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Biomedical subjects

R E Baker

Publications and source records attributed to R E Baker.

At least 37 records · Page 2Linked to original sources
PubMed1997

Cocultured, but not isolated, cortical explants display normal dendritic development: a long-term quantitative study.

Dendritic growth has been studied in long-term organotypic neonatal rat occipital neocortex grown either apart as isolated explants or in tandem as cocultures. Quantitative light microscopic measurement of dendritic and axonal branching patterns within the cortical slice was accomplished using rapid Golgi stained materials. In both isolates and cocultures the overall cellular organization of the slice was maintained over 4 weeks in vitro with morphologically distinguishable pyramidal and nonpyramidal neurons located within the same layers and with the same orientations as observed in situ. Long-term increases in the total length of basal dendrites, apical dendrite and axons were observed only in cocultures and were similar to growth patterns reported for in situ materials. Dendritic growth was mainly due to elongation of terminal dendritic segments. Surprisingly, isolated explants showed no long-term increases in total (basal) dendrites, apical dendrites or axons with time in vitro. A transient decrease in the number of basal dendritic segments and increase in terminal segment lengths at the end of the first week in vitro, however, was observed in nonpyramidal neurons. It is hypothesized that (i) afferent inputs and/or efferent targets develop only in cocultures and provide a crucial conditions for the continued growth of dendritic/axonal arborization for neocortical neurons in vitro, (ii) intrinsic interconnectivity within isolated explants is not sufficient to maintain long-term growth of neuritic arbors, and (iii) remodelling of dendritic arbors within isolated explants occurs at the same time as these explants are showing noticeable increases in the level of spontaneous bioelectric activity, which suggests that dendritic growth and network formation may be function dependent.

Animals↗
PubMed1997

Glutamate dependent dendritic outgrowth in developing neuronal networks of rat hippocampal cells in vitro.

The present study was undertaken to determine whether release of glutamate is capable of influencing dendritic morphology in a developing network of rat hippocampal neurons in vitro. Control cultures developed a dense network of fibers and evinced spontaneous electrical activity from the third day in vitro. Dendrites were examined in cultures maintained for 2 weeks in vitro: the experimental group grown in medium containing the glutamate receptor antagonists AP-5 and DNQX. Dendritic extensions were analyzed as a function of time (days in vitro) using a number of morphometric parameters, vis. the number of processes, the number and length of intermediate and terminal segments, as well as the total length of all segments. We found that the effect of age and treatment was most prominently reflected in the length of the terminal segments. Chronic addition of ionotropic glutamate receptor antagonists from day 2 in culture arrested all dendrite parameters at the prefunctional level. The results suggest that glutamate release is crucial for the onset of dendritic morphological development in hippocampal neurons.

Animals↗
PubMed1997

Plasticity in fast synaptic inhibition of adult oxytocin neurons caused by switch in GABA(A) receptor subunit expression.

We found that magnocellular oxytocin neurons in adult female rats exhibit an endogenous GABA(A) receptor subunit switch around parturition: a decrease in alpha1:alpha2 subunit mRNA ratio correlated with a decrease in allopregnanolone potentiation and increase in decay time constant of the GABA(A) receptor-mediated IPSCs in these cells. The causal relationship between changes in alpha1:alpha2 mRNA ratio and the ion channel kinetics was confirmed using in vitro antisense deletion. Further, GABA(A) receptors exhibited a tonic inhibitory influence upon oxytocin release in vivo, and allopregnanolone helped to restrain oxytocin neuron in vitro firing only before parturition, when the alpha1:alpha2 subunit mRNA ratio was still high. Such observations provide evidence for the physiological significance of GABA(A) receptor subunit heterogeneity and plasticity in the adult brain.

Animals↗
PubMed1995

Suppression of rhythmic discharges of medullary neurones in organotypic cultures of new-born rats by calcium antagonists.

The contribution of transmembrane calcium flux to the generation of periodic bioelectric activity in cultured organotypic medullary tissue of 6 day old rats was determined by adding calcium antagonists (CA) to the recording saline and by lowering the calcium concentration of this saline. Organic CA flunarizine and verapamil (50-100 mumol/l) reversibly suppressed rhythmic discharge and diminished the CO2 response of medullary neurones within 30-60 min. Inorganic CA cobalt and magnesium exerted the same effects within a few minutes. After lowering the calcium concentration rhythmic activity became unstable, but recovered on exposure to increased CO2 concentration, the excitatory effect of which was strongly reduced. These findings point to a significant role for transmembrane calcium flux and intracellular calcium concentration in sustaining both periodic activity and the CO2 response of medullary neurones.

Animals↗
PubMed1995

Antisense oligonucleotide-induced block of individual GABAA receptor alpha subunits in cultured visual cortex slices reduces amplitude of evoked inhibitory postsynaptic currents.

Whole cell patch clamp recordings were made in layer II-IV from organotypic slices of rat primary visual cortex, explanted at postnatal day 6 and maintained in a serum-free medium. Neurons evinced current clamp characteristics typical for stellate cells. Between 7 and 21 days in culture, both glutamate- and GABA-mediated postsynaptic currents were observed. Long-term culturing in the presence of a degenerate 15-mer antisense oligonucleotide directed against the transcripts of all alpha subunits genes of the GABAA receptor resulted in a dose dependent reduction of evoked GABA synaptic currents. This reduction was maximal (80%) at 20 microM. A randomized control oligo had no effect. Evoked glutamatergic excitatory postsynaptic currents were unaffected following oligo treatment. A 15-mer antisense oligo directed against the alpha 1 subunit gave variable effects: in some cells the amplitude of evoked GABAergic inhibitory postsynaptic currents (IPSCs) was reduced by 50-75%, while in other cells recorded from the same slices, there was little or no effect. An antisense oligo, directed against the alpha 2 subunit, however, gave a consistent and robust 80% reduction of the amplitude of evoked IPSCs. A 15-mer 3-base mismatch oligo against alpha 2 had no effect. We conclude that the alpha 2 subunit functions in postsynaptic GABAA receptors located on or close to the cell bodies of stellate cells. The role of the alpha 1 subunit is less clear, but this subunit seems spatially differentiated. The in situ antisense oligo technique should provide further insight into the biophysical and pharmacological consequences of the subunit composition of ligand gated channels at functional synapses.

Animals↗
PubMed1995

Development and isoproterenol-induced regulation of adrenoceptor binding in cultured rat neocortical explants is seen only with the beta-1, not with the beta-2 subtype.

The presence and time-course of beta-adrenoceptor density in cultured explants of neocortex obtained from 6-day-old rat pups were investigated using a [125I]ICYP binding assay. A delayed, but more pronounced, increase in the receptor expression was observed as compared to the situation previously described in vivo. These changes only occurred for the beta 1-subtype of the receptor, whereas the beta 2-subtype binding remained constant up to 3 weeks in vitro. The delay of beta 1-adrenoceptor expression may be due to the incomplete presence of the proper maturational input, and the late enhancement of receptor expression to upregulation related to the absence in vitro of noradrenergic input. Decreased beta-adrenoceptor levels could be induced by chronic treatment of the beta-agonist isoproterenol (1 microM) introduced either for 3 or 13 days. Again, changes in density were found only for the beta 1-adrenoceptor binding sites. There is no reduction of receptor density following return to control conditions for 10 days after a 3-day treatment with isoproterenol, demonstrating the ability of this model to attain its final receptor density notwithstanding the developmental insult.

Animals↗
PubMed1995

Rhythm generation in organotypic medullary cultures of newborn rats.

Organotypic transverse medullary slices (obex level) from six-day-old rats, cultured for two to four weeks in chemically defined medium contained rhythmically discharging neurones which were activated by CO2 and H+. The mechanisms underlying this rhythmicity and the spread of excitation and synaptic transmission within this organotypic tissue were examined by modifying the composition of the external solution. Our findings showed that (1) Exposure to tetrodotoxin (0.2 microM) or to high magnesium (6 mM) and low calcium (0.2 mM) concentrations abolished periodic activity. (2) Neither the blockade of GABAergic potentials with bicuculline methiodide (200 microM) and/or hydroxysaclofen (200 microM) nor the blockade of glycinergic potentials with strychnine hydrochloride (100 microM) abolished rhythmicity. (3) While atropine sulphate (5 microM) was ineffective in modulating periodic discharges nicotine (100 microM) - like CO2-shortened the intervals between the periodic events; hexamethonium (50-100 microM) reduced both periodic and aperiodic activity. (4) Exposure to the NMDA antagonist 2-aminophosphonovaleric acid (50 microM) suppressed periodic events only transiently. In the presence of 2-aminophosphonovaleric acid rhythmicity recovered. However, the AMPA-antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10-50 microM), abolished periodic activity reversibly within less than 5 min. When 6-cyano-7-nitroquinoxaline-2,3-dione and nicotine were administered simultaneously periodic events persisted for up to 10 min. These findings indicate that synaptic excitatory drive is a prerequisite for the generation of rhythmic discharges of medullary neurones in this preparation. This drive may activate voltage-dependent channels or it may facilitate endogenous cellular mechanisms which initiate oscillations of intracellular calcium concentration. To test the latter possibility (5) calcium antagonists were added to the bath saline. The organic calcium antagonists verapamil and flunarizine (50-100 microM each) and the inorganic calcium antagonists cobalt (2 mM) and magnesium (6 mM) suppressed periodic activity and abolished or weakened the chemosensitivity towards CO2/acidosis. (6) Dantrolene (10 microM). an inhibitor of intracellular calcium release decreased the periodicity, while thapsigargin (2 microM) which blocks endoplasmic Ca(2+)-ATPase, transiently accelerated the occurrence of periodic events. (7) Oscillations of intracellular free calcium concentrations in Fura-2 AM-loaded cells were weakened or abolished by cobalt (2 mM). The results of (5)-(7) indicate that transmembrane calcium fluxes as well as intracellular Ca(2+)-release and -clearance mechanisms are a prerequisite for intracellular free calcium oscillations which may be important in the generation of rhythmic discharges in medullary neurones.

Animals↗
PubMed1995

Role of the Saccharomyces cerevisiae general regulatory factor CP1 in methionine biosynthetic gene transcription.

Saccharomyces cerevisiae general regulatory factor CP1 (encoded by the gene CEP1) is required for optimal chromosome segregation and methionine prototrophy. MET16-CYC1-lacZ reporter constructs were used to show that MET16 5'-flanking DNA contains a CP1-dependent upstream activation sequence (UAS). Activity of the UAS required an intact CP1-binding site, and the effects of cis-acting mutations on CP1 binding and UAS activity correlated. In most respects, MET16-CYC1-lacZ reporter gene expression mirrored that of chromosomal MET16; however, the endogenous gene was found to be activated in response to amino acid starvation (general control). The latter mechanism was both GCN4 and CP1 dependent. MET25 was also found to be activated by GCN4, albeit weakly. More importantly, MET25 transcription was strongly CP1 dependent in gcn4 backgrounds. The modulation of MET gene expression by GCN4 can explain discrepancies in the literature regarding CP1 dependence of MET gene transcription. Lastly, micrococcal nuclease digestion and indirect end labeling were used to analyze the chromatin structure of the MET16 locus in wild-type and cep1 cells. The results indicated that CP1 plays no major role in configuring chromatin structure in this region, although localized CP1-specific differences in nuclease sensitivity were detected.

Base Sequence↗
PubMed1995

Total contact casting.

The treatment of diabetic foot ulcerations has been a difficult task for podiatrists. Numerous methods and materials have been used in an attempt to alleviate this frustrating and complex treatment dilemma. However, there is one treatment method that has been used successfully for decades on plantar ulcerations of the neuropathic foot. Total contact casting has been an easily applicable and effective treatment modality for neuropathic ulcerations of the diabetic foot.

Adult↗
PubMed1994

Structural maturation, cell proliferation and bioelectric activity in long-term slice-cultures of immature rat hippocampus.

Explants of transverse slices of the 6-day-old rat hippocampus were grown in a serum-free medium for 2-14 days. Histology performed after various culturing periods demonstrated that these slices maintain a high degree of 3-dimensional organotypy, while undergoing growth and differentiation of the main cellular elements similar to that seen in vivo. Histological indications of continuing cell proliferation were verified by autoradiography showing a labelling of neuroblasts in the dentate gyrus and of glioblasts at the sites of gliogenesis observed in vivo. Spontaneous bioelectric activity and evoked potentials were recorded, both indicating the development of impulse generation and neuronal connectivity within the explant. Silver impregnation and electron microscopic studies lent further support for the presence of neuronal networks intrinsic to the hippocampus. These findings suggest that within the period studied the hippocampal slice cultures mature in a fashion similar to that seen in situ.

Animals↗
PubMed1994

In vivo and in vitro characterization of Allpyral grass pollen extracts.

The Food and Drug Administration, Center for Biologics Evaluation and Research (CBER) has developed methodology to standardize both aqueous and freeze-dried (lyophilized) extracts. Thus far, it has not been determined whether or not this methodology can be used to standardize alum-adsorbed extracts. This study was designed to examine the in vivo and in vitro potency of selected Allpyral grass pollen extracts, including timothy, orchard grass, perennial ryegrass, sweet vernalgrass, and meadow fescue. Puncture testing was performed on highly grass-sensitive subjects with the concentrate of each of the five Allpyral grass extracts. Additionally, puncture testing was done on 22 subjects to compare Allpyral timothy grass with a lyophilized, standardized timothy grass extract. The ID50EAL (Intradermal Dilution for 50 mm sum of Erythema determines the Allergy Unit) skin test method was used to determine allergy units of the Allpyral extracts. Relative potency of the Allpyral timothy extracts to a timothy laboratory standard was determined using an ELISA-inhibition assay. Intradermal tests were also performed to examine the potency of the supernatant obtained after centrifugation of the whole Allpyral timothy extract. The puncture test responses to the Allpyral timothy extracts were less than those to the lyophilized extract. Those 10,000 PNU/mL Allpyral grass pollen extracts tested were determined to contain a calculated 10,000 BAU/mL. By ELISA inhibition, the Allpyral timothy extracts were determined to be approximately 1,000-fold less potent than the laboratory standard. The estimated concentration of the supernatant preparation to elicit a target response was notably (mean = 1,175 times) greater than that of the whole Allpyral timothy extract needed to elicit the same erythema response.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme-Linked Immunosorbent Assay↗
PubMed1993

Mutational analysis of the Saccharomyces cerevisiae general regulatory factor CP1.

The Saccharomyces cerevisiae general regulatory factor CP1, a helix-loop-helix protein that binds the centromere DNA element I (CDEI) of yeast centromeres, is required in yeast for optimal centromere function and for methionine prototrophy. Mutant alleles of CEP1, the gene encoding CP1, were generated by linker insertion, 5'- and 3'-deletion, and random mutagenesis and assayed for DNA binding activity and their ability to confer CP1 function when expressed in yeast. A heterologous CDEI-binding protein, TFEB, was also tested for CP1 function. The results suggested that DNA binding is required for both biological functions of CP1 but is not sufficient. A direct and quantitative correlation was observed between the chromosome loss and nutritional (i.e., Met) phenotypes of strains carrying loss of function alleles, but qualitatively the chromosome loss phenotype was more sensitive to decreased CP1 expression. The data are consistent with a model in which CP1 performs the same general chromatin-related function at centromeres and MET gene promoters and is normally present in functional excess.

Alleles↗
PubMed1992

Effects of spontaneous bioelectric activity and gangliosides on cell survival in vitro.

Chronic suppression of spontaneous bioelectric activity in spinal cord explants in the presence of tetrodotoxin (TTX) during network formation caused a large reduction in cell number (lowered DNA levels). The addition of gangliosides failed to protect against this cell loss. Conversely, the omission of galactose from the growth medium had no effect on DNA levels. It was concluded that the presence or absence of afferent selectivity is unlikely to require the survival of a regionally specific subpopulation of preferred dorsal root ganglion target cells. Neocortical explants also showed a large reduction in DNA levels following chronic TTX treatment, and morphometric analysis confirmed that neuronal survival was affected to the same degree. Chronic ganglioside supplementation failed to influence DNA and cell counts in either control or TTX-treated explants, but one of the added gangliosides (GD1a) stimulated extensive neuritic outgrowth in electrically silenced cultures. Particular ganglioside species, therefore, may exert a growth stimulating influence that can partially compensate for the absence of bioelectric self-stimulation during early development.

Animals↗
PubMed1992

NMDA receptor levels in chronically depolarized long-term neonatal rat neocortical explants.

The levels of the N-methyl-D-aspartate subclass of glutamate receptor were determined in organotypic neocortical explants chronically exposed to a growth medium containing 25 mM potassium (K25). Explants exposed to 25 mM potassium for 2-3 weeks evinced significantly less binding of the non-competitive N-methyl-D-aspartate receptor-associated channel antagonist 125I-MK801 than did age-matched controls. Surprisingly, cultures that were returned to control growth medium for a further 2 or 7 days showed even less binding of the ligand. The Kd values of binding were not affected and were similar to those of fresh postnatal cortex. The maximum number of binding sites did not vary between postnatal day 6 and 14 days in vitro control cultures, but were significantly less than those measured at postnatal day 20 (comparable age: 14 days in vitro). Several conclusions can be drawn from these findings: (i) the density of the N-methyl-D-aspartate subclass glutamate receptor does not attain in vivo levels under the present culturing conditions, but remains at those levels associated with the stage of development at which the tissue was brought into culture, (ii) chronic depolarization results in a drastic reduction in N-methyl-D-aspartate receptor density, which is not compensated for after the return to normal growth conditions, (iii) depolarization selectively inhibits cellular maturation of the neocortex (but not survival, as shown previously), including neurotransmitter receptor production and/or insertion into membranes or assembly.

Animals↗
PubMed1992

Meiosis in Saccharomyces cerevisiae mutants lacking the centromere-binding protein CP1.

CP1 (encoded by the CEP1 gene) is a centromere binding protein of Saccharomyces cerevisiae that binds to the conserved DNA element I (CDEI) of yeast centromeres. To investigate the function of CP1 in yeast meiosis, we analyzed the meiotic segregation of CEN plasmids, nonessential chromosome fragments (CFs) and chromosomes in cep1 null mutants. Plasmids and CFs missegregated in 10-20% of meioses with the most frequent type of aberrant event being precocious sister segregation at the first meiotic division; paired and unpaired CFs behaved similarly. An unpaired chromosome I homolog (2N + 1) also missegregated at high frequency in the cep1 mutant (7.6%); however, missegregation of other chromosomes was not detected by tetrad analysis. Spore viability of cep1 tetrads was significantly reduced, and the pattern of spore death was nonrandom. The inviability could not be explained solely by chromosome missegregation and is probably a pleiotropic effect of cep1. Mitotic chromosome loss in cep1 strains was also analyzed. Both simple loss (1:0 segregation) and nondisjunction (2:0 segregation) were increased, but the majority of loss events resulted from nondisjunction. We interpret the results to suggest that CP1 generally promotes chromatid-kinetochore adhesion.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1992

Possible cross-regulation of phosphate and sulfate metabolism in Saccharomyces cerevisiae.

CP1 (encoded by the gene CEP1) is a sequence-specific DNA binding protein of Saccharomyces cerevisiae that recognizes a sequence element (CDEI) found in both yeast centromeres and gene promoters. Strains lacking CP1 exhibit defects in growth, chromosome segregation and methionine biosynthesis. A YEp24-based yeast genomic library was screened for plasmids which suppressed the methionine auxotrophy of a cep1 null mutant. The suppressing plasmids contained either CEP1 or DNA derived from the PHO4 locus. Subcloning experiments confirmed that suppression correlated with increased dosage of PHO4. PHO4c, pho80 and pho84 mutations, all of which lead to constitutive activation of the PHO4 transcription factor, also suppressed cep1 methionine auxotrophy. The suppression appeared to be a direct effect of PHO4, not a secondary effect of PHO regulon derepression, and was PHO2-dependent. Spontaneously arising extragenic suppressors of cep1 methionine auxotrophy were also isolated; approximately one-third of them were alleles of pho80. While PHO4 overexpression suppressed the methionine auxotrophy of a cep1 mutant, CEP1 overexpression failed to suppress the phenotype of a pho4 mutant; however, a cep1 null mutation suppressed the low inorganic phosphate growth deficiency of a pho84 mutant. The results may suggest that phosphate and sulfate metabolism are cross-regulated.

Acid Phosphatase↗
PubMed1991

Rhythm generation in brainstem cultures grown in a serum-free medium.

Electrophysiological studies were carried out on long term cultured brainstem tissue taken from neonatal rats with the object of investigating mechanisms underlying respiratory rhythm generation. The preparations were derived from 360 microns thick horizontal medullary slices which were explanted into a chemically defined nutrient medium and which remained organotypically intact for ca. 1 month. In 44 of the 50 explants examined both periodic and aperiodic bioelectric activity was detected, the cycle length of the former ranging from 0.5 to 10 s (mean, 2.7 s) at a pH of 7.4 and bath temperature of 32 degrees C. Periodic activity could take several forms, but commonly consisted of regularly repeated, 100-300 ms long, depolarizing (D-) waves or sequences of inhibitory and/or excitatory postsynaptic potentials. Lowering the pH of the superfusate by lowering the bicarbonate concentration, increasing the pCO2 or adding H+ shortened the interval between periodic events, and increased both the amplitude and duration of the D-waves. The interval was also shortened when the bath temperature was increased (Q10: ca.2.5). The mean resting membrane potential of neurons exhibiting periodic activity was -49 mV (n = 62) and not significantly different from that of aperiodically discharging neurons either in the same preparations or in cultured explants from the neocortex. These observations suggest that brainstem cultures constitute a useful 'model' system for studying pH-dependent rhythm generation in small neuronal networks of the medulla.

Action Potentials↗