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Biomedical subjects

R E Baker

Publications and source records attributed to R E Baker.

At least 73 records · Page 4Linked to original sources
PubMed1987

Central neuronal responsiveness to sensory ganglion stimulation is correlated with the incidence of spontaneous bioelectric activity in developing spinal cord cultures.

In spinal cord explants co-cultured with dorsal root ganglion cells for 3-4 weeks in a (horse)serum-containing medium, the spread of ganglion-evoked action potentials from monosynaptic innervation sites ("polysynaptic excitability index") was not correlated with the incidence of neuronal "background" discharges. Moreover, chronic exposure of serum-grown cultures to tetrodotoxin (TTX) in a dose sufficient to reversibly block bioelectric activity, failed to significantly affect this index. For explants grown in a chemically defined medium (CDM) similar excitability scores were obtained only if a low level of spontaneous activity was measured. The most active preparations scored considerably higher, with intermediate values being found in the moderately active cultures. Chronic TTX-exposure in developing CDM-grown cultures reduced their excitability scores to the level found in weakly active, untreated, explants despite a normal incidence of spontaneous activity. The present study indicates that low levels of spontaneous activity in untreated explants were associated with a similar sluggishness of DRG-evoked responses as previously observed after chronic treatment with TTX. These results give additional grounds for confidence that this reduced responsiveness of spinal cord neurons to sensory input is indeed attributable to prolonged reduction of centrally generated excitation during development in vitro.

Aging↗
PubMed1987

Gene size differentially affects the binding of yeast transcription factor tau to two intragenic regions.

Yeast transcription factor tau (transcription factor IIIC) specifically interacts with tRNA genes, binding to both the A block and the B block elements of the internal promoter. To study the influence of A block-B block spacing, we analyzed the binding of purified tau protein to a series of internally deleted yeast tRNA(3Leu) genes with A and B blocks separated by 0 to 74 base pairs. Optimal binding occurred with genes having A block-B block distances of 30-60 base pairs; the relative helical orientation of the A and B blocks was unimportant. Results from DNase I "footprinting" and lambda exonuclease protection experiments were consistent with these findings and further revealed that changes in A block-B block distance primarily affect the ability of tau to interact with A block sequences; B block interactions are unaltered. When the A block-B block distance is 17 base pairs or less, tau interacts with a sequence located 15 base pairs upstream of the normal A block, and a new RNA initiation site is observed by in vitro transcription. We propose that the initial binding of tau to the B block activates transcription by enhancing its ability to bind at the A block, and that the A block interaction ultimately directs initiation by RNA polymerase III.

Binding Sites↗
PubMed1986

Gangliosides restore the specificity of afferent projection patterns in spinal cord explants chronically exposed to tetrodotoxin.

Sensory afferent projection patterns within organotypic explants of fetal mouse spinal cord-dorsal root ganglia (SC-DRG) were mapped out histologically using an HRP-staining method. Cultures grown in tetrodotoxin-containing medium with added gangliosides, in contrast to those grown without the addition of these compounds, showed preferential DRG innervation of the dorsal half of the cord. It therefore appears that gangliosides can compensate for the absence of functional activity during the development of selective innervation patterns.

Animals↗
PubMed1986

Effects of tRNATyr point mutations on the binding of yeast RNA polymerase III transcription factor C.

A physical DNA binding assay was employed to analyze the binding of yeast RNA polymerase III transcription factor C (TFIIIC) to tRNA genes. The assay allowed us to measure the equilibrium constants for specific and nonspecific TFIIIC-DNA binding and to assess the effects of tRNATyr-DNA gene promoter mutations on binding. Sequence alterations in the B block element of the promoter greatly affect the equilibrium constant for specific TFIIIC binding (K8). Mutations which decrease tRNATyr homology to the recognized B block consensus sequence drastically reduce K8 (43- to 370-fold), while mutations which increase homology increase K8 (4- to 5-fold). By contrast, point mutations in the A block element of the tRNATyr promoter have less than 2-fold effects on K8; however, total deletion of A block sequences reduces K8 2- to 5-fold. These results indicate that TFIIIC-rTNA gene binding involves interactions with both A block and B block sequences, but B block interactions dominate, and the relative contribution of A block interactions is small. Since A block sequences are absolutely required for active tRNA gene transcription, the binding results suggest that the role of A block sequences in transcription is not confined solely to TFIIIC binding.

Base Sequence↗
PubMed1986

Some functional effects of suppressing bioelectric activity in fetal mouse spinal cord-dorsal root ganglion explants.

Organotypic explants of fetal mouse spinal cord-dorsal root ganglia were grown for 3 weeks in the presence of 10 mM magnesium ion, which effectively eliminated all recordable bioelectric activity throughout the culturing period. When tested in minimal essential medium, the chronically silenced explants had significantly fewer points from which spontaneous neuronal activity could be recorded. In addition, fewer points could be found that showed dorsal root ganglion-evoked responses, resulting from a greater tendency for the spinal cord activity to be restricted to the vicinity of the dorsally entering DRG fibers. These findings, therefore, support the hypothesis that spontaneous bioelectric activity is required for functional as well as structural maturation of neural networks.

Animals↗
PubMed1985

Horseradish peroxidase tracing of dorsal root ganglion afferents within fetal mouse spinal cord explants chronically exposed to tetrodotoxin.

Afferent projection patterns within organotypic explants of fetal mouse spinal cord-dorsal root ganglia (DRG) were mapped out histologically using an HRP-staining method. The role of spontaneous bioelectric activity in the development of cord-DRG connections was studied using tetrodotoxin (TTX) to eliminate action potential discharges in the experimental cultures. Cultures grown in TTX-supplemented medium had a significantly lower proportion of DRG afferents within the dorsal half of the cord explant than did untreated cultures. In addition, afferent fiber entrances were made predominantly on the dorsolateral aspect in control cord explants, in contrast with the more diffuse entrance pattern displayed by the experimental group. Comparison of those cultures in both groups where the fiber entrances were chiefly dorsal revealed a greater tendency in the TTX group for DRG afferents to grow ventrally after penetrating the cord. Thus, it appears that bioelectric activity of the target neurons may be required by DRG afferents for the development of selective innervation patterns.

Action Potentials↗
PubMed1984

Structural features of yeast tRNA genes which affect transcription factor binding.

Transcription of yeast tRNA genes in vitro requires, in addition to RNA polymerase III, two accessory factors which are resolved by ion-exchange chromatography. One of these transcription factors (factor C) binds to tRNA genes. The stability of factor C-tRNA gene complexes is gene-dependent: the tRNAAGGArg gene forms a highly stable complex while tRNA3Leu and tRNATyr gene complexes are unstable under our standard assay conditions. To determine how differences in tRNA gene structure affect factor C binding, mutant tRNATyr genes, internally deleted tRNA3Leu genes and hybrid transcription units containing both tRNATyr and tRNA3Leu segments were compared in their abilities to stably bind factor C. Sequence changes in either of the two highly conserved promoter elements (A block and B block) affect factor C complex stability. Changes towards the consensus sequence increase complex stability while changes away from the consensus sequence drastically reduce stability. Also, the distance separating the A and B blocks affects complex stability; 34-53 bp gives highest stability. These results indicate that the stable binding of transcription factor C to tRNA genes involves interactions with both A block and B block sequences.

Base Sequence↗
PubMed1984

Effects of chronic suppression of bioelectric activity on the development of sensory ganglion evoked responses in spinal cord explants.

Dorsal root ganglion (DRG) afferent terminals were identified, using electrophysiological techniques, within fetal mouse spinal cord cross-sections cultured in vitro. Afferent distribution patterns were monitored in explants grown for 3 to 6 weeks either in a serum-supplemented or in a serum-free, chemically defined medium (CDM). Bioelectrically active control explants from both series were compared with explants which had been reversibly silenced by chronic exposure to tetrodotoxin (TTX). The control (serum-grown) cultures showed a significant dorsal cord innervation preference, whereas in the corresponding TTX series there was an equal dorsoventral distribution. In the CDM series the mean number of DRG evoked responses was lower at first in TTX-grown than in control cultures, but with age in vitro there was a rise in excitability to normal levels. Spontaneous neuronal activity was abnormally low in cultures (serum as well as CDM-grown) which had been exposed to TTX. It is concluded that bioelectric activity may be an important factor in the proper regulation of synaptic connectivity and functional responsiveness in the developing spinal cord.

Animals↗
PubMed1983

Effects of chemical additives on functional innervation patterns in mouse spinal cord-ganglion explants in serum-free medium.

The distribution of sensory evoked bioelectric activity was examined in spinal cord-dorsal root ganglion (SC-DRG) preparations cultured in a chemically defined, serum-free medium (CDM). DRG afferents showed no preferential innervation of dorsal cord regions in this CDM, on the basis of electrophysiological mapping of the distribution of evoked responses at 4 weeks in vitro. Addition of chondroitin sulfate, galactose-1-phosphate or D(+)-galactose (but not glucose-1-phosphate) to the CDM resulted in a significant increase in presumed monosynaptic connections within the dorsal cord, thus mimicking the results observed in serum-supplemented medium [1,6]. Inasmuch as D(+)-galactose bears no negative charges yet restores the selective functional innervation, whereas glucose-1-phosphate (a highly charged molecule) fails to do so, it is concluded that it is galactose utilization, rather than the charged nature of the chondroitin sulfate and galactose-1-phosphate molecules, which is responsible for the effect.

Animals↗
PubMed1983

Effects of gangliosides on the development of selective afferent connections within fetal mouse spinal cord explants.

Monosynaptic evoked responses were used to localize dorsal root ganglion (DRG) afferent terminals within organotypic fetal mouse spinal cord explants which had been chronically exposed to purified bovine gangliosides during early development. Ganglioside grown cultures showed a highly significant increase in dorsal cord innervation preferences in comparison with control cultures. The amount of evoked activity was significantly lower than was observed in controls, suggesting that the formation of 'incorrect' functional connections was blocked by specific chemical factors. Exposure to N-acetylgalactosamine, a major ganglioside amino sugar, also resulted in an increased dorsal cord innervation preference by the DRG afferents.

Acetylgalactosamine↗
PubMed1983

A cross-national study of differences in length of stay of patients with cardiac diagnoses.

To determine the reason for large regional differences in average hospital length of stay shown in federal discharge abstract data, the medical records of 482 cardiac patients from hospitals belonging to two metropolitan area PSROs of Baltimore, Maryland, and 438 cardiac patients from hospitals in the Metropolitan area PSRO from Portland, Oregon, were reviewed, stratified by diagnosis and complications, and compared for length of stay. Cardiac patients were hospitalized between 2.5 and 7 days longer in Baltimore than in Portland. Federal data on length of hospital stay were basically correct for the diagnostic categories studied. Length-of-stay differences could not be explained by patient differences and appeared to be due to differences in physician practice patterns.

Aged↗
PubMed1982

Nucleotide sequences of yeast genes for tRNA(2), tRNA(2) and tRNA(1): homology blocks occur in the vicinity of different tRNA genes.

Three members of a collection of pBR322-yeast DNA recombinant plasmids containing yeast tRNA genes have been analyzed and sequenced. Each plasmid carries a single tRNA gene: pY44, tRNA(2); pY41, tRNA(2); pY7, tRNA(1). All three genes are intronless and terminate in a cluster of Ts in the non-coding strand. The sequence information here and previously determined sequences allow an extensive comparison of the regions flanking several yeast tRNA genes. This analysis has revealed novel features in tRNA gene arrangement. Blocks of homology in the flanking regions were found between the tRNA genes of an isoacceptor family but, more interestingly, also between genes coding for tRNAs of different amino-acid specificities. Particularly, three examples are discussed in which sequence elements in the neighborhood of different tRNA genes have been conserved to a high degree and over long distances.

Journal Article↗
PubMed1982

The termination of cutaneous nerves in the dorsal horn of the spinal cord in normal and in skin-rotated frogs.

Following transganglionic transport of cobaltic lysine applied to the cut end of cutaneous nerves, the central terminal arbours were filled to such an extent that it was possible to visualize the dermatomal projection upon the dorsal horn in unoperated and in skin-rotated frogs. Sensory nerves of the trunk skin terminate in an oval-shaped area which probably corresponds to laminae 1-3 (or 1-4) in the mammalian spinal cord. One type of terminating collateral had a restricted terminal field either in the medial, or in the lateral, part of the oval-shaped area. Another type ended with a large terminal arbour covering almost the total extent of the oval shaped area. These terminal arbours were found for both the dorsal and the ventral cutaneous nerves. Dorsal cutaneous nerves projected preferentially to the lateral part, while ventral cutaneous nerves projected to the medial part, but both nerves sent a significant contingent of collaterals to the whole extent of the oval-shaped area. A rearrangement of the central projection patterns of the dorsal cutaneous nerves, consistently mediating misdirected responses, was not observed in skin-rotated frogs. Collaterals of trunk skin nerves terminated exclusively in the thoracic segments; they did not enter the limb-innervating (brachial and lumbosacral) segments of the spinal cord. The results corroborate the crossed pattern of sensory input in the dermatomal projection. The large, extensively overlapping, areas of termination of individual skin nerves suggest that wiping responses mediated by these nerves should be regarded as complex behaviour released from a preprogrammed centre rather than a reflex arc in which the presence specificity would be determined by wiring specificity.

Animals↗
PubMed1982

Influence of growth medium, age in vitro and spontaneous bioelectric activity on the distribution of sensory ganglion-evoked activity in spinal cord explants.

The role of serum added to the culture medium and of spontaneous bioelectric activity in the development of sensory afferent connections was studied, employing fetal mouse spinal cord explants with attached dorsal root ganglia (DRG) as an in vitro model system. Afferent DRG terminals in the cord explants were localized on the basis of 'fixed-latency' DRG-evoked action potentials, which were anatomically verified in several experiments using horseradish peroxidase histology. In serum-supplemented medium (HSSM), but not in chemically defined medium (CDM), those DRG fibers which grew into the dorsal side of the cord terminated predominantly within the dorsal cord region, and remained there throughout the experimental period (18-33 days in vitro). In contrast, ventrally entering fibers terminated equally in both the dorsal and the ventral cord regions in young cultures (18-24 days in vitro) but were no longer observed after 27 days in vitro. Cultures grown in HSSM with the addition of xylocaine, in order to chronically suppress spontaneous bioelectric activity, essentially corresponded (at 25-32 days in vitro) to the picture seen in the control series at the same age. On the basis of polysynaptic DRG-evoked responses in the cord, developmental changes in local neuronal networks were inferred which resulted in less spread of DRG-evoked activity with age in HSSM, and more spread with age in CDM-grown cultures. It is concluded that for the formation of selective DRG connections in the spinal cord: (i) a serum-borne factor plays a role: and (ii) functional activity is not required.

Animals↗
PubMed1981

Chemically defined medium enhances bioelectric activity in mouse spinal cord-dorsal root ganglion cultures.

Co-cultures of mouse spinal cord with dorsal root ganglion (DRG) cultures were grown either in horse serum (HS)-supplemented medium or in a serum-free, chemically defined medium (CDM). The cytoarchitecture of cord--DRG explants was fully retained in CDM, with little or no distortion due to flattening of the explant, as is invariably observed in HS-supplemented cultures. Functional properties such as bioelectric activity and DRG--spinal cord interconnectivity were well sustained in CDM.

Animals↗
PubMed1981

Peripheral reinnervation patterns and dorsal root ganglion cell topography in skin-grafted Rana pipiens frogs.

Behavioral responses to mechanical stimulation of the skin were observed in unoperated, 180 degrees skin-rotated and sham-grafted Rana pipiens frogs before and after crushing various cutaneous nerve bundles. In the two control groups, wiping responses directed towards the animal's dorsum were mediated solely via dorsomedial (DM) and dorsolateral (DL) nerve trunks. In skin-rotated frogs, DM and DL nerve trunks were responsible for almost all of the misdirected responses elicited from dorsal cutaneous areas. Six frogs (2 sham- and 4 skin-rotated) possessed some areas of plical skin from which responses could only have been mediated via more ventrally located nerve trunks. However, such dorsal expansions of ventral receptive fields never included the midline skin areas from which misdirected responses had been elicited in the intact animal. The topographic arrangement of cells within the DRG appears to be weakly polarized in cobalt-stained sections, but with large individual variations in size and observed number of neuronal profiles. For the skin-rotated group the ratio of cobalt-filled profiles in the dorsal as compared with the ventral half of the ganglion was 1.6, which did not differ significantly from the control value of 1.9. Taken as a whole, the results of the present study indicate that selective (re)innervation of displaced skin by the original nerve fibers cannot explain the origin of misdirected responses in Rana pipiens.

Animals↗
PubMed1981

Immunocytochemical identification of phenylalanine hydroxylase and albumin in cultured hepatoma cells and isolated rat hepatocytes.

Rhodamine-conjugated antibodies specific for phenylalanine hydroxylase and serum albumin were employed as cytochemical probes to identify these two proteins in H4 hepatoma cells and in isolated rat hepatocytes. Each fluorescent antibody stained the cells specifically and in a distinctive manner. In both cell types, albumin staining was discretely localized in cytoplasmic and in H4 cultures varied somewhat from cell to cell. Evidence from cultures of REB15 cells, a strain derived by cloning H4 cells in tyrosine-free medium, suggested that the staining variability of H4 cells could reflect a variability in phenylalanine hydroxylase content. Hydrocortisone-treated H4 cells and REB15 cultures contain increased amounts of phenylalanine hydroxylase; and all cells in the culture appear to be induced by the hormone. Evidence was presented to show that the albumin visualized within the isolated hepatocytes had been synthesized by these cells, and, furthermore, that quantitatively nearly all intracellular albumin in the isolated rat hepatocytes appeared to be entrained in the secretion pathway (analogous data already exist for H4 cells [Baker, R.E., and R. Shiman. 1979. J. Biol. Chem. 254:9633-9639]). By scoring specific fluorescence, 86 and 98% of the H4 cells and 89 and 98% of the isolated hepatocytes were found to contain phenylalanine hydroxylase and albumin, respectively. Therefore, almost all cells in each population appeared to synthesize both proteins. An implication of these findings is that in rat virtually all liver parenchymal cells must synthesize both phenylalanine hydroxylase and albumin.

Animals↗