Biomedical subjects

C A Miller

Publications and source records attributed to C A Miller.

At least 19 recordsLinked to original sources

PubMed2000

A newly emerging toxic dinoflagellate, Pfiesteria piscicida: natural ecology and toxicosis to fish and other species.

Pfiesteria, a toxic dinoflagellate, recently has emerged as a cause of fish kills near the East Coast. Recent research into one species. Pfiesteria piscicida, has revealed a complex life cycle of at least 24 stages. Metamorphosis of one stage to another often depends on presence or absence of fish. Growth of P piscicida is promoted both directly and indirectly by nutrients such as inorganic phosphate and nitrate, as well as organic phosphate, and may be related to effluent-induced blooms. Sewage and agricultural runoff flowing into estuaries often provide these nutrients and may be correlated with the majority of fish kills in the Atlantic coastal region of the US (5). P piscicida is extremely toxic, with a low density capable of killing fish within 3 minutes (1,3,12). Fish exposed to sublethal doses of the toxin have prominent lesions. The syndrome leads to population level death losses and associated economic losses in local fisheries.

Animals↗

PubMed1999

A human aryl hydrocarbon receptor signaling pathway constructed in yeast displays additive responses to ligand mixtures.

An optimized signal transduction pathway that reproduces the response of human aryl hydrocarbon (Ah) receptor to ligands has been established in Saccharomyces cerevisiae. Ligand treatment induced a 50-fold increase in beta-galactosidase activity from a reporter plasmid in yeast engineered to express human Ah receptor and Ah nuclear translocator (Arnt) proteins. The archetypal Ah receptor ligand, 2,3,7,8-tetrachlorodibenzo(p)dioxin, activated Ah receptor and induced lacZ reporter activity at concentrations of >/=0.3 nM. Mixtures of halogenated and nonhalogenated Ah receptor ligands produced additive signaling responses in this yeast bioassay. These results were consistent with the existence of a common binding site and mechanism of ligand-mediated Ah receptor activation. Although yeast have no natural counterpart to the Ah receptor pathway, expression of human Ah receptor and Arnt under the appropriate conditions provides a functional model system for studying Ah receptor activation and signal transduction.

Aryl Hydrocarbon Receptor Nuclear Translocator↗

PubMed1999

The inefficient replication origin from yeast ribosomal DNA is naturally impaired in the ARS consensus sequence and in DNA unwinding.

Ribosomal DNA (rDNA) replication origins of Saccharomyces cerevisiae are known to function inefficiently, both in the context of the tandem rDNA repeats in the chromosome and as single copy autonomously replicating sequences (ARSs) in plasmids. Here we examined components of the rDNA ARS that might contribute to inefficient extrachromosomal replication. Like the efficient H4 ARS, the rDNA ARS requires a match to the 11 bp ARS consensus sequence (ACS) and a broad non-conserved region that may contain multiple elements, including a DNA unwinding element (DUE). Using a single-strand-specific nuclease hypersensitivity assay and by determining the superhelical density required for stable DNA unwinding, we found that the DNA of the rDNA ARS is not as easily unwound as the H4 ARS. Unwinding of the rDNA ARS required additional energy, similar to the unwinding of mutations in the H4 ARS that stabilize the double helix in the DUE region and impair replication. In vivo extrachromosomal replication of the rDNA ARS was cold sensitive, like H4 ARS mutants that require additional energy to unwind the DUE region but unlike the easily unwound, wild-type H4 ARS. Impairment of replication function at reduced temperature suggests that the elevated energy requirement for DNA unwinding inherent in the wild-type rDNA ARS contributes to inefficient replication function. We also examined the essential ACS match in the rDNA ARS, which is known to be imperfect at one position. A point mutation in the essential ACS that corrects the imperfect match increased the efficiency of extrachromosomal replication. Our results reveal that the essential ACS element and DNA unwinding in the rDNA ARS are naturally impaired, suggesting that inefficient function of the rDNA replication origin has a biological purpose.

Cold Temperature↗

PubMed1999

Human c-Jun N-terminal kinase expression and activation in the nervous system.

Differential expression and localization of c-Jun N-terminal kinases (JNKs) in the human brain may reflect transduction of a variety of extracellular stimuli to selective cellular responses. Of the three JNKs, JNK1 and 2 are widely distributed in tissues and JNK3 is predominantly restricted to brain where it is expressed in neurons. Although there is considerable molecular conservation among all three JNKs, we distinguished expression of each by in situ hybridization, immunoblot analysis with a panel of antibodies, and stress-activation using c-Jun as substrate. In the human central nervous system (CNS), there are at least 10 isoforms: JNK3alpha1 and JNK1alpha1 were the major JNK isoforms expressed; JNK2 was not detected. On immunoblots of brain homogenates, antibody selectivity identified JNK3alpha1 as a 45-kDa protein, JNK1alpha1, a slightly lower band at 44 kDa, and a 50-kDa band of unknown specificity. Recombinant human JNK3alpha1, transfected either into CHO, COS-1, or Neuro2A (N2A) cells, was strongly expressed as a 45-kDa protein in each. Transfected JNK3alpha1, and endogenous JNK1, each immunoprecipitated from N2A cells, phosphorylated recombinant forms of human c-Jun. Kinase activity of each JNK was modestly stimulated in N2A cells by anisomycin but not by ceramide, UV irradiation, or heat shock. Endogenous JNK activation, especially at a low level, may reflect a chronic and cumulative stress process that contributes to hyperphosphorylation of cytoskeletal proteins such as those found in Alzheimer's disease (AD), and ultimately, induction of apoptosis.

Animals↗

PubMed1999

Tuberculin skin testing among economically disadvantaged youth in a federally funded job training program.

Low income, medically underserved communities are at increased risk for tuberculosis. Limited population-based national data are available about tuberculous infection in young people from such backgrounds. To determine the prevalence of a positive tuberculin skin test among economically disadvantaged youth in a federally funded job training program during 1995 and 1996, the authors evaluated data from medical records of 22,565 randomly selected students from over 100 job training centers throughout the United States. An estimated 5.6% of students had a documented positive skin test or history of active tuberculosis. Rates were highest among those who were racial/ethnic minorities, foreign born, and (among foreign-born students) older in age (p < 0.001). Weighted rates (adjusting for sampling) were 1.3% for white, 2.2% for Native American, 4.0% for black, 9.6% for Hispanic, and 40.7% for Asian/Pacific Islander students; rates were 2.4% for US-born and 32.7% for foreign-born students. Differences by geographic region of residence were not significant after adjusting for other demographic factors. Tuberculin screening of socioeconomically disadvantaged youth such as evaluated in this study provides important sentinel surveillance data concerning groups at risk for tuberculous infection and allows recommended public health interventions to be offered.

Adolescent↗

PubMed1999

Electrically evoked single-fiber action potentials from cat: responses to monopolar, monophasic stimulation.

We recorded action potentials from single auditory-nerve fibers of cats using monophasic current pulses delivered by a monopolar intracochlear electrode. These simple stimuli provided a means of investigating basic properties and hypotheses of electrical excitation. Standard micropipette recording techniques were used. Responses to anodic (positive) and cathodic (negative) stimulus pulses were recorded separately to evaluate stimulus polarity effects. Mean spike (action potential) latency was polarity dependent, with greater latencies for cathodic stimulation. Threshold stimulus level was also polarity dependent, with relatively lower cathodic thresholds. Both effects are consistent with trends reported in the compound action potential. Variability in single-fiber latency (i.e., jitter) was dependent upon stimulus polarity. In contrast, the slope of single-fiber input-output functions failed to show a clear polarity dependence, although such trends have been seen in the compound action potential data. We also observed a relatively greater degree of adaptation over time with anodic stimulation. Bimodal post-stimulus-time histograms were recorded in a small number (2%) of fibers, supporting the hypothesis that both the peripheral (dendritic) and central axonal processes are excitable with the same stimulus polarity, in a limited number of cases. This observation, together with analyses of interactions among measures of latency, threshold, and jitter, is consistent with the hypothesis that, with monopolar intracochlear stimulation, most fibers are stimulated at axonal (modiolar) sites and a minority of fibers nearest the electrode are stimulable at their peripheral processes.

Action Potentials↗

PubMed1999

An empirically based model of the electrically evoked compound action potential.

The relationship between electrically evoked single-fiber action potentials and the electrically evoked compound action potential of the auditory nerve is of interest to those attempting to model such responses with computational techniques. It also relates to efforts to exploit the gross potentials that can now be recorded by some implantable cochlear prostheses. In this paper, we develop a computational model of the auditory nerve response to single, pulsatile, electrical stimuli based upon the response characteristics obtained from 230 single fibers of 13 cats. These fibers were stimulated by brief (39s) monophasic cathodic stimuli delivered by a monopolar intracochlear electrode. The data were pooled to obtain an estimate of the distribution of fiber thresholds. Post-stimulus time histograms were modeled using Poisson functions and adjusted to account for empirically determined latency and jitter characteristics. The probabilistic nature of single-fiber input-output functions (i.e. Verveen's (1961) 'relative spread') was also modelled. PST histograms from 5000 modelled fibers were then summed and convolved with an estimated 'unit potential' following the method of Goldstein and Kiang (1958). This convolution produced modelled compound action potentials, which were then compared with experimentally obtained data. Manipulations of model parameters affecting threshold, jitter, and relative spread suggest that the most important determinant of the shape of the EAP amplitude-level function is the threshold distribution. A model based solely on threshold distribution produces an EAP input-output function similar to one that accounts for probabilistic single-fiber input-output functions. Discrepancies between these two models do occur if the threshold distribution function is compressed significantly, as might be the case in pathological cochleae with altered distributions or numbers of nerve fibers.

Algorithms↗

PubMed1999

How do cochlear prostheses work?

The past two decades have witnessed a revolution in the treatment of sensorineural hearing loss. Cochlear prostheses have evolved from laboratory experiment to a commercial technology that has benefited over 20,000 people. Paralleling this phenomenal development has been a substantial increase in our understanding of the biophysical, physiological and psychophysical mechanisms underlying the function of these devices.

Animals↗

PubMed1999

A mechanical energy analysis of gait initiation.

The analysis of gait initiation (the transient state between standing and walking) is an important diagnostic tool to study pathologic gait and to evaluate prosthetic devices. While past studies have quantified mechanical energy of the body during steady-state gait, to date no one has computed the mechanical energy of the body during gait initiation. In this study, gait initiation in seven normal male subjects was studied using a mechanical energy analysis to compute total body energy. The data showed three separate states: quiet standing, gait initiation, and steady-state gait. During gait initiation, the trends in the energy data for the individual segments were similar to those seen during steady-state gait (and in Winter DA, Quanbury AO, Reimer GD. Analysis of instantaneous energy of normal gait. J Biochem 1976;9:253-257), but diminished in amplitude. However, these amplitudes increased to those seen in steady-state during the gait initiation event (GIE), with the greatest increase occurring in the second step due to the push-off of the foundation leg. The baseline level of mechanical energy was due to the potential energy of the individual segments, while the cyclic nature of the data was indicative of the kinetic energy of the particular leg in swing phase during that step. The data presented showed differences in energy trends during gait initiation from those of steady state, thereby demonstrating the importance of this event in the study of locomotion.

Adult↗

PubMed1999

Activation of silent replication origins at autonomously replicating sequence elements near the HML locus in budding yeast.

In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity. Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302). Another, ARS303, resides between HML and the CHA1 gene, and its function is not known. Here we further localized and characterized ARS303 and in the process discovered a new ARS, ARS320. Both ARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III. However, at their native locations, where the two ARSs are in a cluster with ARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene, trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), or cis-acting mutations that delete the E and I silencers containing ARS elements. These results suggest that, for the HML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share key cis- and trans-acting components. Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at the HML ARS cluster, as well as at ARS301, the E silencer. Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances from HML in the chromosome. The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins.

Base Sequence↗

PubMed1998

Overlapping 3'-end formation signals and ARS elements: tightly linked but functionally separable.

3'-End formation signals are closely associated with autonomous replicating sequences (ARSs) in Saccharomyces cerevisiae in that ARSs frequently contain signals that direct 3'-end formation (Chen et al., 1996). Mutationally-inactivated ARSs that co-reside with 3'-end formation sequences do not disrupt 3'-end formation, thus demonstrating that replication function does not affect termination function. To test the corollary possibility that 3'-end formation is important for replication function, we made point mutations in ARS305 that increase readthrough of the 3'-end formation signals and determined plasmid replication efficiency. Replication efficiency, as assessed by plasmid stability assays, was not altered by mutations affecting 3'-end formation when transcription through the ARS was either absent or highly-induced. Under conditions of high-level transcription through the ARS, the rate of plasmid loss in both wild-type and mutated terminators increased over five-fold from rates observed during transcriptionally repressed conditions. This result indicates that the native 3'-end formation signal is incapable of protecting the replication function when high levels of transcription are directed into the ARS. Thus, the compact nature of the S. cerevisiae genome, rather than a functional inter-dependence, may account for close association of transcription terminators and ARSs.

Base Sequence↗

PubMed1998

Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids: expressionvectors with bi-directional promoters for use in Saccharomyces cerevisiae.

The pBEVY (bi-directional expression vectors for yeast) plasmids were designed with constitutive and galactose-induced bi-directional promoters to direct the expression of multiple proteins in Saccharomyces cerevisiae . Using human estrogen receptor as a test gene, relatively balanced expression levels from each side of a bi-directional promoter were observed. Expression of a functional heterodimeric transcription factor composed of human aryl hydrocarbon receptor (Ahr) and aryl hydrocarbon receptor nuclear translocator (Arnt) proteins was accomplished using a single pBEVY plasmid. Previous studies suggest that inhibitory cross-talk between the estrogen receptor and the Ahr/Arnt complex may occur and that Hsp90-Ahr complex formation is important for Ahr-mediated signal transduction. Evidence for functional interaction among these proteins was investigated using pBEVY plasmids in a yeast system. No inhibitory cross-talk was observed in signaling assays performed with yeast that co-expressed Ahr, Arnt and estrogen receptor. In contrast, Ahr/Arnt-mediated signal transduction was reduced by 80% in a temperature-sensitive Hsp90 strain grown under non-permissive conditions. We conclude that pBEVY plasmids facilitate the examination of multiple protein interactions in yeast model systems.

Dimerization↗

PubMed1998

A splicing variant of a death domain protein that is regulated by a mitogen-activated kinase is a substrate for c-Jun N-terminal kinase in the human central nervous system.

The mitogen-activated kinase activating death domain protein (MADD) that is differentially expressed in neoplastic vs. normal cells (DENN) was identified as a substrate for c-Jun N-terminal kinase 3, the first demonstration of such an activity for this stress-activated kinase that is predominantly expressed in the brain. A splice isoform was identified that is a variant of MADD. A protein identical to MADD has been reported to be expressed differentially in neoplastic vs. normal cells and is termed "DENN." We demonstrated differential effects on DENN/MADD in a stressed vs. basal environment. Using in situ hybridization, we localized both the substrate and the kinase to large pyramidal neurons in the human hippocampus. It was interesting that, in four of four patients with neuropathologically confirmed acute hypoxic changes, we detected a unique translocation of DENN/MADD to the nucleolus. These changes were apparent only in neurons sensitive to hypoxia. Moreover, in those cells, translocation of the substrate was accompanied by nuclear translocation of JNK3. These findings place DENN/MADD and JNK in important hypoxia insult-induced intracellular signaling pathways. Our conclusions are important for future studies for understanding these stress-activated mechanisms.

Alternative Splicing↗

PubMed1998

Chondroitin sulfate proteoglycan specific to retinal horizontal neurons.

ABSTRACT Proteoglycans (PGs) are a diverse group of highly glycosylated macromolecules that are implicated in the development and maintenance of neuronal circuitry. With its highly ordered, layered structure, the retina ideally serves to define the synthesis, processing, and distribution of these molecules within a specific cellular subpopulation. In retinal sections, monoclonal antibody (MAb) 6A2 immunostained a horizontal cell-specific antigen. Antigen 6A2 was expressed within abundant processes in the outer plexiform layer and in rare neurites that extend across the inner nuclear layer to the inner plexiform layer. Ultrastructurally, the antigen was localized to cisternae within horizontal cell somata, along tubulovesicular structures in dendrites, and in the perisynaptic space encircling presynaptic terminals of the cone photoreceptor triad. These findings suggest that this PG is synthesized within the horizontal cells, transported to the terminals, and released into the extracellular spaces just proximal to the synapse. Based on the focal stain in the adjacent photoreceptor cell, it is possible that antigen is pinocytosed by this cell and is concentrated at the ribbon synapse. In Western immunoblots of retinal homogenates, MAb 6A2 recognized a heterogeneous chondroitin sulfate (CS) PG (CSPG) of approximately 400-500 kDa. After sequential enzymatic removal of CS glycosaminoglycans, a major broad band of 300-500 kDa was identified by MAb 1B5, which detects CSPGs that bear uronic acid linked to unsulfated N-acetylgalactosamine as the initial disaccharide in the CS chain. Localization of this PG around presynaptic terminals of the horizontal neuron and at the ribbon synapse suggests that it may play a modulatory and sustaining role at the synapse.

Animals↗

PubMed1998

Peptides bind to eosinophils in the rat stomach.

BACKGROUND: An immunological role for eosinophils has been well established. However, roles for eosinophils in the physiological functions of the organs they populate are little explored. METHODS: Fixed, frozen, then vibratomed sections of rat stomach were exposed to biotinylated 1-17 gastrin (bG17), biotinylated gastrin-releasing peptide (bGRP), biotinylated neuromedin C (bNC), biotinylated vasoactive intestinal peptide (bVIP), and biotinylated substance P (bSP). Binding sites were identified using an avidin-biotin-glucose oxidase complex and tetranitroblue tetrazolium staining. RESULTS: bG17, bGRP, and bNC all bound to cells in the lamina propria and to a lesser extent in the submucosa. Neither bVIP nor bSP bound to cells in these sections. Stained cells were identified as eosinophils in the light microscope on the basis of their distribution and staining properties using the Luna stain for eosinophils and in the transmission electron microscope (TEM) on the basis of a light/TEM matching process. Plastic sections viewed in the light microscope showed that stain was localized to a granular component in the cytoplasm of the eosinophils. No other cell type, specifically neither mast cells nor plasma cells, stained. G17 competed for the bG17 binding site better than did NC. A competition study in which polyglutamic acid failed to compete with bG17 for the binding site, and the observation that bG17, bGRP, and bNC did not bind to other positively charged sites (e.g., collagen, red blood corpuscles), demonstrated that binding was not due to nonspecific electrostatic interactions alone. Binding of bG17 to a CCK(B)/gastrin-type receptor was ruled out when specific receptor antagonists failed to block binding. CONCLUSIONS: The particulate nature of the binding site suggests a secretory substance. If so, eosinophils might use that substance to destroy, neutralize, or control the activity of peptide hormones bound to it in the extracellular space.

Animals↗

PubMed1998

Frontotemporal dementia: neuropil spheroids and presynaptic terminal degeneration.

We compared the neuropathological changes of 6 patients with clinically diagnosed lobar dementia. In the 4 patients with histopathology consistent with frontotemporal dementia (FTD), moderate neuronal loss contrasted with marked cortical and subcortical gliosis. We found silver-positive, carbohydrate-rich, spheroidal enlargements of presynaptic terminals within the neuropil of specific central nervous system regions. The spheroids were immunopositive for the cytoskeletal proteins tau, the high molecular weight neurofilament subunit (NF-H), and beta-tubulin. No abnormal tau phosphorylation and only rare ubiquitin immunoreactivity were detected in these structures. Carbohydrate modifications, including advanced glycation end products within the spheroid matrix, were confirmed by lectin binding, and by anti-pentosidine immunoreactivity, an indicator of oxidative stress. Ultrastructurally, the spheroids consisted of unmyelinated, membrane-enclosed structures filled with randomly arrayed pairs of filaments, approximately 8 to 10 nm in diameter, that were sparsely labeled with antibodies to unmodified tau (T14) and NF-H. Despite clinical overlap, the central nervous systems of our FTD patients are distinguishable from those of other patients with other lobar dementias, including Pick's disease and corticobasal degeneration, by the absence of abnormally phosphorylated neuronal or glial tau inclusions. The neuropil spheroids are dystrophic changes of certain selectively vulnerable presynaptic terminals, suggesting a retrograde degenerative process in FTD.

Adult↗

PubMed1998

Electrically evoked compound action potentials of guinea pig and cat: responses to monopolar, monophasic stimulation.

We recorded electrically evoked compound action potentials (EAPs) from guinea pigs and cats using monophasic current pulses delivered by a monopolar intracochlear electrode. By using simple stimuli, we sought results that could shed light on basic excitation properties of the auditory nerve. In these acute experiments, the recording electrode was placed directly on the auditory nerve. Responses to anodic and cathodic stimulus pulses were recorded separately to evaluate stimulus polarity effects. Several polarity-dependent properties were observed. Both EAP morphology and latency were polarity-dependent, with greater latencies for cathodic stimulation. Threshold stimulus level was also polarity-dependent, but in different directions in the two species: cats had lower cathodic thresholds while guinea pigs had lower anodic thresholds. We also observed that the slopes of the EAP amplitude-level functions depended upon stimulus polarity. In most cases where EAP saturation amplitude could be measured, that amplitude was similar for anodic and cathodic stimuli, suggesting that either stimulus polarity can recruit all fibers, or at least a comparable numbers of fibers. The common findings (e.g., EAP morphology and polarity-dependent latency) observed in these two species suggest results that can be extrapolated to responses obtained in humans, while the species-specific findings (e.g., dependence of threshold on polarity) may point to underlying anatomical differences that caution against overgeneralization across species. Some of our observations also bear upon hypotheses of how electrical stimuli may excite different sites on auditory nerve fibers.

Action Potentials↗

PubMed1998

It takes two to tango: understanding and acquiring symmetrical verbs.

Children's ability to understand symmetrical verbs was investigated, along with adults' use of linguistic and visual cues to learn novel symmetrical verbs. Symmetrical verbs encode a relationship r between two entities such that X r Y entails Y r X. In Experiment 1, sixteen children (mean age 4;8) acted out two types of sentences with symmetrical and asymmetrical verbs. Eight adult judges viewed videotapes of the children's performance and tried to guess what sentence type was being enacted. Judges' performance was predicted (p < .05) by the verb type, symmetrical or asymmetrical. In Experiment 2, seventy-two adult subjects received visual and linguistic cues to the meanings of novel verbs. Both cue types affected subjects' judgments about whether the new verbs were symmetrical or asymmetrical (p < .05).

Adult↗