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S S Smith

Publications and source records attributed to S S Smith.

At least 127 records · Page 7Linked to original sources
PubMed1989

Sex steroids modulate motor-correlated increases in cerebellar discharge.

Adult female rats implanted with a microelectrode drive unit were trained to walk on a computer-controlled treadmill apparatus (10 s on every 20 s for 2 h) during recording of single Purkinje neurons in the paravermal area of the anterior cerebellum. Vigorous increases in the firing rate of individual units were found to be correlated with movement of specific limbs in particular stages of the step cycle during treadmill locomotion. Both spontaneous and motor-evoked discharge of individual Purkinje neurons were monitored before and after s.c. injection of either 17 beta estradiol (E2, 100 ng/kg) or progesterone (P, 50 micrograms). The percent increase in firing rate during locomotion versus rest was determined as a measure of the evoked:spontaneous discharge ratio. Drug-induced changes in this ratio indicate differential effects on the individual parameters, rather than simple excitatory or inhibitory effects. For all neurons tested, E2 augmented the movement-evoked discharge over pre-E2 control levels. The onset for this effect occurred at 15 min post-steroid, with a peak response noted at 30-35 min post-steroid. By 60-90 min, a partial recovery of the evoked:spontaneous ratio was noted, although absolute increases in both parameters were still observed, indicating long-term effects on neuronal activity. These effects were independent of the stage of the estrous cycle. In contrast, P decreased absolute firing rates of Purkinje cells during stationary and locomotor phases. However, the evoked:spontaneous ratio was decreased to an even greater degree. The latency for this effect was 9-12 min, with recovery to control levels of response seen at 30 min post-steroid. This response was typical of cells tested on estrus and diestrus 1. Cells tested on proestrus or diestrus 2, when E2 levels are increasing, were not modulated by P using the above paradigm.

Action Potentials↗
PubMed1989

Binding and transport of transthyretin-gold by the endothelium of the rat choriocapillaris.

The photoreceptors of the neural retina require retinol for synthesis of rhodopsin. In the plasma, retinol is bound to retinol binding protein which is carried by transthyretin (TTR; formerly called prealbumin). It is unknown whether, or how, retinol carrier proteins cross the endothelium of the choriocapillaris, the blood supply to the outer neural retina. This was examined in the present study with TTR-gold probes perfused into rats and localized by electron microscopic techniques. TTR-gold, often in clusters, was localized to diaphragmed fenestrae, parajunctional areas, coated pits, transendothelial channels, multivesicular bodies, and to vesicles close to the Golgi apparatus. The probe was also identified at the luminal and abluminal fronts and the interior of transendothelial channels in an apparent sequence of transit. TTR-gold was also found in a series of interconnected vesicles adjacent to the abluminal side of the endothelium. Localizations were not seen when rat albumin fraction V was substituted for TTR and when the rats were perfused with Pronase E before labeling with TTR-gold. These observations indicate that binding and receptor mediated-like transport of TTR by the endothelium of the choriocapillaris is present. This is similar to the processing of heparin-gold by this endothelium.

Animals↗
PubMed1989

Essential fatty acid deficiency delays the onset of puberty in the female rat.

This study assessed the effect of a dietary deficiency in the essential fatty acids (EFA) linoleic and linolenic acids on the onset of female puberty. EFA deficiency was produced in female rats by means of a semipurified diet and was biochemically documented by analyzing serum and erythrocyte fatty acid levels of more than 30 fatty acids, including all members of the n-6 and n-3 series. Levels of linoleic acid (18:2 n-6) and all n-6 derivatives, particularly arachidonic acid, were strikingly reduced. A less pronounced but clear-cut decrease in n-3 fatty acids, including docosahexaenoic acid (22:6 n-3) was also found. The times of puberty and first ovulation, as assessed by the ages at vaginal opening and first diestrus, were significantly delayed in EFA-deficient rats. The mechanisms underlying this delay appear to reside at both hypothalamic and ovarian sites. Simulation of preovulatory plasma estradiol (E2) levels via implantation of E2-containing Silastic capsules evoked a LH surge 30 h later in control juvenile rats, but not in EFA-deficient animals, indicating a delay in the development of the hypothalamic component of E2-positive feedback in the latter group. This delay appears to be due at least in part to reduced prostaglandin E2 (PGE2) synthesis, as the ability of the neurotransmitter norepinephrine to induce PGE2 release from median eminence nerve terminals was markedly reduced in EFA-deficient rats compared with that in controls. The decrease in hypothalamic PGE2 release was related to the EFA deficiency and not to reduced PG synthase activity, as determined by HPLC analysis of PG synthase products derived from exogenous [14C]arachidonic acid. Basal and hCG-stimulated PGE2 synthesis was also compromised in ovaries from EFA-deficient rats. Depressed gonadal function resulting from the EFA deficiency was further evidenced by a reduced gonadotropin receptor content, a blunted E2 response to hCG in vitro, and an increase in mean serum FSH levels. These results suggest that the delay in puberty resulting from EFA deficiency is due to a reduced availability of arachidonic acid for synthesis of bioactive metabolites. This results in delayed development of both the hypothalamic and ovarian components of the reproductive axis.

Animals↗
PubMed1988

Locally applied estrogens potentiate glutamate-evoked excitation of cerebellar Purkinje cells.

Ongoing studies in our laboratory have demonstrated that systemically administered sex steroids 17 beta-estradiol (E2) and progesterone (P) alter cerebellar Purkinje cell responses to the amino acid neurotransmitter glutamate (Glu) in the urethane-anesthetized, ovariectomized adult rat. In the present study, we have examined the effects of locally pressure ejected E2 (0.5 microM) on Purkinje cell responsiveness to microiontophoretically applied Glu. The inactive stereoisomer of E2, 17 alpha-E2 (0.5 microM), estradiol benzoate (EB, 0.5 microM), and estrone (E1, 0.5 microM) were also tested (vehicle: 0.01% propylene glycol-saline, pH 7.4). Extracellular activity of single Purkinje neurons was recorded using multibarrel glass micropipets. Spontaneous firing rate and neuronal responses to microiontophoretic pulses (10 s every 40 s at 10-50 nA) of Glu were examined before, during and after continuous local pressure application of the steroids (1-5 psi, 10-15 min). Local E2 administration increased Glu response by 86% within 2-3 min after the onset of steroid application, with no recovery apparent by 30 min after termination of steroid administration. As such, local E2 application mimicked the effect of systemic injection of this steroid. The inactive estrogen isomer, 17 alpha-E2, failed to significantly enhance Glu responsiveness. Both EB and E1, however, significantly potentiated Glu responsiveness in a manner similar to locally applied E2. In addition, EB administration produced long-lasting increases in background discharge, unlike E2, and eventual recovery of Glu responses to control pre-steroid levels. In summary, this study provides a demonstration of local sex steroid actions on neuronal responsiveness in a model extrahypothalamic CNS area. These effects were specific, as the inactive 17 alpha-E2 isomer did not alter neuronal physiology. The results presented here suggest that the neuronal effects of systemic estrogen may be mediated by local actions of E2 or E1.

Action Potentials↗
PubMed1988

RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase.

In vitro methylation of Bluescribe plasmid DNA (pBS) with human placental DNA methyltransferase to 6% 5-methylcytosine (mC) reduced transformation efficiencies in rglB+ host strains C600 and DS410 by almost 2 orders of magnitude. By contrast, the rglB- derivative of DS410 showed no reduction in transformation efficiency with methylation while the rglB- derivative of C600 was partially tolerant to methylation. Further, we show that the 1.8 kilobase (kb) and 1.2 kb KpnI fragments derived from the human L1 repeat have respectively 18.3% and 2.3% mC in vivo. Using these hyper- and hypo-methylated genomic segments ligated into the pBS plasmid, transformants with the highly methylated 1.8 kb L1 insert were recovered at 17 to 40 fold higher frequency with the rglB- host strains than with the rglB+ hosts. In addition, recombinant phage (lambda 2001) containing inserts of plant genomic DNA with 26.7% mC (from Petunia hybrida) when plated on rglB- hosts gave titres up to 222 times higher than on the rglB+ strains.

Cell Line↗
PubMed1988

Polyamines, DNA methylation and cell differentiation.

The cellular concentration of AdoMet is normally very much higher than that of dcAdoMet, the aminopropyl group donor in polyamine synthesis. However, when cells are depleted of their putrescine and spermidine, i.e. the aminopropyl group acceptors, the dcAdoMet concentration increases dramatically, to a level that may greatly exceed that of AdoMet. Using a highly purified DNA methyltransferase and its preferred substrates, a defined hemimethylated duplex oligodeoxynucleotide or poly(dI-dC)-poly(dI-dC), we demonstrate that dcAdoMet is a poor methyl group donor, and that it starts to inhibit DNA methylation when its concentration exceeds that of AdoMet. At a dcAdoMet/AdoMet ratio of 5:1 there is very little methyl transfer. This study suggests that the antiproliferative and differentiative effects brought about by inhibitors of polyamine synthesis may be partly attributable to dcAdoMet-mediated inhibition of DNA methylation.

Cell Differentiation↗
PubMed1988

Use of etomidate, temporary arterial occlusion, and intraoperative angiography in surgical treatment of large and giant cerebral aneurysms.

The operative management of large and giant aneurysms is complicated by their typically atheromatous and thick walls, frequent intramural thrombosis with calcification, and broad-based necks that often incorporate perforating and other vital vessels. Not infrequently, it is necessary to at least focally arrest the intracranial circulation and open or excise these aneurysms to facilitate vascular reconstruction. This maneuver, in patients whose disease processes have destroyed autoregulatory function or who have inadequate sources of anatomical collateral supply, may cause the threshold for permanent ischemic injury to be exceeded. The authors have recently treated 14 such patients while under electroencephalographic monitoring to document electrical burst suppression induced by the administration of etomidate, followed by temporary clipping to permit vascular repair and intraoperative angiography to document patency of parent arteries. Up to 60 minutes of internal carotid artery occlusion, 35 minutes of middle cerebral artery occlusion, 19 minutes of upper basilar artery occlusion, and 4 1/2 minutes of lower basilar artery occlusion have been well tolerated using this protocol. In such situations, etomidate may be effective in protecting the cerebral circulation without the detrimental cardiotoxicity observed with protective doses of barbiturates.

Adult↗
PubMed1988

Non-small cell lung cancer in autogenous subcutaneous bronchial grafts in dogs.

The progression of preneoplasia into lung cancer can be serially studied in a new canine model that is simpler and more cost effective than previously reported methods of orthotopic endobronchial carcinogenesis. Short segments of bronchus, obtained by pneumonectomy, were placed on the back of 10 dogs in the form of subcutaneous bronchial autografts. These autografts (12 to 14 per dog) became vascularized and lined with normal respiratory epithelium. Four to 12 weeks after autograft implantation, 10% methylcholanthrene in crystalline form was put into 57 autografts and 10% methylcholanthrene in a silicone polymer sustained-release implant was placed into 54 autografts. Ten autografts without carcinogen (one per dog) served as controls. Serial samplings of each autograft during 9 to 97 weeks of carcinogen exposure showed the neoplastic progression from normal bronchial cells to invasive cancer through stages such as atypical squamous metaplasia and carcinoma in situ. To date, cancers have been histologically proved in 60 autografts; 36 were induced by implants and 24 by the crystalline form. Thirty-nine cancers were epidermoid, and the remainder were either adenocarcinomas (n = 3) or poorly differentiated spindle cell cancers (n = 18). The sustained-release implant method resulted in larger autografts with a greater tendency to progress to cancer than the crystalline carcinogens (p greater than 0.025). Therefore, the sustained-release implant is now considered the preferred method. Measurement of nuclear deoxyribonucleic acid by image analysis of nine histologic cancers demonstrated hyperploidy. Deoxyribonucleic acid from the L1 repeated sequence family was demonstrably hypomethylated in spindle cell tumors. Curettement of individual autografts yielded sheets of respiratory epithelium from which 43.5 to 409.5 micrograms of deoxyribonucleic acid was isolated. For the first time, deoxyribonucleic acid from each stage of the neoplastic progression in non-small cell lung cancer is available in adequate quantities for serial biochemical and therapeutic analysis.

Animals↗
PubMed1988

Biochemical and cytogenetic studies of human lung cancers.

In ongoing studies, we have tested resected lung cancers from 41 men and 49 women; of those with primary lung cancer, 46 patients are free of disease and 35 have died of cancer or have persistent disease. Measurements and studies were as follows: total cellular deoxyribonucleic acid content by image analysis (n = 77); total genomic deoxyribonucleic acid methylation state and banding patterns from probed Southern blots (n = 36); radioimmunoassay for motilin, bombesin, gastrin, vasoactive intestinal peptide, and cholecystokinin (n = 18); and cytogenetic analysis (n = 39). All lung cancers were hyperploid. Adenocarcinomas and epidermoid carcinomas were generally hexaploid to nearly septaploid; comparisons by stage and histologic features suggested potential prognostic correlations. There was general hypomethylation of deoxyribonucleic acid (p less than 0.001). Deoxyribonucleic acid digests from restriction endonuclease Hpa II, when probed with deoxyribonucleic acid homologous to KPN, showed banding patterns that separated histologically indistinguishable primary adenocarcinomas and metastatic adenocarcinomas from one another. Cancers studied with radioimmunoassay were all negative for polypeptide hormones. Five cancers grew adequately in vitro to permit study of 190 detailed karyotypes (20 to 50 per tumor). Chromosome modal numbers ranged from 49 to 109. There were from 4 to 20 clearly abnormal marker chromosomes per tumor; abnormality derived from chromosome 1 was prevalent. Ten of 19 tumors xenotransplanted to nude mice were carried through two to five transplant generations without a change in histologic patterns.

Animals↗
PubMed1987

Sex steroid effects on extrahypothalamic CNS. I. Estrogen augments neuronal responsiveness to iontophoretically applied glutamate in the cerebellum.

The purpose of this study was to test whether 17 beta-estradiol (E2) could alter neuronal activity or responsiveness to iontophoretically applied amino acid neurotransmitters in an area not reported to contain classical E2 receptors. Such a region is the cerebellum, which was selected as a model system for these studies because it has been well characterized electrophysiologically. Extracellular activity of cerebellar Purkinje neurons was recorded from urethane-anesthetized, adult, ovariectomized rats using multibarrel glass micropipets. Spontaneous firing rate and responses of single units to microiontophoretic pulses (10 s pulses every 40 s) of GABA (10-50 nA) or glutamate (GLUT, 3-40 nA) were examined before, during and after iontophoretic (0.25 mM 17 beta-estradiol hemisuccinate) or jugular i.v. (100, 300 or 1000 ng/kg 17 beta-estradiol) administration of E2. Both modes of E2 administration resulted in a significant increase in Purkinje cell excitatory responses to GLUT, independent of the direction of change in spontaneous firing rate. This effect was seen as early as one minute after iontophoretic application of E2 and 10-40 min following i.v. E2. In all cases, recovery to the control level of response was not observed by 2 h following E2 administration. 17 alpha-E2 (300 ng/kg) resulted in a less pronounced, transient increase in GLUT response, while a lower dose (100 ng/kg) did not have any effect. Prior administration of the anti-estrogen tamoxifen did not prevent any of the observed E2 effects. In addition, estrogen-priming did not alter E2-induced potentiation of GLUT responses. In contrast to the effect of E2 on GLUT responsiveness, GABA-mediated inhibition of Purkinje cells was either increased, antagonized or unchanged following E2 application. In summary, this study suggests the hypothesis that circulating levels of E2 may alter neuronal sensitivity to specific neurotransmitter substances within the cerebellar circuitry.

Action Potentials↗
PubMed1987

Sex steroid effects on extrahypothalamic CNS. II. Progesterone, alone and in combination with estrogen, modulates cerebellar responses to amino acid neurotransmitters.

In a preliminary report we have shown that both intravenous and local application of progesterone (P) are capable of increasing cerebellar Purkinje cell responsiveness to microiontophoretically applied gamma-aminobutyric acid (GABA) and decreasing responsiveness to glutamate (GLUT) in the urethane-anesthetized, ovariectomized adult rat. In the present study we have examined the time course of effects of several doses of P and different combinations of both E2 and P on responses of individual Purkinje cells to GABA and GLUT. Extracellular activity of single Purkinje neurons was recorded using multibarrel glass micropipets. Spontaneous firing rate and responses of neurons to microiontophoretic pulses (10 s pulses every 40 s) of GABA (10-50 nA) and GLUT (3-40 nA) were examined before and after jugular i.v. administration of P or E2/P combinations to ovariectomized rats. In some cases animals received s.c. injections of E2 (2 micrograms) at 24 and 48 h before the day of recording. This injection schedule results in maximal reproductive effects of P. Within 5-15 min after P administration (5,50 or 500 micrograms) to ovariectomized rats, Purkinje cell responses to GLUT were decreased by 87%, and inhibitory responses to GABA were increased by 50%, with no associated change in spontaneous firing rate. In addition, the magnitude of the change in amino acid response was directly proportional to the dose of P. In most cases, complete recovery was observed 20-45 min after P administration. E2 pretreatment did not alter these P-induced effects. Combinations of E2 (300 ng/kg) and P (50 or 500 micrograms) injected simultaneously resulted in effects on GLUT responsiveness which were similar to those seen with P alone, while effects similar to E2 alone were observed with administration of E2 plus P at 5 micrograms. The administration of a protein synthesis inhibitor, anisomycin (30 mg/kg, i.v.), 20 min before the recording session did not prevent any of the above steroid effects. These results indicate that sex steroids can act to alter neuronal responsiveness to putative neurotransmitters in a CNS region not known to contain steroid receptors and that the particular combination of steroids will determine the neuronal response. These findings further suggest that the observed steroid-induced alterations in Purkinje cell responsiveness do not appear to require genomic mechanisms.

Action Potentials↗
PubMed1987

Human DNA (cytosine-5)methyltransferase selectively methylates duplex DNA containing mispairs.

The presence of the C.C mispair in a defined duplex oligodeoxynucleotide enhanced its capacity to serve as a substrate for highly purified human DNA methyltransferase. Analysis of tritiated reaction products showed that the C.C mispair acted as a "methylation acceptor" in that it was itself rapidly methylated. The m5C.G base pair also enhanced the capacity of the oligodeoxynucleotide to serve as a substrate for the enzyme. However, this complementary base pair was found to act as a "methylation director". That is, the presence of the m5C in one strand induced the enzyme to rapidly methylate at the cytosine residue on the opposite strand in an adjacent C.G base pair.

Base Composition↗
PubMed1987

Structural organization and DNA methylation patterning within the mouse L1 family.

We have studied stable differences in patterns of DNA methylation seen in the repeated sequences of mouse cells. A cloned 1330-base pair fragment of mouse repetitive DNA (pFS-13) was used as a probe in Southern blotting experiments. Mouse spleen and L1210 lymphoma DNA appeared to be normally methylated at HpaII sites probed by this sequence. Friend erythroleukemia cell, and Sp2 cell DNA both showed an abnormal banding pattern in HpaII digests. Hybridization in situ to metaphase chromosomes showed that probed sequences were broadly interspersed along the arms of each mouse chromosome. The DNA sequence of the 1330-base pair insert in the clone was determined; a copy of the R sequence of L1 was found at its 5' end. Walking experiments using M13 subclones from pFS-13 permitted the construction of a map for d(pCCGG) sites at the 3' end of the mouse L1 family. The unmethylated d(pCCGG) sites in Sp2 and Friend cells could then be assigned to polymorphic-repeated sequence groups within L1, homologous to the region spanned by BAM5 and R. Since there are several thousand copies of each of the fragments seen in autoradiographs, these sequences must possess a common methylation state at many genomic locations. Concerted (nonrandom) hypomethylation of certain subfamilies of L1 appears to be a stable characteristic of several cell lineages. These findings suggest that certain L1 families possess commonalities that permit and perhaps require differential DNA methylation in established cell lineages.

Animals↗
PubMed1987

Size of the directing moiety at carbon 5 of cytosine and the activity of human DNA(cytosine-5) methyltransferase.

M13 DNAs in which carbon 5 of each deoxycytidine residue in one strand is replaced with a bulky group are very good substrates for human DNA (cytosine-5) methyltransferase. Rate enhancements of up to 35 fold are obtained depending on the size of the moiety at C-5. The enzyme appears optimally suited to sense a methyl group in one strand at this position. Alkaline density gradient analyses of the distribution of methyl groups applied to 5-BrdCyd or 5-IdCyd substituted DNA reveal that these groups serve to direct the enzyme to methylate the unsubstituted strand.

Cytosine↗