone levels remained elevated (1.2-1.5 ng/ml) until they dropped sharply to basal levels on the day of birth. This pattern of progesterone secretion during delayed gestation is similar to that seen in other marsupials, such as the tammar wallaby. There was no significant difference between the progesterone profiles of the two experimental groups. We deduce, then, that lactation had no effect on corpus luteum function (as assessed by plasma progesterone levels) in the pregnant bettong.

Analysis of Variance↗
PubMed1992

Sex differences in odor-stimulated flank marking in the golden hamster (Mesocricetus auratus).

To determine whether sex differences exist in the frequency of odor-stimulated flank marking, intact male and female hamsters were exposed to the recently vacated home cages of male stimulus hamsters for a 10-min test on 4 consecutive days. Females were found to mark at significantly higher levels than males. To investigate the role of gonadal hormones in the sex differences in flank marking, gonadectomized male and female hamsters were implanted with Silastic capsules containing estradiol or testosterone. Females exhibited twofold higher levels of odor-stimulated flank marking than males, and the amount of flank marking was significantly higher when the hamsters were administered testosterone than when they were administered estradiol. These data demonstrate that sex differences exist in the frequency of flank marking stimulated by the odors of male hamsters, and that these sex differences do not appear to result from the typical sex-specific patterns of circulating levels of estradiol and testosterone.

Animals↗
PubMed1992

Atomic interactions in protein-carbohydrate complexes. Tryptophan residues in the periplasmic maltodextrin receptor for active transport and chemotaxis.

We have refined the 1.9 A resolution crystal structures of two maltodextrin receptor mutants in which tryptophan residues 230 and 232 have been changed to alanine and compared these structures with the refined 1.7 A structure of the wild-type protein. In the wild-type structure, Trp230, which is located in the maltodextrin-binding groove, stacks against the B-face of the reducing sugar of the bound maltose. Trp232, which is located near the protein surface, does not participate directly in sugar binding. Relative to the wild-type structure, neither mutation caused a significant rearrangement in the overall protein structure or in the mode of binding maltose. Although the position once occupied by Trp230 remains empty, a new water molecule has moved near the void. In contrast, a new water molecule has entered into the space once occupied by Trp232. Whereas one hydrogen bond is formed with the water molecule near the Trp230 void, no hydrogen bond is associated with the water molecule occupying the space vacated by Trp232. The three van der Waals' contacts between Trp230 and maltose in the wild-type structure that are lost in the W230A mutation could contribute to the 12-fold decrease in ligand-binding activity of the mutant protein. The W232A mutation causes little change in binding activity. The structures of these mutant proteins also provide some insight into the complicated tryptophan fluorescence spectra of the maltodextrin binding-protein. The change in fluorescence due to the deletion of Trp230 can readily be explained as resulting directly from loss of Trp230 in the sugar-binding site. The change in fluorescence due to deletion of Trp232, however, is ascribed to the modification of local interactions mediated by the binding of maltodextrin since the tryptophan is not directly involved in any sugar-binding interaction.

Bacterial Proteins↗
PubMed1996

Determinants of sleepiness in automobile drivers.

Governmental agencies do not systematically investigate the presence of daytime sleepiness as a determinant of driving accidents. We surveyed automobile drivers traveling on summer vacations and driving long distances on a European highway. We evaluated their subjective daytime sleepiness while driving and any sleep deprivation just prior to departure. Five-hundred sixty-seven automobile drivers (mean age 37.7 +/- 11 years) were interviewed at a roadside rest-stop. Questions covered the sleep/wake schedule during the year, sleep habits, and the presence of symptoms frequently associated with sleep-disordered breathing. Sleep behavior just prior to departure was compared to the usual sleep schedule during the year. Fifty percent of the responders had a sleep restriction just prior to departure (mean -203 minutes) compared to usual total sleep time during the year; 10% had no nocturnal sleep prior to departure. Drivers younger than 30 years were significantly more acutely sleep deprived than other drivers. Economic migrants (subjects with low economic status) also experienced significant acute sleep restriction.

Accidents, Traffic↗
PubMed1979

Papain-induced asthma--physiological and immunological features.

Increasing reports of respiratory disease associated with exposure to papain prompted clinical, physiological, and immunological studies of the supervisor of a meat tenderizer factory who developed asthma after long-term contact with papain dust. His symptoms were worse at work and better on weekends and vacations. Bronchial inhalation challenges produced both immediate and late asthma to papain but not to the other ingredients in the food product. Immunological studies revealed the presence of specific IgE antibodies by direct and passive transfer skin tests and the radioallergosorbent test, and specific precipitating antibodies by immunodiffusion tests. These findings are indicative of a dual type I and III hypersensitivity. Papain acting as an allergen in an occupational setting is a risk factor for eliciting asthma even in a nontropic individual.

Administration, Intranasal↗
PubMed1984

Ultraviolet difference spectroscopy of bovine carbonic anhydrase substituted with various divalent metals.

The ultraviolet (uv) difference spectra of M(II)-apocarbonic anhydrase at pH 5-9 are reported. For Zn(II) at all pH's and Co(II) at pH greater than or equal to 7.65 identical protein difference spectra are seen and a positive 300 nm feature is interpreted as consistent with interaction of a metal-bound hydroxyl with a Trp chromophore near the active site. Hg(II), Cu(II), and Cd(II) do not provoke a positive 300 nm band even at alkaline pH (although a Cd(II) spectral band at 300 nm becomes less negative, i.e., more like the holoenzyme with increasing pH) and the 280-292 nm spectral region is generally different from that of Zn(II) and high pH Co(II). A specific orientation of M-OH and, hence, an ordered solvent structure in the enzyme site is implied for enzyme activation. Ni(II) appears to bind to the vacated zinc site slowly, at low pH, in a manner similar to zinc. At higher pH's Ni(II) may be displaced toward a Tyr residue in the active site of apocarbonic anhydrase.

Animals↗
PubMed1985

Mechanistic flexibility in the reduction of copper(II) complexes of aliphatic polyamines by mercapto amino acids.

The reactions of copper(II)-aliphatic polyamine complexes with cysteine, cysteine methyl ester, penicillamine, and glutathione have been investigated, with the goal of understanding the relationship between RS- -Cu(II) adduct structure and preferred redox decay pathway. Considerable mechanistic flexibility exists within this class of mercapto amino acid oxidations, as changes in the rate law could be induced by modest variations in reductant concentration (at fixed [Cu(II)]0), pH, and the structure of the redox partners. With excess cysteine present at 25 degrees C, pH 5.0, I = 0.2 M (NaOAc), decay of 1:1 cys-S- -Cu(II) transient adducts was found to be first order in both cys-SH and transient. Second-order rate constants characteristic of Cu(dien)2+(6.1 X 10(3) M-1 sec-1), Cu(Me5dien)2+ (2.7 X 10(3) M-1 sec-1), Cu(en)22+ (2.1 X 10(3) M-1 sec-1), and Cu(dien)22+ (4.7 X 10(3) M-1 sec-1) are remarkably similar, considering substantial differences in the composition and geometry of the oxidant first coordination sphere. A mechanism involving attack of cysteine on the coordinated sulfur atom of the transient, giving a disulfide anion radical intermediate, is proposed to account for these results. Moderate reactivity decreases in the cysteine-Cu(dien)2+, Cu(Me5dien)2+ reactions with increasing [H+] (pH 4-6) reflect partial protonation of the polyamine ligands. A very different rate law, second order in the RS- -Cu(II) transient and approximately zeroth order in mercaptan, applies in the pH 5.0 oxidations of cysteine methyl ester, penicillamine, and glutathione by Cu(dien)2+ and Cu(Me5dien)2+. This behavior suggests the intermediacy of di-mu-mercapto-bridged binuclear Cu(II) species, in which a concerted two-electron change yields the disulfide and Cu(I) products. Similar hydroxo-bridged intermediates are proposed to account for the transition from first- to second-order transient dependence in cysteine oxidations by Cu(dien)2+ and Cu(Me5dien)2+ as the pH is increased from 5 to 7. Yet another rate law, second order in transient and first order in cysteine, applies in the pH 5.0 oxidation of cysteine by Cu(Me6tren)2+ (k(25 degrees C) 7.5 X 10(7) M-2 sec-1, I = 0.2M). Steric rigidity of this trigonal bipyramidal oxidant evidently protects the coordinated sulfur atom from attack in a RSSR- -forming pathway. Formation of a coordinated disulfide in the rate-determining step is proposed, coupled with attack of a noncoordinated cysteine molecule on a vacated coordination position to stabilize the (Me6tren)Cu(I) product.

Amino Acids, Sulfur↗
PubMed1993

Temperature dependent coordination effects in base-off adenosyl and methylcobalamin by X-ray edge spectroscopy.

Examination of the role of base-off cobalamin species (where the 5,6-dimethylbenzimidazole ligand coordinated to cobalt is detached by protonation of the imidazole nitrogen) in differentiation between homolytic and heterolytic cobalt-carbon bond cleavage mechanisms is a primary step in better understanding B12-dependent enzyme catalysis. X-ray absorption edge spectroscopy provides the first direct structural evidence of five-coordination in base-off adenosyl- and base-off methylcobalamin complexes at room temperature. Integration of 1s-3d pre-edge transitions of the base-off species reveals the dependence of coordination number on temperature. Gradual increases in 1s-3d transition intensities, as the temperature is increased from 180 K to 298 K, reflect a change in the coordination number from six (where a water molecule is presumed to occupy the coordination site vacated by the 5,6-dimethylbenzimidazole ligand) to primarily five-coordinate. Base-off configurations that strengthen the Co-C bond may be both decreasing the tendency for homolytic cleavage while increasing the tendency for hetrolytic Co(I) B12 formation.

Cobamides↗
PubMed1995

Contingent tolerance to the anticonvulsant effects of carbamazepine: relationship to loss of endogenous adaptive mechanisms.

Contingent tolerance to the anticonvulsant effects of carbamazepine on amygdala kindled seizures develops when the drug is repeatedly given prior to but not after the electrical stimulation. Such tolerance can be reversed by kindling the rats for several days without drug or even by continuing to give the drug but after each seizure has occurred. Contingent tolerance can be slowed by reducing the electrical stimulus intensity and by chronic continuous (as opposed to repeated paired) drug administration. Contingent cross-tolerance has been demonstrated from carbamazepine to PK11195 (a drug active at peripheral-type benzodiazepine receptors) and valproate, but not to clonazepam and diazepam (two drugs active at central-type benzodiazepine receptors) or phenytoin. Endogenous physiological changes occur in conjunction with contingent tolerance, exemplified by the decrease in seizure threshold that returns to normal upon reversal of tolerance. We suggest that contingent tolerance is associated with a loss of seizure-induced adaptations, since many biochemical changes that occur following seizures (or in non-tolerant animals given drug after seizures) are not observed in tolerant animals. These include a loss of seizure-induced up-regulation of GABAA receptors and a loss of increases in mRNA expression for corticotropin-releasing-factor (CRF), thyrotropin-releasing-hormone (TRH), neuropeptide Y (NPY), glucocorticoid receptors and brain-derived neurotrophic factor (BDNF). Thus, several putative seizure-induced anticonvulsant adaptations, such as increases in GABAA receptors and TRH and NPY mRNA fail to occur in tolerant animals. These findings are consistent with the novel observations that, paradoxically, seizures themselves appear to facilitate the anticonvulsant effects of carbamazepine or diazepam on amygdala kindled seizures. That is, animals given a 'vacation' from seizures show a decreased response to these agents, a phenomenon we have called the 'time-off seizure' effect. Thus, seizures are postulated to induce adaptive changes that influence seizure thresholds and potentiate the anticonvulsant effects of exogenously administered drugs such as carbamazepine and diazepam. Taken together, these data suggest that seizures are associated with endogenous adaptations lasting days to weeks and that a selective failure of some of these to occur during contingent drug administration may underlie the development of contingent tolerance. These observations suggest tht endogenous illness-related mechanisms may participate both in the therapeutic responses of some agents and that their failure to occur could relate to loss of drug efficacy via tolerance; these processes may reveal new potential targets for therapeutic intervention.

Adaptation, Physiological↗
PubMed1996

Enzyme reactions in a multicompartment electrolyzer with isoelectrically trapped enzymes.

UNLABELLED: The possibility of performing bioconversions under an electric field is here reported. A system is described by which the enzyme is trapped by an isoelectric mechanism between two zwitterionic membranes having pI values encompassing the isoelectric point of the enzyme. The enzyme is loaded into a multicompartment electrolyzer and kept operating under an electric field, which will continuously harvest the reaction product. Since, under focusing conditions, all buffering ions will vacate the reaction chamber at steady state, the buffering ion is trapped into the enzyme chamber by using amphoteric buffers co-isoelectric with the enzyme. As an example of such "isoelectrically immobilized' reactor, the enzyme beta-hydroxysteroid dehydrogenase is blocked into an isoelectric trap delimited by a pI 8.0 and a pI 6.5 membranes. 100 mM histidine (pI 7.47) is co-immobilized by the same isoelectric mechanism into the enzyme chamber. The dehydrocholic acid substrate (3,7,12-trioxo-5 beta-cholanoic acid) and reduced co-factor (NADH) are continuously infused into the enzyme chamber and the product (3 beta-hydroxy-7,12-dioxo- 5 beta-cholanoic acid, a compound of pharmaceutical interest) and the oxidized co-factor (NAD+) collected, separately, into the two neighbouring chambers at the anodic side. ADVANTAGES: in a soluble form, the enzyme maintains the reaction kinetics of the free soluble form. Additionally, the reaction product and exhausted co-factor can be recovered by electrophoretic transport.

3-Hydroxysteroid Dehydrogenases↗
PubMed1981

Postnatal development of ganglionic neurons in the absence of preganglionic input: morphological observations on synapse formation.

Synapse formation during postnatal development of the superior cervical sympathetic ganglion was studied in rats after neonatal ganglionic denervation, when reinnervation was either permitted or prevented. In both groups of operated animals, 90% of the synapses were lost by the fourth postoperative day. In the rats in which reinnervation was permitted, restoration of synapse numbers began by one month after surgery and reached 50% of control by two months. There was no further synapse restoration after this time. In the animals in which reinnervation was prevented, synapse numbers increased, but at all times were approximately 10% of controls. Thus the intrinsic ganglionic synapses underwent their normal postnatal developmental increase in number, but did not sprout to any significant degree in response to the massive deafferentation caused by removal of the preganglionic input. Vacant postsynaptic membrane thickenings (those not apposed by presynaptic terminals) appeared in both experimental groups. There was no significant loss of these vacant thickenings in either group over the course of 3 months. Therefore, the vacated postsynaptic sites do not appear to be recontacted during reinnervation, while the reinnervating axons appear to cause the formation of new postsynaptic sites on the ganglion cells.

Animals↗
PubMed1994

Sprouting of CA3 pyramidal neurons to the dentate gyrus in rat hippocampal organotypic cultures.

The understanding of the mechanisms of a functional synaptic plasticity in the hippocampus has been expanded greatly by the use of in vitro slice preparations. The question addressed in the present study was whether morphological plasticity observed in vivo can also be reproduced in hippocampal slices. In vivo, hippocampal commissural and association fibers are known to sprout and occupy synaptic sites vacated by deafferentation of the dentate gyrus (DG). In hippocampal slice preparations, the major input to the DG is eliminated, so that the DG is deafferented. Might intrinsic neurons sprout to the DG if the slice preparation is maintained for weeks? In this study hippocampal slices obtained from 6-day-old rats were cultured. Stimulation of the dentate stratum moleculare produced antidromic field potentials in the CA3 of the slices cultivated for more than 1 week. The antidromic response was not observed in CA1 pyramidal neurons. The CA3 to DG projection response was also observed in a CA3 mini-slice placed near a co-cultured whole hippocampal slice, when the DG in the latter was stimulated. Moreover, stimulation of the CA3 mini-slice induced synaptic responses in the DG of the whole-slice. The conclusion drawn is that deafferentation could induce axonal sprouting in a neuron-specific manner in hippocampal organotypic culture. This preparation would be potentially useful for the screening of chemical factors that influence sprouting of central neurons.

2-Amino-5-phosphonovalerate↗
PubMed1993

A survey of attitudes, beliefs, and behavior regarding tanning bed use, sunbathing, and sunscreen use.

BACKGROUND: Although cosmetic tanning and unprotected solar exposure are common, little is known about general attitudes, beliefs, and behavior regarding sunbathing, sunscreen use, and tanning salon use. OBJECTIVE: We sought to determine the frequency of UV exposure in a select sample and to assess the knowledge and beliefs of the effects of UV irradiation. METHODS: A written, anonymous questionnaire was distributed to a sample of 477 persons in a shopping mall, at a social gathering, and on a vacation cruise ship. The instrument explored demographic information, sunscreen use, sunbathing habits, tanning bed use, and cutaneous solar effects. RESULTS: Forty-two percent of respondents seldom or never used sunscreen, and 33% sunbathed at least once a week. Although the three sample populations differed in education, sunbathing habits, sunscreen use, and tanning bed use, they were equally informed about UV light hazards. Compared with those who had not used tanning beds, tanning bed users were more likely to be female and more knowledgeable about the long-term effects of UV. Tanning beds were most commonly used in tanning or hair salons, (mean 23 +/- 7 minutes at 2.3 +/- 1.1 times per week). Reported positive psychologic sequelae from tanning bed use were more common than negative physical sequelae. At least 10% would continue to use tanning beds if these were proved to cause skin cancer. CONCLUSION: In this select sample, sunbathing and tanning bed use were common. No group surveyed universally practiced sun protection and avoidance. Clientele of tanning beds may be aware of the damaging effects of the sun, but may not be aware that tanning bed use is associated with skin damage.

Adolescent↗