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Biomedical subjects

D Brown

Publications and source records attributed to D Brown.

At least 307 records · Page 17Linked to original sources
PubMed1997

Vacuolar H+-ATPase in ocular ciliary epithelium.

The mechanisms controlling the production of aqueous humor and the regulation of intraocular pressure are poorly understood. Here, we provide evidence that a vacuolar H+-ATPase (V-ATPase) in the ocular ciliary epithelium is a key component of this process. In intracellular pH (pHi) measurements of isolated ciliary epithelium performed with 2',7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF), the selective V-ATPase inhibitor bafilomycin A1 slowed the recovery of pHi in response to acute intracellular acidification, demonstrating the presence of V-ATPase in the plasma membrane. In isolated rabbit ciliary body preparations examined under voltage-clamped conditions, bafilomycin A1 produced a concentration-dependent decrease in short-circuit current, and topical application of bafilomycin A1 reduced intraocular pressure in rabbits, indicating an essential role of the V-ATPase in ciliary epithelial ion transport. Immunocytochemistry utilizing antibodies specific for the B1 isoform of the V-ATPase 56-kDa subunit revealed localization of V-ATPase in both the plasma membrane and cytoplasm of the native ciliary epithelium in both rabbit and rat eye. The regional and subcellular distribution of V-ATPase in specific regions of the ciliary process was altered profoundly by isoproterenol and phorbol esters, suggesting that change in the intracellular distribution of the enzyme is a mechanism by which drugs, hormones, and neurotransmitters modify aqueous humor production.

Animals↗
PubMed1997

Modelling the activity of the Ultrabithorax parasegment-specific regulatory domains around their anterior boundaries.

The Drosophila Ultrabithorax (Ubx) gene was one of the first homeotic genes to be characterised and it specifies the phenotypic characteristics of parasegments 5 and 6. The intricate pattern of Ubx expression in these parasegments is conferred by enormous regulatory regions which, together with the structural gene, constitute the Ubx domain. A genetic, "open-for-business", model based on classical mutational analysis proposed that the regulatory regions were in fact composite structures, consisting of parasegment-specific regulatory domains that contain arrays of cell-specific enhancers which drive the intricate pattern of Ubx expression. Here, we propose a molecular mechanism, based on a survey of numerous transgenic studies, of the setting up of these parasegment-specific regulatory domains in the appropriate parts of the late blastoderm embryo. We construct a mathematical model of this mechanism using the molecular characteristics of a single initiator element, the distributions of segmentation gene products and simple competitive binding equations. We attempt to fit this mathematical model to the idealised patterns of activity and closure of the parasegment-specific domains derived from the genetic model. The resulting fitted pattern of activity and closure obtained with the simplest version of our competitive binding model shows some deviations from the idealised pattern based on the genetic model. Comparison of the predictions with recent experimental evidence suggests that the competitive binding model more accurately reflects certain features of the Ubx expression pattern--features not predicted by the genetic model.

Animals↗
PubMed1997

Prognostic significance of Bcl-2 expression and p53 nuclear accumulation in colorectal adenocarcinoma.

The products of bcl-2 and p53 genes are involved in the regulation of apoptosis and proliferation and have been associated with prognosis in several malignancies, including colorectal adenocarcinoma. Although 2 European studies have reported a prognostic significance of Bcl-2 expression in colorectal adenocarcinomas, a study from the United States did not observe such an association. Therefore, we used immunohistochemistry to evaluate the prognostic significance of Bcl-2 expression, p53 nuclear accumulation and their concomitant expression in 134 US patients with colorectal adenocarcinoma. Antigen retrieval was required for adequate detection of Bcl-2 expression. Fifty percent of the colorectal tumors were classified as expressing Bcl-2, and Bcl-2 expression was associated with longer patient survival. Antigen retrieval was not necessary for detecting nuclear accumulation of p53 by immunohistochemistry. Nuclear accumulation of p53 was detected in 44% of colorectal adenocarcinomas and was associated with decreased patient survival. Tumors that did not express detectable levels of Bcl-2 but exhibited nuclear accumulation of p53 were associated with the shortest patient survival (log rank, p = 0.001). Multivariate Cox regression analysis demonstrated that Bcl-2 expression (p = 0.018), p53 nuclear accumulation (p = 0.024) and regional lymph-node metastasis (p = 0.005) were independent prognostic factors. Although a trend toward an inverse correlation between Bcl-2 and p53 expression was observed, the prognostic value of Bcl-2 expression was independent of p53 status. Thus, assessment of both Bcl-2 and p53 status may be valuable in predicting the prognosis of patients with colorectal adenocarcinomas.

Aged↗
PubMed1997

Single-stranded RNA recognition by the bacteriophage T4 translational repressor, regA.

The T4 protein, RegA, is a translational repressor that blocks ribosome binding to multiple T4 messages by interacting with the mRNAs near their respective AUG start codons. Other than the AUG, there are no obvious similarities between the affected mRNAs. High affinity RNA ligands to RegA were isolated using SELEX (systematic evolution of ligands by exponential enrichment). The selected RNAs exhibited the consensus sequence 5'-AAAAUUGUUAUGUAA-3'. The AUG was invariant, suggesting that it is the primary effector of binding specificity. The UU immediately 5' to the AUG and the upstream poly(A) tract were highly conserved among the selected RNAs. Boundary and footprinting experiments are consistent with the consensus sequence defining the RegA-binding site. Interestingly, chemical modification and nuclease digestion data indicate that the RNA-binding site is single-stranded, as if RegA discriminates between targets based on their primary sequence, not their secondary structure. Minor variations from the consensus at positions other than the universally conserved AUG have little effect on RegA binding, but accumulation of mutations has a profound effect on the interaction. Comparison of the in vivo targets for RegA to the SELEX-generated consensus suggests a repression pattern whereby the translation of individual messages is sequentially halted until the least similarly affected message, the regA gene itself, is repressed.

Bacteriophage T4↗
PubMed1997

The nucleotide sequence of Saccharomyces cerevisiae chromosome IX.

Large-scale systematic sequencing has generally depended on the availability of an ordered library of large-insert bacterial or viral genomic clones for the organism under study. The generation of these large insert libraries, and the location of each clone on a genome map, is a laborious and time-consuming process. In an effort to overcome these problems, several groups have successfully demonstrated the viability of the whole-genome random 'shotgun' method in large-scale sequencing of both viruses and prokaryotes. Here we report the sequence of Saccharomyces cerevisiae chromosome IX, determined in part by a whole-chromosome 'shotgun', and describe the particular difficulties encountered in the random 'shotgun' sequencing of an entire eukaryotic chromosome. Analysis of this sequence shows that chromosome IX contains 221 open reading frames (ORFs), of which approximately 30% have been sequenced previously. This chromosome shows features typical of a small Saccharomyces cerevisiae chromosome.

Base Composition↗
PubMed1997

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIII.

Systematic sequencing of the genome of Saccharomyces cerevisiae has revealed thousands of new predicted genes and allowed analysis of long-range features of chromosomal organization. Generally, genes and predicted genes seem to be distributed evenly throughout the genome, having no overall preference for DNA strand. Apart from the smaller chromosomes, which can have substantially lower gene density in their telomeric regions, there is a consistent average of one open reading frame (ORF) approximately every two kilobases. However, one of the most surprising findings for a eukaryote with approximately 6,000 genes was the amount of apparent redundancy in its genome. This redundancy occurs both between individual ORFs and over more extensive chromosome regions, which have been duplicated preserving gene order and orientation. Here we report the entire nucleotide sequence of chromosome XIII, the sixth-largest S. cerevisiae chromosome, and demonstrate that its features and organization are consistent with those observed for other S. cerevisiae chromosomes. Analysis revealed 459 ORFs, 284 have not been identified previously. Both intra- and interchromosomal duplications of regions of this chromosome have occurred.

Base Composition↗
PubMed1997

Cloning and characterization of the urea transporter UT3: localization in rat kidney and testis.

Urea transport in the kidney plays an important role in urinary concentration and nitrogen balance. Recently, three types of urea transporters have been cloned, UT1 and UT2 from rat and rabbit kidney and HUT11 from human bone marrow. To elucidate the physiological role of the latter urea transporter, we have isolated the rat homologue (UT3) of HUT11 and studied its distribution of expression and functional characteristics. UT3 cDNA encodes a 384 amino acid residue protein, which has 80% identity to the human HUT11 and 62% identity to rat UT2. Functional expression in Xenopus oocytes induced a large (approximately 50-fold) increase in the uptake of urea compared with water-injected oocytes. The uptake was inhibited by phloretin (0.75 mM) and pCMBS (0.5 mM) (55 and 32% inhibition, respectively). Northern analysis gave a single band of 3.8 kb in kidney inner and outer medulla, testis, brain, bone marrow, spleen, thymus, and lung. In situ hybridization of rat kidney revealed that UT3 mRNA is expressed in the inner stripe of the outer medulla, inner medulla, the papillary surface epithelium, and the transitional urinary epithelium of urinary tracts. Co-staining experiments using antibody against von Willebrand factor showed that UT3 mRNA in the inner stripe of the outer medulla is expressed in descending vasa recta. These data suggest that UT3 in kidney is involved in counter current exchange between ascending and descending vasa recta, to enhance the cortico-papillary osmolality gradient. In situ hybridization of testis revealed that UT3 is located in Sertoli cells of seminiferous tubules. The signal was only detected in Sertoli cells associated with the early stages of spermatocyte development, suggesting that urea may play a role in spermatogenesis.

Amino Acid Sequence↗
PubMed1997

A PMLRARalpha transgene initiates murine acute promyelocytic leukemia.

The malignant cells of acute promyelocytic leukemia (APL) contain a reciprocal chromosomal translocation that fuses the promyelocytic leukemia gene (PML) with the retinoic acid receptor alpha gene (RAR alpha). To test the hypothesis that the chimera PMLRAR alpha plays a role in leukemogenesis, we expressed a PMLRAR alpha cDNA in myeloid cells of transgenic mice. PMLRAR alpha transgenic mice exhibited impaired neutrophil maturation early in life, which progressed at a low frequency over the course of several months to overt APL. Both the preleukemic state and the leukemia could be transplanted to nontransgenic mice, and the transplanted preleukemia could progress to APL. The APL recapitulated features of the human disease, including a response to retinoic acid. Retinoic acid caused the leukemic cells to differentiate in vitro and in vivo, eliciting remissions of both the preleukemic state and APL in mice. Our results demonstrate that PMLRAR alpha impairs neutrophil differentiation and initiates the development of APL. The transgenic mice described here provide an apparently accurate model for human APL that includes clear evidence of tumor progression. The model should be useful for exploring the molecular pathogenesis of APL and the mechanisms of the therapeutic response to retinoic acid, as well as for preclinical studies of therapeutic regimens.

Animals↗
PubMed1997

Libraries for genomic SELEX.

An increasing number of proteins are being identified that regulate gene expression by binding specific nucleic acidsin vivo. A method termed genomic SELEX facilitates the rapid identification of networks of protein-nucleic acid interactions by identifying within the genomic sequences of an organism the highest affinity sites for any protein of the organism. As with its progenitor, SELEX of random-sequence nucleic acids, genomic SELEX involves iterative binding, partitioning, and amplification of nucleic acids. The two methods differ in that the variable region of the nucleic acid library for genomic SELEX is derived from the genome of an organism. We have used a quick and simple method to construct Escherichia coli, Saccharomyces cerevisiae, and human genomic DNA PCR libraries that can be transcribed with T7 RNA polymerase. We present evidence that the libraries contain overlapping inserts starting at most of the positions within the genome, making these libraries suitable for genomic SELEX.

Binding Sites↗
PubMed1997

The epidemiology of measles in England and Wales since the 1994 vaccination campaign.

The incidence of measles in England and Wales has fallen since the national vaccination campaign in November 1994, in which 92% of children aged 5 to 16 years were vaccinated. A total of 148 confirmed cases with onsets in the 18 months from January 1995 to June 1996 have been ascertained. Notified cases did not provide a reliable measure of incidence: 11,343 suspected cases were notified in the same period, 6426 (57%) of whom were tested for salivary antibody. Only 90 (1.4%) of cases tested were confirmed. Many confirmed cases occurred in small clusters; 12 imported cases were identified. The pattern of small, local clusters is what would be expected from the introduction of imported cases into a population with herd immunity. Serological surveillance showed that the campaign produced a significant fall in the proportion of 5 to 16 year old children with low levels of measles antibody: the proportion with levels < 50 mIU/ml fell from 8.4% to 2.1%; the proportion with levels < 100 mIU/ml fell from 15.7% to 6.6%. About 15% of 2 to 4 year old children had antibody levels < 100 mIU/ml before and after the campaign. The addition of a routine second dose of measles vaccine (as measles, mumps, and rubella vaccine) to the vaccination schedule will provide another opportunity to immunise these children before they start school. The two dose vaccination programme should maintain the herd immunity of the population and the elimination of endemic measles transmission.

Adolescent↗
PubMed1997

Mumps surveillance in England and Wales supports introduction of two dose vaccination schedule.

Sentinel surveillance in general practice and laboratory reports to the PHLS Communicable Disease Surveillance Centre show that the incidence of mumps has fallen to very low levels since vaccination against measles, mumps, and rubella was introduced in 1988. Hospital admissions for mumps show a 92% decline compared with the prevaccination era, to a rate of 0.2 per 100,000 population per year. Serological surveillance has shown an increase in the proportion of school age children who have no detectable antibody to mumps, which is consistent with the reduction in mumps virus transmission. The proportion of children aged 11 to 15 years with no detectable antibody is expected to peak at 19% in 1997. Mathematical models suggest that this increase in susceptibility is unlikely to allow a large resurgence of mumps in the short term but that school outbreaks may become more common. Outbreaks in universities and military establishments are possible in the medium term. Analysis of efficacy data for mumps vaccine indicates that mumps is unlikely to be eliminated with a single dose of vaccine at current coverage rates. A second dose of vaccine, which is now being offered to preschool children, will reduce morbidity and should eventually eliminate mumps if coverage is high enough.

Adolescent↗
PubMed1997

The epidemiology of rubella in England and Wales before and after the 1994 measles and rubella vaccination campaign: fourth joint report from the PHLS and the National Congenital Rubella Surveillance Programme.

The national immunisation campaign carried out in the United Kingdom in November 1994 was designed to give children aged 5 to 16 years of age a single dose of a combined measles and rubella vaccine. Its main objective was to prevent an epidemic of measles predicted in school age children. The rubella component of the vaccine was included in order to reduce the high level of susceptibility to rubella in young adult males and thus reduce the risk of transmission from this group to pregnant women. Susceptibility to rubella in children aged 5 to 16 years has fallen from 15.7% to 3.4% since the measles and rubella campaign. Despite this the incidence of laboratory confirmed rubella rose substantially in 1996, largely on account of cases among males aged 17 to 24 years, who were not vaccinated in the 1994 campaign and about 16% of whom are susceptible. The impact of the resurgence on the incidence of infection in pregnancy has been relatively limited, due to the low level of susceptibility in the antenatal population (2% in nulliparous and 1.2% in parous women for 1994/5). No cases of congenital rubella arising from administration of measles and rubella vaccine during the campaign have been identified. The numbers of babies born with congenital rubella and terminations of pregnancy for rubella arising from the 1996 resurgence are expected to be similar to those that followed the 1993 resurgence. The reduction in susceptibility in future cohorts of young men who received measles and rubella vaccine in the 1994 campaign should prevent future resurgences after the year 2000. If a second dose of measles, mumps, and rubella (MMR) vaccine had not been introduced, susceptibility levels in the school age population would have risen to about 12% in the future. The effect of the second dose of MMR vaccine introduced for children aged 4 to 5 years in October 1996 will be assessed through serological surveillance.

Adolescent↗
PubMed1997

From oligonucleotide shapes to genomic SELEX: novel biological regulatory loops.

The SELEX method and oligonucleotide combinatorial chemistry discovery process yields high-affinity/high-specificity ligands for virtually any molecular target. Typically, the enormous starting libraries used in the SELEX process contain 10(14)-10(15) sequences. We now ask if the smaller sequences, complexity of extant organisms, and evolutionary history provide useful interactions between oligonucleotides and at least some unexpected targets. That is, do organisms contain a robust "linkage map" between their oligonucleotides and proteins and/or small molecules that enriches life?

Base Sequence↗
PubMed1997

Surgical salvage after radiotherapy for advanced laryngopharyngeal carcinoma.

OBJECTIVE: To comment on the use of surgery after the failure of radiotherapy in patients with advanced laryngeal, oropharyngeal, and hypopharyngeal carcinomas. DESIGN: Randomized, controlled, clinical trial, with a mean follow-up period of 3.1 years. SETTING: The Princess Margaret Hospital, Toronto, Ontario. PATIENTS: Patients with advanced laryngopharyngeal carcinoma (T3 or T4 or N+). INTERVENTION: Three hundred thirty-six patients who met the eligibility criteria were enrolled in a randomized, controlled, clinical trial and treated with primary radiotherapy using either the standard fractionation regimen or the hyperfractionation regimen. One hundred eight patients with recurrent disease underwent salvage surgery and were observed prospectively, with careful documentation of surgical and tumor data, complications, recurrences, and survival. MAIN OUTCOME MEASURES: Surgical complication rate and survival. RESULTS: Of the 108 patients who underwent surgery, 29 (27%) had complications. One third of these had multiple complications. There was no statistical difference between the surgical complication rates of the 2 radiotherapy groups (16 patients [28%] in the standard fractionation radiotherapy group and 13 [25%] in the hyperfractionation radiotherapy group). Survival after surgery was statistically correlated with the TNM system for the stage of the recurrent tumor, the pathologic nodal status, and the surgical margin status. The overall 3-year survival rate, which was calculated from the date of surgery, for the group of patients who underwent surgery was 22%. CONCLUSIONS: In this patient population, the hyperfractionation regimen was not associated with a higher surgical complication rate. Using the policy of primary radiotherapy and reserving surgery for the management of failures of radiotherapy, 140 patients (71%) at highest risk (laryngeal and hypopharyngeal primary tumor sites) retained an intact larynx until the end of the study or death. Since it is the stage of the recurrent tumor that correlates with survival rather than the stage of the tumor at initial presentation, we suggest that tumors be restaged at the time of recurrence.

Adult↗
PubMed1997

Future pathways for combinatorial chemistry.

Investment in combinatorial chemistry (combichem) in the pharmaceutical industry is being driven by the need for increased efficiency. Results from pioneers in the field have demonstrated where mixture or discrete compound synthesis is useful, and what mixture sizes and compound concentrations are appropriate. To make the techniques of combichem of general utility in drug discovery, a broad range of advances is still required. Conversion of organic chemistry to solid phase conditions is key, as are developments in linkers and resins. Library design methodology requires further development. Combinatorial biosynthesis of focused libraries of natural products holds great promise for capitalising on hardwon natural product leads. Miniaturisation of screens is required to reduce the cost of screening combinatorial libraries. Developments in the processes preceding and following synthesis are required to enable the flow of increased numbers of compounds without new bottlenecks developing. The impact of combinatorial chemistry will be greatly enhanced by synergy with ongoing parallel developments in genetic technologies, screening technologies and bioinformatics.

Biotechnology↗
PubMed1997

Induction of carcinoembryonic antigen expression in a three-dimensional culture system.

MIP-101 is a poorly differentiated human colon carcinoma cell line established from ascites that produces minimal amounts of carcinoembryonic antigen (CEA), a 180 kDa glycoprotein tumor marker, and nonspecific cross-reacting antigen (NCA), a related protein that has 50 and 90 kDa isoforms, in monolayer culture. However, MIP-101 produces CEA when implanted into the peritoneum of nude mice but not when implanted into subcutaneous tissue. We tested whether three-dimensional (3D) growth was a sufficient stimulus to produce CEA and NCA 50/90 in MIP-101 cells, because cells grow in 3D in vivo rather than in two-dimensions (2D) as occurs in monolayer cultures. To do this, MIP-101 cells were cultured on microcarrier beads in 3D cultures, either in static cultures as nonadherent aggregates or under dynamic conditions in a NASA-designed low shear stress bioreactor. MIP-101 cells proliferated well under all three conditions and increased CEA and NCA production three- to four-fold when grown in 3D cultures compared to MIP-101 cells growing logarithmically in monolayers. These results suggest that 3D growth in vitro simulates tumor function in vivo and that 3D growth by itself may enhance production of molecules that are associated with the metastatic process.

Animals↗