Biomedical subjects
Publications and source records attributed to D A Wheeler.
We have developed a faculty research interests resource by "mining" MEDLINE for relationships that are not directly queryable through the normal MEDLINE schema. Faculty citations are retrieved and World-Wide Web pages built to interconnect authors, their citations, and the MeSH terms that have been assigned to these citations. The design and development of the resource are discussed and examples of the results illustrated.
Comparison of the primary amino acid sequence of the human X-linked voltage-dependent anion channel, with other sequences in data base searches, identified regions of similarity in streptococcal streptokinase and bovine bactericidal permeability increasing protein. These regions of similarity were in different areas of the protein and were relatively short. However, examination of an empirically derived structural model of the channel showed that each region of similarity in streptokinase and bovine bactericidal permeability increasing protein corresponded to contiguous transmembrane domains within the channel protein. We speculate that these transmembrane domains may be functionally significant for streptokinase and bovine bactericidal permeability increasing protein. These investigations demonstrate the need for incorporation of information regarding secondary, tertiary, and quaternary structures, as well as function, in algorithms used for database searches.
QS-21, a purified Quillaja saponaria saponin immunologic adjuvant, contains two functional groups that we hypothesized to be involved in the adjuvant mechanism of action through charge or Schiff base interaction with a cellular target. Derivatives, prepared by modification of these sites, were prepared and tested for their ability to augment the immunogenicity of the antigen ovalbumin (OVA) in C57BL/6 mice. QS-21 derivatives that were modified at the carboxyl group on an anionic sugar, glucuronic acid, retained adjuvant activity for antibody stimulation, inducing relative increases in antibody titers similar to those induced by QS-21, although the minimum adjuvant dose required for this stimulation was increased several fold relative to the dose of unmodified QS-21. One of these derivatives also retained significant activity for induction of OVA-specific cytotoxic T-lymphocytes. In contrast, QS-21 derivatives modified at an aldehyde on the triterpene did not show adjuvant activity for antibody stimulation or for induction of cytotoxic T-lymphocytes, suggesting that this functional group may be involved in the adjuvant mechanism.
Infection with the human immunodeficiency virus is now a worldwide pandemic, transmitted through contact with infected body fluids, particularly during heterosexual intercourse. Infection in adults is followed by an asymptomatic period that lasts several years, after which continued destruction of the immune system renders the individual susceptible to multiple opportunistic infections. Antiviral therapy has been shown to prolong patient survival. However, viral resistance has limited the options available to clinicians. Until a truly effective antiviral agent or immune-modulator is developed, efforts to change risky sexual practices will remain the most important resource in limiting the spread of this infection.
A powerful and versatile system for the identification of novel response elements for members of the intracellular receptor family is presented as applied to the human estrogen receptor. In the past, a limited number of estrogen response elements (EREs) have been functionally identified in the promoter regions of estrogen-regulated genes. From these a consensus ERE has been defined that is identical to the ERE of the Xenopus laevis vitellogenin gene, i.e., 5'-GGTCA NNN TGACC-3'. In order to investigate without bias the range of sequences that could function as EREs in vivo, we have developed a genetic selection in yeast expressing the human estrogen receptor (hER) and transformed with a random oligonucleotide library in a vector where expression of a selectable marker requires insertion of an upstream activating sequence. More than 1,000,000 transformants were screened and of 726 clones that contained activating sequences, 65 were found to be hormone-dependent. Sequencing revealed that the majority contained at least one 4/5 match to a canonical ERE half-site, but only one contained a full consensus ERE as previously defined. Some contained half-sites arranged as direct repeats. Twelve elements were further characterized to compare estrogen activation in yeast and mammalian cells and in vitro binding to hER. The results of these studies reveal that sequences that bind weakly to hER in vitro are fully functional as EREs in yeast and are conditionally responsive to estrogen in mammalian cells. In addition, an element was identified that is more sensitive to the partial agonist activities of tamoxifen and nafoxidine than is the consensus ERE, indicating that not only promoter context but the sequence of the binding site itself can allow distinction between receptor activated by agonist and that activated by antagonist.
Rapid genomic scanning methods are required to identify expressed sequences and we report an efficient, sensitive and specific approach which relies upon hybridization of an amplified, labeled cDNA library to digested cosmid DNA. We identified expressed sequences within a cosmid in the glycerol kinase (GK) "critical region" of Xp21 that had impressive similarity to prokaryotic GKs. We used this genomic sequence information to clone the human hepatic GK cDNA. Independent confirmation of the identity of this gene was obtained by functional complementation of GK deficient E. coli mutants with a construct containing the complete human X-linked GK coding sequence.
Certain of the rhythm mutations in Drosophila melanogaster lead to arrhythmic locomotor activity (and aperiodic eclosion) in constant conditions. In light-dark (LD) cycles, however, such mutants exhibit clear fluctuations between high levels of activity when the lights are on and much lower ones when they are off. Our data, in contrast to some previous conclusions, strongly suggest that period0 (per0) adults are, in LD conditions, merely being "forced" into exhibiting periodic behavior. These experiments involved application of 8-, 12-, 16-, and 24-hr LD cycles, in which the arrhythmic mutant could have any of these periodicities imposed upon it, whereas wild-type flies tended to exhibit periods of about 24 hr in cycling conditions whose T values were > 8 hr different from 24. In phase-shift experiments, it was found that Drosophila expressing genotypes associated with rhythmicity achieved a 5-hr advance over a 2-day period following an advanced lights-on; per0 adults altered the phase of their locomotor peaks more rapidly. Against a background of the fact that eyeless or blind flies exhibit normal entrainment, it was hypothesized that double-mutant flies--carrying such visual mutations and per0 as well--should not synchronize to LD cycles, if the forced rhythms seen in the latter single-mutant type are mediated solely by light input through the external photoreceptors. Since an appreciable proportion of the double mutants did synchronize (to LD 12:12), it is thus suggested that the visual cues involved in forcing rhythmicity could be input through the same extraocular photoreceptors that, in general, subserve the fly's rhythm system.
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.
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.
Drosophila males modulate the interpulse intervals produced during their courtship songs. These song cycles, which are altered by mutations in the clock gene period, exhibit a species-specific variation that facilitates mating. We have used chimeric period gene constructs from Drosophila melanogaster and Drosophila simulans in germline transformation experiments to map the genetic control of their song rhythm difference to a small segment of the amino acid encoding information within this gene.
We have amplified and sequenced the complete coding region of bovine hexokinase isoenzyme 1 (HK1) from brain RNA with PCR primers selected for sequence conservation. The sequence information was analyzed to evaluate the evolutionary and structure-function relationships among the mammalian and yeast HK isoenzymes. Structure to function analysis identified an unduplicated, invariant N-terminal domain involved in HK1 outer mitochondrial membrane targeting, as well as putative carbohydrate and nucleotide-binding sites in the regulatory and catalytic halves of HK1 essential to enzyme function. The ATP-binding site in the catalytic half of the HK1 protein resembles nucleotide-binding regions from protein kinases, with the single amino acid replacement (lysine to glutamate) in the ATP-binding site of the amino half explaining the loss of HK1 catalytic function in the regulatory domain. Sequence comparisons suggest that the 50-kDa mammalian and yeast glucokinases arose separately in evolution. In addition to providing valuable phylogenetic and structure-function insights, this work provides an efficient strategy for rapid cloning and sequencing of the coding regions for other HKs and related proteins.
Scientific and clinical research in AIDS has not only contributed to an understanding of the disease itself, but also expanded our understanding of issues that span the length and breadth of the medical and biological sciences.
Spectral analyses were applied to the courtship songs of the mutants cacophony (cac), dissonance (diss), fruitless (fru), and period (per), as well as to the double mutant cac diss. Aberrant intervals between song pulses were observed in diss, cac, cac diss, and fru songs. diss males displayed a defect in song hums manifested by an irregular sine wave, although the fundamental frequencies were normal. Sine song frequencies and intrapulse frequencies were aberrant in cac diss males. Two per mutant alleles (pero1 and pers) were associated with normal song [corrected] pulses and hums. These findings are discussed with regard to the mechanisms of song production and the role of these sounds in Drosophila reproduction.
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The clinical and pathologic features observed in five children with spontaneous liver cell adenoma are described. All tumors showed benign biologic behavior. Two patients had untreated tumors that were followed for 5 years and 26 years, respectively, and showed no evidence of malignant transformation; the latter tumor unprecedentedly showed signs of spontaneous regression over time. Unusual physical findings associated with the liver cell adenomas included generalized osteoporosis, urticaria, and koilonychia. Microscopically, the liver cell adenomas were composed of cords, rarely sheets, of neoplastic hepatocytes with negligible pleomorphism and rare to absent mitoses; bile ducts, portal tracts, and central veins were absent. A fibrous capsule was present in all cases. Previously undescribed histologic features of this tumor type included focal extramedullary hematopoiesis and multinucleate giant cell formation in association with tumor necrosis. Benign neoplasms of the type described in this and previous studies probably should be managed conservatively.
P-element-mediated transformations involving DNA fragments from the period (per) clock gene of Drosophila melanogaster have shown that several subsegments of the locus restore rhythmicity to per0 or per- mutants. Such fragments overlap in a genomic region complementary to one transcript, a 4.5-kb RNA which is probably the per message, in that it is necessary and (in terms of expression from this X-chromosomal locus) sufficient for the fly's circadian rhythms. It is also at least necessary for the high-frequency oscillations normally produced by courting males as they vibrate their wings. The entirety of the 4.5-kb transcript is not necessary for rather strong rhythmicity; nor does it seem to be sufficient, in transformants, for wild-type behavioral phenotypes. A 0.9-kb RNA, homologous to genomic region immediately adjacent to the source of the 4.5-kb species, oscillates in its abundance over the course of a day; but coverage of this transcript source in several transformants carrying a per0 mutation--which eliminates the 0.9-kb RNA's oscillation--does not restore rhythmicity. All of the independently isolated arrhythmic mutations tested were covered by the same array of overlapping per+-derived DNA fragments, implying that the only portion of the locus which has mutated to arrhythmicity is complementary to the 4.5-kb transcript.
This study of 17 cases describes a homogeneous clinicopathologic group of tumors previously subsumed under the general term, hepatobiliary cystadenoma. This unique group of neoplasms that the authors have termed "cystadenoma with mesenchymal stroma" (CMS), occurred exclusively in women and showed two essential tumor components: a cyst lining of columnar to cuboidal, mucin secreting epithelium; and a moderately to densely cellular stroma composed of spindle (rarely oval) cells. The stromal element appeared similar to primitive mesenchyme, both on light and electron microscopic study, showing variable differentiation toward fibroblasts, smooth muscle, adipose tissue and capillaries. Cases of hepatobiliary "cystadenoma" in the literature with histology similar to CMS had similar sex and age incidences, while histologically dissimilar cases, lacking the mesenchymal stroma, had significantly different parameters. Four cases of CMS in this series showed malignant change that was manifested as papillary adenocarcinoma, suggesting the need for complete surgical removal of these premalignant neoplasms.