Showing papers by "University of Colorado Denver published in 1985"

Functional coupling of γ-aminobutyric acid receptors to chloride channels in brain membranes

Abstract: gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, is believed to act by increasing membrane conductance of chloride ions. In this study it was found that GABA agonists increased the uptake of chloride-36 by cell-free membrane preparations from mouse brain. This influx was rapid (less than 5 seconds), and 13 micromolar GABA produced a half-maximal effect. The GABA antagonists (bicuculline and picrotoxin) blocked the effect of GABA, whereas pentobarbital enhanced the action. This may be the first demonstration of functional coupling among GABA and barbiturate receptors and chloride channels in isolated membranes. The technique should facilitate biochemical and pharmacological studies of GABA receptor-effector coupling.

Primary malignant melanoma of the oral cavity. A review of 177 cases

Abstract: Malignant melanoma of the oral cavity is rare, accounting for about 1% to 8% of all melanomas. There have been no prospective studies of melanoma in this location, and all previous papers have reported small numbers of cases or have retrospectively reviewed case reports from the literature. The authors report six new cases and review 171 cases published since the last major review in 1975. The classification of oral melanomas with radial growth phases is discussed.

Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments.

Abstract: In the murine coronavirus mouse hepatitis virus, a single glycoprotein, E2, is required both for attachment to cells and for cell fusion. Cell fusion induced by infection with mouse hepatitis virus strain A59 was inhibited by the addition of monospecific anti-E2 antibody after virus adsorption and penetration. Adsorption of concentrated coronavirions to uninfected cells did not cause cell fusion in the presence of cycloheximide. Thus, cell fusion was induced by E2 on the plasma membrane of infected 17 Cl 1 cells but not by E2 on virions grown in these cells. Trypsin treatment of virions purified from 17 Cl 1 cells quantitatively cleaved 180K E2 to 90K E2 and activated cell-fusing activity of the virions. This proteolytic cleavage yielded two different 90K species which were separable by sodium dodecyl sulfate-hydroxyapatite chromatography. One of the trypsin cleavage products, 90A, was acylated and may be associated with the lipid bilayer. The other, 90B, was not acylated and yielded different peptides than did 90A upon limited digestion with thermolysin or staphylococcal V8 protease. Thus, the cell-fusing activity of a coronavirus required proteolytic cleavage of the E2 glycoprotein, either by the addition of a protease to virions or by cellular proteases acting on E2, which was transported to the plasma membrane during virus maturation. There is a striking functional similarity between the E2 glycoprotein of coronavirus, which is a positive-strand RNA virus, and the hemagglutinin glycoprotein of negative-strand orthomyxoviruses, in that a single glycoprotein has both attachment and protease-activated cell-fusing activities.

Substantia nigra transplants into denervated striatum of the rat: ultrastructure of graft and host interconnections.

Abstract: A number of recent experiments suggest that grafted dopaminergic neurons provide functional input to a host caudoputamen which previously had been deprived of its dopaminergic input. The purpose of the present study was to determine whether tyrosine hydroxylase immunoreactive processes which originate in the graft participate in morphologically identifiable synapses in the host neuropil. Prior to transplantation, adult Sprague-Dawley rats received unilateral injections of 6-hydroxydopamine into the medial forebrain bundle. Animals were screened for the success of striatal denervation by a test of apomorphine-induced rotation. Transplants of fetal substantia nigra then were placed into cavities in the caudoputamen. After a 6–8 month survival period, animals were perfused and prepared for tyrosine hydroxylase immunocytochemistry. No evidence of sprouting of the host catecholoaminergic system was observed, even after long survival times. Both pre- and postsynaptic immunoreactive elements were clearly present in the host caudoputamen. Immunoreactive axons made synaptic contact with unlabeled dendrites; immunoreactive dendrites were postsynaptic to unlabeled axon terminals. The present results suggest that both host-to-graft and graft-to-host synapses are present in the host caudoputamen.

Evidence of aluminum loading in infants receiving intravenous therapy.

Abstract: To investigate the possibility that premature infants may be vulnerable to aluminum toxicity acquired through intravenous feeding, we prospectively studied plasma and urinary aluminum concentrations in 18 premature infants receiving intravenous therapy and in 8 term infants receiving no intravenous therapy. We also measured bone aluminum concentrations in autopsy specimens from 23 infants, including 6 who had received at least three weeks of intravenous therapy. Premature infants who received intravenous therapy had high plasma and urinary aluminum concentrations, as compared with normal controls: plasma aluminum, 36.78 +/- 45.30 vs. 5.17 +/- 3.1 micrograms per liter (mean +/- S.D., P less than 0.0001); urinary aluminum:creatinine ratio, 5.4 +/- 4.6 vs. 0.64 +/- 0.75 (P less than 0.01). The bone aluminum concentration was 10 times higher in infants who had received at least three weeks of intravenous therapy than in those who had received limited intravenous therapy: 20.16 +/- 13.4 vs. 1.98 +/- 1.44 mg per kilogram of dry weight (P less than 0.0001). Creatinine clearances corrected for weight did not reach expected adult values until 34 weeks of gestation. Many commonly used intravenous solutions are found to be highly contaminated with aluminum. We conclude that infants receiving intravenous therapy have aluminum loading, which is reflected in increased urinary excretion and elevated concentrations in plasma and bone. Such infants may be at high risk for aluminum intoxication secondary to increased parenteral exposure and poor renal clearance.

Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories

Abstract: A major problem of cytogenetics testing in mammalian cells is lack of agreement of results among laboratories. Our objective was to develop a sensitive in vitro test protocol that was applicable to large-scale chemical screening and yielded comparable results in two laboratories. We used sister chromatid exchange (SCE) and chromosome aberration (CAb) tests in Chinese hamster ovary (CHO) cells. The initial protocol used standard cell densities, medium, batch of rat liver S9 for metabolic activation; positive, negative, and solvent controls; staining and scoring techniques; and fixation times. Treatment without S9 was for 8-12 hr (CAb) or 26 hr (SCE), and with S9 for 2 hr in serum-free medium. Bromodeoxyuridine (BrdUrd) (10 microM) was added to SCE cultures only, 2 hr after addition of the test chemical. Doses were based on the 50% toxicity level in a preliminary test of cell survival 24 hr after treatment. One hundred cells (CAb) or 50 cells (SCE) were scored from each control and from five dose levels. Five clastogens were tested in the first two-laboratory comparison: mitomycin-C, triethylenemelamine, N-methyl-N'-nitro-N-nitrosoguanidine, cyclophosphamide, and benzo(alpha)pyrene. There was quite good agreement between laboratories. Seventeen compounds were then tested "blind" in the two laboratories. As testing proceeded, some discrepancies occurred between the laboratories, and the protocol was modified in attempts to improve the resolution of marginal responses and make dose selection more consistent. The preliminary test for cell survival was omitted. A 10(5) dose range in a half-log series was tested, and cells were scored at the highest dose at which sufficient mitotic cells were obtained, and at the next two lower doses. By delaying fixation times, SCE and CAb were scored at doses that inhibited cell cycle progression. This protocol gave comparable results in the two laboratories in many cases and by testing up to a maximum dose, limited by solubility and/or toxicity, should detect a high proportion of clastogens and SCE inducers.

Sequence dependence of the curvature of DNA: a test of the phasing hypothesis.

Abstract: Certain DNA molecules derived from both prokaryotic and eukaryotic organisms display markedly abnormal electrophoretic behavior on polyacrylamide gels. These molecules share a common element of sequence which involves collections of A/T residues that are approximately in phase with the helix repeat. This sequence periodicity has led to the suggestion that such phasing is important in generating the abnormal behavior. We have demonstrated that such phasing is, in fact, essential, thus ruling out alternative models which invoke any form of isotropic or centrosymmetric flexibility as the source of the phenomenon. We have also shown that the abnormal behavior is not a simple consequence of marginal thermodynamic stability. The most plausible explanation for the observed behavior is that stable, local distortions of the helix axis result in macroscopic curvature when such distortions are propagated in phase with the helix repeat.

Statistical Techniques in AERJ Articles, 1979–1983: The Preparation of Graduate Students to Read the Educational Research Literature:

Abstract: The statistical techniques used in American Educational Research Journal (AERJ) research articles between 1979 and 1983 were coded by (a) type of technique, and (b) "major" or "minor" importance for comprehending the research findings. Twenty-seven types of technique were identified and further classified as "basic," "intermediate," or "advanced" level, in terms of typical statistics course progression. Of the "major" techniques, 33%, 37%, and 17% were basic, intermediate, and advanced level,, respectively. A review of statistics texts currently in use revert led that basic texts predominate and that the content coverage in both the basic-and intermediate-level texts generally matched well the techniques found in the AERJ articles. The findings suggest that students with both a basic-and intermediate-level knowledge of statistics would understand most of the techniques encountered in AERJ. Other topics addressed included an examination of trends in use of statistical techniques over time, and a comparison...

Ultrastructure of mouse vallate taste buds. I. Taste cells and their associated synapses

Abstract: The ultrastructural features of murine vallate taste bud cells and their associated synapses have been examined in thin and thick sections with conventional transmission electron microscopy and high-voltage electron microscopy. Computer-assisted reconstructions from serial sections were utilized to aid in visualization of taste bud cell-nerve fiber synapses. We have classified taste bud cells on the basis of previously established criteria-namely, size of the nucleus, shape and density of chromatin, density of cytoplasm, and presence or absence of dense-cored or clear vesicles, other cytoplasmic organelles, and synaptic foci. Both dark cells and light cells are present, as well as cells with intermediate morphological characteristics. Synapses were observed from taste bud cells onto nerve fiber processes. In virtually all instances, synapses are associated with the nuclear region of the taste cell. These synapses are characterized by the presence of 40-70 nm clear vesicles embedded in a thickened presynaptic membrane separated from the postsynaptic membrane by a 16-30 nm cleft. Synapses are not unique to any particular cell type. Dark, intermediate, and light cells all synapse onto nerve fibers. Two general types of synapses exist: spot (or macular) and fingerlike. In the latter, the postsynaptic region of the neuronal process protrudes into an invagination of the taste cell membrane. Differences in synaptic morphology are not correlated with taste cell type. In some cases a single taste cell was observed to possess both macular and fingerlike synapses adjacent to one another, forming a synaptic complex onto a single neuronal process. On the basis of the presence of synaptic contacts, we conclude that both "dark" and "light" cells are gustatory receptors.

Susceptibility of Campylobacter isolates to the bactericidal activity of human serum.

Abstract: Although Campylobacter jejuni and related thermophilic organisms are more common human pathogens than are Campylobacter fetus, most bloodstream or systemic isolates are C. fetus. To understand the pathophysiology related to this observation, the authors studied susceptibility to the bactericidal activity of normal human serum of Campylobacter coli, C. jejuni, and C. fetus isolates from feces and blood. In standardized assays, 10 of 15 C. jejuni and related isolates showed 90% kill (mean, 90.6% +/- 5.9); under more stringent conditions, the relatively resistant strains were completely killed. In contrast, all C. fetus strains were highly serum resistant under both standard and stringent conditions. Killing of C. jejuni was ablated by heating serum to 56 C but restored by addition of complement. Both classical and alternative complement pathways may contribute to killing, and adsorption studies demonstrated antibody dependence. Serum resistance may permit systemic infection by C. fetus, whereas complement- and antibody-mediated serum sensitivity of C. jejuni may account for the relative infrequency of systemic invasion.

The symbolic and object play of children with autism: a review

Abstract: The unique characteristics of autistic children's symbolic and object play are presented and discussed in the context of a literature review covering research since 1964. Several theoretical issues are highlighted: the relationship of play in facilitating language and cognition, play as an intervention, and play as an assessment tool. Difficulties in research methodology due to pooling autistic and schizophrenic subject are raised, as well as possible difficulties inherent in ignoring severity levels within the autistic population. The appropriateness of play therapy is questioned, and evidence is presented to provide encouragement for further inquiry into the study of autistic play.

Carnitine deficiency, organic acidemias, and Reye's syndrome.

Abstract: Relative carnitine deficiency is important in the pathophysiology of several disorders, including Reye's syndrome and organic acidemias. In acute clinical crises, carnitine serves as a "buffer," trapping toxic acyl compounds. Mitochondrial failure develops in carnitine deficiency when there is insufficient tissue carnitine available to buffer toxic acyl-CoA metabolites. Toxic levels of acyl-CoA impair the citrate cycle, gluconeogenesis, the urea cycle, and fatty-acid oxidation. Carnitine replacement therapy is safe and induces excretion of toxic acyl groups in the urine.

Augmented growth equation for cell wall expansion.

Abstract: The Growth Equation representing the relative rate of irreversible wall expansion is augmented with an elastic expansion component. Some of the utility of this augmented Growth Equation is demonstrated through selected applications.

Pituitary-gonadal function in Klinefelter syndrome before and during puberty.

Abstract: Serum concentrations of follicle-stimulating hormone, luteinizing hormone, testosterone, and estradiol were determined at intervals before and during puberty in 40 individuals with Klinefelter syndrome (47,XXY karyotype), of whom 27 had been detected in neonatal cytogenetic screening programs. Prior to the appearance of secondary sexual changes, basal serum hormone concentrations and acute responses to stimulation with gonadotropin-releasing hormone and human chorionic gonadotropin were normal. The timing of the onset of clinical puberty was normal. Early pubertal boys showed initial testicular growth and normal serum testosterone levels, while serum follicle-stimulating hormone and estradiol concentrations were significantly elevated. By midpuberty, the Klinefelter subjects were uniformly hypergonadotropic and their testicular growth had ceased. Serum testosterone concentrations after age 15 remained in the low-normal adult range. Serum estradiol levels remained high, irrespective of the presence or absence of gynecomastia. Exaggerated responses to gonadotropin-releasing hormone are seen in pubertal subjects with elevated basal gonadotropin values.

Improved neurologic function after long-term correction of vitamin E deficiency in children with chronic cholestasis.

Abstract: We studied the effect of long-term correction of vitamin E deficiency on neurologic function in 14 children with chronic cholestasis. Vitamin E repletion was achieved in all, either by large oral doses (up to 120 IU per kilogram of body weight per day) or by intramuscular administration of dl-alpha-tocopherol (0.8 to 2.0 IU per kilogram per day). With early institution of therapy, neurologic function remained normal in two asymptomatic children below the age of three years after 15 and 18 months of therapy. Neurologic function became normal in three symptomatic children below age three after 18 to 32 months of therapy. Restitution of neurologic function was more limited in nine symptomatic children 5 to 17 1/2 years old after 18 to 48 months of therapy. We conclude that vitamin E repletion therapy should be initiated at an early age in children with chronic cholestasis complicated by vitamin E deficiency, to prevent irreversible neurologic injury.

Measurement of substance P metabolites in rat CNS.

Abstract: A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.

Topographic and laminar organization of the vagal gustatory system in the goldfish, Carassius auratus

Abstract: The large majority of intraoral taste buds in goldfish are located on the gill arches and on the palatal organ, a muscular organ situated on the roof of the mouth. These taste buds are innervated by branches of the vagus nerve which terminate in a laminated vagal lobe, itself being an enlargement of the special visceral sensory column of the medulla. The tracer horseradish peroxidase (HRP) was used to determine the connectivity of the various branches of the vagus nerve that innervate the oropharyngeal gustatory surfaces. The entire oral cavity is mapped onto the vagal lobe so that the anterior end of the palatal organ and the most anterior gill arch are represented anteriorly in the vagal lobe; progressively more posterior oral structures are represented progressively more posteriorly in the lobe. The medial part of the palatal organ and the opposing gill arch surface, i.e., the ventromedial portion, are represented ventrally in the vagal lobe. The dorsolateral portions of the palatal organ and gill arches are represented dorsomedially in the vagal lobe. The topographic representation of the oral structures is similar for both the motor and sensory systems. In addition to this overall topographic organization, the different oropharyngeal structures are represented differentially in the layers of the vagal lobe. Palatal organ inputs reach layers VI and IX while gill arch inputs terminate in layers II, IV, and IX. The overall organization of the vagal lobe suggests a highly organized reflex system which is involved in the separation of food from substrate, especially during bottom feeding.

Renal cortical brush-border and basolateral membranes: cholesterol and phospholipid composition and relative turnover.

Abstract: A new procedure for the rapid isolation of renal cortical brush-border and basolateral membranes from the same homogenate is described. Brush-border membranes isolated using Mg2+-EGTA precipitation were enriched 18-fold for leucine aminopeptidase and had a recovery of 32.5%. Basolateral membrane fractions were isolated using a discontinuous sucrose gradient and showed an enrichment of 10.7-fold and recovery of 12.8% using (Na+,K+)-ATPase as a marker enzyme. Lipid analysis using two-dimensional TLC separation of phospholipids and gas liquid chromatography for cholesterol showed marked differences in the lipid composition of the brush-border and basolateral membranes. The brush-border membrane had increased sphingomyelin, phosphatidylserine, ethanolamine plasmalogens, and an increased cholesterol-to-phospholipid and sphingomyelin-to-phosphatidylcholine ratio compared to the basolateral membrane. The relative turnover of total membrane and individual phospholipid species using a double isotope ratio method was carried out. Phospholipids were labeled with either phosphorus 32 and 33 or acetate (3H, 1-14C). The relative turnover of phospholipid species and cholesterol differed strikingly. Phosphatidylcholine showed a high turnover, phosphatidylethanolamine and phosphatidylinositol had intermediate values and sphingomyelin, phosphatidylserine and cholesterol had low relative turnover rates. The order of phospholipid class relative turnover was independent of the labeled precursor used. The brush-border membrane had a significantly reduced relative turnover rate for total membrane phospholipids, sphingomyelin and cholesterol compared to the basolateral membrane. These data show marked differences in the lipid composition and relative turnover rates of the phospholipid species of the brush-border and basolateral membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Na channels in skeletal muscle concentrated near the neuromuscular junction

Abstract: Neuronal function depends crucially on the spatial segregation of specific membrane proteins, particularly the segregation associated with sites of synaptic contact. Understanding the factors governing this localization of proteins is a major goal of cellular neurobiology. A conspicuous example of synaptic specialization is the almost exclusive localization of vertebrate skeletal muscle acetylcholine (ACh) receptors to the subsynaptic membrane of the neuromuscular junction (for example, refs 1,2). The localization of other membrane proteins in skeletal muscle has been much less studied, but a knowledge of their distribution is crucial for understanding the factors governing regional specialization. We have explored the distribution in muscle of the voltage-gated Na channel responsible for the action potential using the loose patch-clamp technique, and have measured Na currents in 5-10 micron-diameter membrane patches as a function of distance from the end plate region of snake and rat muscle fibres. Here we report that the Na current density immediately adjacent to the endplate is 5-10-fold higher than at regions away from the endplate. The increased Na current density falls off rapidly with distance, reaching the background level 100-200 micron from the endplate. Although one might expect ACh receptors to be concentrated near the region of ACh release, such a concentration for Na channels, which propagate the impulse throughout the length of the cell, is surprising and suggests that factors similar to those responsible for concentrating ACh receptors at the endplate also operate to concentrate Na channels.