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Biomedical subjects

J C Hall

Publications and source records attributed to J C Hall.

At least 19 recordsLinked to original sources

Circadian oscillations in period gene mRNA levels are transcriptionally regulated.

The period (per) gene is involved in regulating circadian rhythms in Drosophila melanogaster. The per gene is expressed in a circadian manner, where fluctuations in per mRNA abundance are influenced by its own translation product, which also cycles in abundance. Since per gene expression is necessary for circadian rhythmicity, we sought to determine how certain features of this feedback loop operate. The results of this study reveal that fluctuations in per mRNA are primarily controlled by fluctuations in per gene transcription, that per mRNA has a relatively short half-life, and that sequences sufficient to drive per mRNA cycling are present in 1.3 kilobases of 5' flanking sequences. These and other results indicate that the per feedback loop has all of the basic properties necessary to be a component of a circadian oscillator.

Animals

Identification of novel RNA-binding proteins that interact in the coding region of protein D sense RNA in vitro.

Ultraviolet (UV)-cross-linking and sodium dodecyl sulfate(SDS) polyacrylamide gel electrophoretic (PAGE) analysis were used to identify proteins of nuclear and cytosolic (S100) origin that specifically bind to an in vitro transcribed mRNA sequence for protein D. The coding region of the protein D cDNA was subcloned, in vitro transcribed to [32P]RNA, and incubated with nuclear and cytosolic extracts of enzymatically dispersed epididymal cells. As revealed by UV-cross-linking and SDS-PAGE analysis, two proteins exhibiting a molecular weight mass of approximately 2.5 and approximately 35 Kd that specifically recognize and bind to the in vitro transcribed mRNA sequence for protein D. Our findings suggest that the regulation of protein D gene expression in the rat epididymis may involve novel RNA-binding proteins.

Animals

A casemix of patients undergoing abdominal surgery.

OBJECTIVE: To construct a casemix for patients undergoing abdominal surgery. DESIGN: A prospective longitudinal study. Wound infection was chosen as the target adverse event. A cluster analysis identified seven risk factors, which were then incorporated into a Boolean cluster (the casemix). SETTING: Royal Perth Hospital. PATIENTS: 1238 adults. MAIN OUTCOME MEASURES: Wound infection, recurrent intraperitoneal sepsis, hospital stay, admission to the intensive care unit, and death. RESULTS: Measures of the severity of illness and the extent of co-morbidity were more important in determining the risk of a poor outcome than was the identity of the diseased organ. CONCLUSIONS: Estimates of severity of illness and extent of co-morbidity should be included in any casemix based system for patients undergoing abdominal surgery.

Abdomen

Protein D is differentially expressed and regulated in the rat epididymis.

We report the use of a sensitive and specific enyzme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) to study the expression of protein D, a major androgen-regulated sperm-binding glycoprotein at the protein and mRNA level in different anatomical regions of the rat epididymis. The concentration of protein D in the caput, corpus and cauda region of the epididymis was 10.2 +/- 0.67, 7.3 +/- 0.61 and 22.8 +/- 1.34 ng/micrograms total protein, respectively. The total RNA extracted from the caput, corpus and cauda regions of the rat epididymis was amplified by PCR with oligonucleotide primers specific for the 5' and 3' portion of protein D cDNA. Compared to the caput and cauda region, a significant reduction (greater than 82 +/- 3%) in the expression of protein D mRNA levels was observed for corpus epididymal RNA. This data demonstrates regional differences in the concentration of protein D and suggests that protein D expression may be regulated at the level of mRNA within the corpus epididymidis.

Animals

Behavioral and molecular analyses suggest that circadian output is disrupted by disconnected mutants in D. melanogaster.

Mutations in the disconnected (disco) gene act to disrupt neural cell patterning in the Drosophila visual system. These mutations also affect adult locomotor activity rhythms, as disco flies are arrhythmic under conditions of constant darkness (DD). To determine the state of the circadian pacemaker in disco mutants, we constructed with pers double mutants (a short period allele of the period gene) and assayed their behavioral rhythms in light-dark cycles (LD), and their biochemical rhythms of period gene expression under both LD and DD conditions. The results demonstrate that disco flies are rhythmic, indicating that they have an active circadian pacemaker that can be entrained by light. They also suggest that disco mutants block or interfere with elements of the circadian system located between the central pacemaker and its outputs that mediate overt rhythms.

Activity Cycles

Evolution of the threonine-glycine repeat region of the period gene in the melanogaster species subgroup of Drosophila.

The Threonine-Glycine (Thr-Gly) region of the period gene (per) in Drosophila was compared in the eight species of the D. melanogaster subgroup. This region can be divided into a diverged variable-length segment which is flanked by more conserved sequences. The number of amino acids encoded in the variable-length region ranges from 40 in D. teissieri to 69 in D. mauritiana. This is similar to the range found within natural populations of D. melanogaster. It was possible to derive a Thr-Gly "allele" of one species from that of another by invoking hypothetical Thr-Gly intermediates. A phylogeny based on the more conserved flanking sequences was produced. The results highlighted some of the problems which are encountered when highly polymorphic genes are used to infer phylogenies of closely related species.

Amino Acid Sequence

Artificial neural network classification of Drosophila courtship song mutants.

Courtship songs produced by Drosophila males--wild-type, plus the cacophony and dissonance behavioral mutants--were examined with the aid of newly developed strategies for adaptive acoustic analysis and classification. This system used several techniques involving artificial neural networks (a.k.a. parallel distributed processing), including learned vector quantization of signals and non-linear adaption (back-propagation) of data analysis. "Pulse" song from several individual wild-type and mutant males were first vector-quantized according to their frequency spectra. The accumulated quantized data of this kind, for a given song, were then used to "teach" or adapt a multiple-layered feedforward artificial neural network, which classified that song according to its original genotype. Results are presented on the performance of the final adapted system when faced with novel test data and on acoustic features the system decides upon for predicting the song-mutant genotype in question. The potential applications and extensions of this new system are discussed, including how it could be used to screen for courtship mutants, search novel behavior patterns or cause-and-effect relationships associated with reproduction, compress these kinds of data for digital storage, and analyze Drosophila behavior beyond the case of courtship song.

Algorithms

An immunoassay for metolachlor detection in river water and soil.

An indirect enzyme-linked immunosorbent assay (EIA) for metolachlor (2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamid e) detection in river water and soil was developed using serum obtained from rabbits immunized against the acid of metalaxyl ((N-(2,6-dimethylphenyl)-N-(methoxy-acetyl)-DL-alanine methyl ester) conjugated to bovine serum albumin. The assay had a linear working range from 1 to 50 ng/ml with a mean I50 value of 13.6 ng/ml and a lower detection limit of 2.0 ng/ml. Both the mean interwell and interassay coefficients of variation were less than 4% over the range of the standard curves for samples which had been prepared in phosphate buffered saline (PBS), river water, or soil extract. Assay cross-reactivity to the following four structurally related chloro-acetanilide pesticides were: propachlor (0%), metazachlor (0%), alachlor (23%), and metalaxyl (5,000%). Mean recoveries of metolachlor in spiked (2.0 to 32.0 ng/ml range) PBS, river water, and soil extract were 102%, 103%, and 110%, respectively. Soil samples were taken over a 56-d period from field plots treated with metolachlor and analyzed by GC and EIA. The correlation coefficient for comparison of the two methods was 0.96 with the slope of the linear regression line being 0.78. Furthermore, no statistical difference (P less than 0.05) was found between the dissipation curves of metolachlor derived from GC data versus EIA data.

Acetamides

Selective embolisation of intrahepatic aneurysms.

The radiological techniques that can be used to selectively embolise small arteries have improved markedly in the past few years. This article discusses the use of transcatheter embolisation in the management of three patients with aneurysms involving the intrahepatic arterial tree.

Adult

Effects of a simulated microgravity model on cell structure and function in rat testis and epididymis.

A tail-suspension (TS) rat model used to simulate microgravity was tested for its effects on the anatomy, cell structure, and function of the testis and epididymis in sexually mature male rats. Rats suspended for 7 days without inguinal canal ligation exhibited a significant (P less than or equal to 0.05) reduction in testis weight compared with controls (1.55 +/- 0.04 to 1.1 +/- 0.02 g). Except for the liver, epididymis, and adrenals of TS rats and TS rats allowed to recover for 7 days, no significant (P less than or equal to 0.05) change was observed in the weight of other body and accessory sex organs. A histological examination of the testes and epididymides of model animals revealed disorganized seminiferous tubules and accumulation of large multinucleated cells and spermatids in the lumen of the epididymis. A significant (P less than or equal to 0.05) increase in serum luteinizing hormone (53.1 +/- 6.7 to 66.2 +/- 10.1 ng/ml) and follicle-stimulating hormone (257 +/- 25 to 305 +/- 38 ng/ml) was observed in TS nonligated rats, whereas serum prolactin and testosterone levels were observed to decline from 8.3 +/- 1.3 to 5.1 +/- 0.29 and 7.1 +/- 1.3 to 3.8 +/- 0.25 ng/ml, respectively. Decreases in testis protein content and testosterone levels of the testis, interstitial fluid, and epididymis were also observed in model animals. These data demonstrate that the suspension procedure used in the National Aeronautics and Space Administration TS model results in the testis and epididymis translocating into the abdominal cavity, causing cellular degeneration and organ dysfunction.

Animals

Mapping the clock rhythm mutation to the period locus of Drosophila melanogaster by germline transformation.

The Clock (Clk) mutation shortens circadian rhythms of locomotor activity and eclosion from ca. 24 h to 22.5-23 h. Clk was previously mapped, by meiotic recombination, very close to the period(per) locus on the X chromosome. To determine whether Clk is a mutation within the per gene or if the former is separate from the latter, two overlapping genomic fragments were cloned from Clk flies to produce a per-containing 13.2 kb construct, per01 flies (which by themselves are arrhythmic)--when transformed with this construct--expressed short-period rhythms. This indicates that the Clk mutation is contained within this 13.2 kb region and is almost certainly a new "fast-clock" allele of per.

Alleles

The analysis of new short-period circadian rhythm mutants suggests features of D. melanogaster period gene function.

A number of new period gene (per) mutants were generated by in vitro mutagenesis and germ line transformation. Missense mutations were made at amino acid 589, which is altered in the 19 h short-period (per(s)) mutant, and insertion mutations were generated with peptides commonly used for epitope tagging. Most of these new per mutants had short behavioral rhythms. Flies with heteroallelic combinations of these new mutant per genes were found to have "hybrid" periods, i.e., they had values that were usually in between those of the individual alleles. These findings suggest that short-period per mutants are not unusual gain-of-function mutants but rather more traditional loss-of-function mutants that are unable to influence the circadian pacemaker in a proper manner. The data also suggest that the per protein may engage in important intermolecular interactions.

Animals

An antibody to the Drosophila period protein labels antigens in the suprachiasmatic nucleus of the rat.

Cell bodies in the rat suprachiasmatic nucleus (SCN) were labeled with an antibody against a small domain of the period (per) protein, the product of a gene in Drosophila that regulates circadian rhythms. In immunoblots of SCN protein extracts, the antibody recognized a band of approximately 115 kD, as well as a heterogeneous antigen ranging from 160 kD to 170 kD. The antibody was found in previous studies to label putative circadian pacemaker neurons in Aplysia and Bulla, as well as the cellular sites of per expression in flies. Taken together, these results suggest that the region of the per protein recognized by this antibody may be widely conserved in neuronal circadian pacemakers.

Animals

Expression of the period clock gene within different cell types in the brain of Drosophila adults and mosaic analysis of these cells' influence on circadian behavioral rhythms.

The product of the period (per) gene of Drosophila melanogaster is continuously required for the functioning of the circadian pacemaker of locomotor activity. We have used internally marked mosaics to determine the anatomical locations at which per expression is required for adult rhythmicity, and thus where the fly's circadian pacemaker is likely located in this holometabolous insect. We first provide a detailed description of the distribution and nature of per-expressing cells in the fly's CNS. Using an antibody to the per gene product, or to that of a reporter of per expression, in conjunction with an antibody to the embryonic lethal-abnormal visual system (elav) gene product--which is used as a marker of neuronal identity--we have experimentally confirmed previously proposed assignments of per-expressing cells to the neuronal and glial classes. Thus, we found that per expression and elav immunoreactivity colocalized in large cells located in the lateral cortex of the central brain, as well as in more dorsally located cells in the posterior central brain. In contrast, we found that cells located at the margins of the cortex and the neuropil, and within the neuropil, as well as smaller cortical cells found throughout the brain's cortex, were elav negative, supporting the notion that they are glial in nature. Using internally marked mosaics, we find that the pacemaker is located in brain but is not exclusive to the eyes, the ocelli, or the optic lobes, which is consistent with previous reports obtained in this and other insects of this class. Although the pacemaker may be a paired structure, we show that the functioning of one of them is sufficient for rhythmicity. Finally, we report that glial expression is sufficient for some behavioral rhythmicity to be manifest. However, the rhythmicities of animals for which per expression was confined to glia were weak, suggesting that neuronal per expression as well may be required for normal pacemaker function.

Animals

Purification and characterization of N-acetyl-beta-D-hexosaminidase in different anatomical regions of the adult rat epididymis.

The purpose of the present study was to purify and kinetically characterize N-acetyl-beta-D-hexosaminidases A and B (EC 3.2.1.52) from the caput, corpus and caudal regions of the adult rat epididymis. The molecular mass of the purified native enzyme was approximately 250,000 and approximately 223,000 daltons for the A and B isozymes, with a subunit molecular mass of approximately 63,000 and approximately 56,000 daltons, as determined by size exclusion chromatography and gel electrophoresis under reducing conditions. The apparent Michaelis-Menten constant and maximum velocity values were 0.60, 1.55 and 0.68 mM and 0.54, 3.20 and 2.30 microM/min./mg protein for the enzyme purified from the caput, corpus and caudal regions, respectively. These values were determined by using p-nitrophenyl-N-acetyl-beta-D-glucosaminide as the substrate. These data suggest that the enzyme may be more active in the corpus region of the epididymis than in the caput and caudal regions.

Animals

The period gene encodes a predominantly nuclear protein in adult Drosophila.

The period gene of Drosophila melanogaster (per) is important for the generation and maintenance of biological rhythms. Previous light microscopic observations indicated that per is expressed in a variety of tissues and cell types and suggested that the per protein (PER) may be present in different subcellular compartments. To understand how PER influences circadian rhythms, it is important to define its subcellular location, especially in adult flies where inducible promoter experiments suggested that it is most relevant to circadian locomotor activity rhythms. To this end, we report the results of an immunoelectron microscopic analysis of wild-type flies and per-beta-galactosidase (beta-gal) fusion gene transgenics using a polyclonal anti-PER antibody or an anti-beta-gal antibody, respectively. Most of the PER antigen and the fusion gene product were located within nuclei, suggesting that PER acts in that subcellular compartment to affect circadian rhythms. The results are discussed in terms of per's possible biochemical functions.

Animals