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Biomedical subjects

J Bryan

Publications and source records attributed to J Bryan.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of the murine thymidine kinase-encoding gene and analysis of transcription start point heterogeneity.

We have determined the molecular organization and transcription start points (tsp) for the murine gene (TK) encoding thymidine kinase. The exon/intron structure and sequences present at the splice junctions of the mammalian TK genes have been highly conserved; however, the promoter sequences of these genes have diverged widely. Both the human and Chinese hamster TK promoter regions contain CCAAT and TATA consensus motifs, whereas the mouse promoter has neither element. This difference between species is reflected in that, unlike the hamster and human TK genes, transcription initiates from numerous specific tsp within a 100-bp region in the mouse TK gene. The complex pattern of tsp seen in the endogenous gene was not maintained in transfected cell lines containing TK promoter::beta-globin (HBB) fusions. Transcription from the murine TK:HBB fusion genes initiated from a small number of tsp that were clustered downstream from the ATG in hybrids containing TK coding sequences, and in the HBB 5' UTR in hybrids that did not. Few or no specific tsp were detected from the upstream sites used in the endogenous mouse TK gene.

Amino Acid Sequence↗
PubMed1992

Localization and characterization of a 7.3-kDa region of caldesmon which reversibly inhibits actomyosin ATPase activity.

Cleavage of caldesmon with chymotrypsin yields a series of fragments which bind both calmodulin and actin and inhibit the binding of myosin subfragments to actin and the subsequent stimulation of ATPase activity. Several of these fragments have been purified by cation exchange chromatography and their amino-terminal sequences determined. The smallest fragment has a molecular mass of about 7.3 kDa and extends from Leu597 to Phe665. This polypeptide inhibits the actin-activated ATPase of myosin S-1; this inhibition is augmented by smooth muscle tropomyosin and relieved by Ca(2+)-calmodulin. The binding of the 7.3-kDa fragment to actin is competitive with the binding of S-1 to actin. Thus, this polypeptide has several of the important features characteristic of intact caldesmon. However, although an intact caldesmon molecule covers between six and nine actin monomers, the 7.3-kDa fragment binds to actin in a 1:1 complex. Comparison of this fragment with others suggests that a small region of caldesmon is responsible for at least part of the interaction with both calmodulin and actin.

Actins↗
PubMed1992

Specificity of photolabeling of beta-cell membrane proteins with an 125I-labeled glyburide analog.

The interaction between sulfonylureas and membrane proteins from a hamster insulin-secreting tumor (HIT) cell line has been examined. Four HIT cell membrane proteins were covalently linked to an 125I-labeled glyburide analog by photolabeling. Three photolabeled polypeptides of M(r) 65,000, 55,000, and 30,000 were identified as low affinity "glyburide receptors." These proteins appear to be of similar abundance, when quantitated by photolabeling, with half-maximal displacements (Ki values) by glyburide, glipizide, and tolbutamide in the low micromolar range. The glyburide analog is more tightly bound to a M(r) 140,000 protein with dissociation constants, determined by filtration binding assays and by photolabeling, of 7 and 9.0 nM, respectively. The labeled analog was displaced from the M(r) 140,000 protein by glyburide, glipizide and tolbutamide with Ki values of 3.3 nM, 103 nM, and 25 microM, respectively, as estimated by photolabeling. Optimal conditions established for visualizing the M(r) 140,000 band on autoradiograms prepared after UV cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis include irradiating the radioligand-receptor complex at 1.5 J/cm2 at 312 nm, followed by heating samples in pH 9.0 sodium dodecyl sulfate-gel sample buffer. With receptor sites partially occupied (5 nM radioligand), approximately 0.75% of the protein is photocoupled to the radioligand and visualized by autoradiography. Our results confirm that the M(r) 140,000 polypeptide contains the beta-cell high affinity glyburide binding site and show that the second generation sulfonylurea antidiabetic drugs have a selective increase in affinity for this receptor.

Animals↗
PubMed1992

Co-expression of sulfonylurea receptors and KATP channels in hamster insulinoma tumor (HIT) cells. Evidence for direct association of the receptor with the channel.

Cell membranes isolated from hamster insulinoma (HIT T15) cells at passages 65-74 contain high and low affinity receptors for a sulfonylurea derivative, 5-[125I]iodo,2-hydroxyglyburide (KD values of approximately 7 nM and 16 microM). Between passages 75 and 85, the estimated B(max) for the high affinity receptor decreases approximately 10-fold from approximately 1.6 to 0.16 pmol/mg membrane protein. By contrast, the density of low affinity binding sites, 800-1000 pmol/mg, is unaltered. The drop in high affinity receptors is paralleled by a decrease in the density of KATP channels assessed using patch-clamp and 86Rb(+)-efflux techniques. These results strongly support the idea that the high affinity sulfonylurea receptor is an integral part of the KATP channel.

ATP-Binding Cassette Transporters↗
PubMed1992

Cloning of cDNAs encoding human caldesmons.

Caldesmon (CDM) is a potential actomyosin regulatory protein found in smooth muscle and nonmuscle cells. Domain mapping and physical studies suggest that CDM is an elongated molecule with an N-terminal myosin/calmodulin-binding domain and a C-terminal tropomyosin/actin/calmodulin-binding domain separated by a 40-nm-long central helix. An 1100-nucleotide (nt) cDNA probe encoding the C terminus of avian caldesmon (aCDM) was used to screen a human aorta library and clone smooth-muscle and non-muscle CDM-encoding cDNAs (CDM). The human (h) smooth-muscle hCDM is 3050-3630 nt long, having variation in length in the 3'-untranslated region. The predicted hCDM protein has a high degree of identity, greater than 90%, to aCDM in the N- and C-terminal-binding domains. The central helical domain is more variable, but retains characteristic repeated peptides and an 'i, i + 4' acidic/basic amino acid (aa) motif found in aCDM which can form intra-helical salt bridges to stabilize the central helix. The predicted smooth-muscle protein is 793 aa long (93,262 Da) with a calculated pI of 5.75. As is the case for the chicken, nonmuscle hCDM is missing the central helical domain, 256 aa overall. Our nonmuscle clone is not full length, but the C-terminal end is identical to the smooth-muscle form. If the N-terminal domain is identical, as it is in the chicken, the predicted protein is 537 aa (62,558 Da). Examination of the 'junctions' at either end of the deleted central domain gives a clear indication of the splice sites and suggests that the nonmuscle form is generated by exon skipping.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Use of the ASI titanium stent in the management of bladder outflow obstruction due to benign prostatic hyperplasia.

An expandable titanium intraprostatic stent was inserted into 30 patients with infravesical obstruction due to benign prostatic hyperplasia (BPH). All of the men were considered unsuitable for transurethral resection of the prostate as a result of comorbid conditions. In 25 patients effective micturition was reestablished with this technique. In 21 of these men, who have been followed for longer than 1 year, the mean maximum flow rate at 1 year was 10.8 ml. per second and the mean residual urine was 56 ml. Although urinary tract infections occurred subsequent to stent insertion in 10 individuals, these resolved after appropriate antibiotic treatment and no stents have had to be removed for this reason. Followup cystoscopy or examination by electron microscopy of those stents that have been removed has shown partial epithelialization of the stent surface in a proportion of patients, and a minor degree of incrustation occurred in 1 case. We conclude that an expandable intraprostatic titanium stent is an acceptable alternative to transurethral resection of the prostate or long-term catheterization in this particular group of high risk patients.

Aged↗
PubMed1992

Finasteride in the treatment of benign prostatic hyperplasia. A urodynamic evaluation.

A group of 69 men with bladder outflow obstruction due to benign prostatic hyperplasia (BPH) were treated in a double-blind, placebo-controlled study with finasteride (Proscar, MK-906), a 5-alpha reductase inhibitor, 5 mg or 10 mg/day or placebo for 3 months; subsequently, 20 patients received finasteride 5 mg/day for a further 9 months in an open extension study. In treated patients dihydrotestosterone declined by over 60%, remaining unchanged with placebo. Symptom scores fell significantly in all 3 groups. Mean maximum flow rates fell slightly in placebo-treated patients but improved by 1.5 ml/s in the 10 mg group and by 3.3 ml/s in the 5 mg group. After 1 year's treatment, the reduction in symptom score and increase in flow rate were well maintained; the mean prostate volume was reduced by 14% and prostatic specific antigen declined by 28%. It was concluded that finasteride shows some efficacy in the treatment of BPH, with minimal toxicity, but 12 months of therapy or longer may be necessary to achieve maximal effect.

Aged↗
PubMed1991

Knowledge and beliefs about cancer in a socioeconomically disadvantaged population.

Americans living in poverty experience a higher incidence of and greater mortality from cancer than the nonpoor. At least 50% of the difference in mortality is believed to be due to delay in diagnosis, although risk-promoting lifestyles and behaviors also contribute to decreased survival. A potential exacerbating factor among the poor is inadequate information and knowledge about cancer and its treatment. Interviews were conducted with 128 cancer patients from a socioeconomically disadvantaged population to assess knowledge of cancer and its treatment and to evaluate care-seeking behaviors. Results indicated that although patients relied primarily on their physicians for information about their disease and treatment, a number of misconceptions regarding cancer existed in this population. Notably, nearly 50% of the patients surveyed either denied or did not know that smoking was related to the development of cancer. Additionally, patients frequently reported inappropriate care-seeking behaviors when asked to respond to a series of common disease-related signs or symptoms. These findings suggest that misinformation and misconceptions regarding cancer and its treatment among patients in this sample may contribute to inappropriate care-seeking behaviors.

Consumer Behavior↗
PubMed1991

Role of the N- and C-terminal actin-binding domains of gelsolin in barbed filament end capping.

Gelsolin is a bivalent Ca(2+)-modulated actin-binding protein that severs, nucleates, and caps filaments. In order to gain a better understanding of the capping mechanism we have studied N- and C-terminal gelsolin fragments, 14NT and 41CT, each of which contains a single functional actin-binding site. The very tight binding measured between gelsolin and the barbed filament end requires gelsolin to greatly decrease the dissociation rate constant of the terminal actin from this end. A mechanism that could account for the observed decrease in dissociation is one in which gelsolin links two actin monomers so that they dissociate more slowly as a dimer. This cannot be the only mechanism, however, since, as shown here, 14NT and 41CT, fragments with single actin-binding sites, decrease the dissociation rate of the capped terminal actin molecule. The observations suggest that these fragments induce a conformational change in the actin monomer that either increases the affinity or alters the kinetics of the terminal actin-actin bond. The available data argue for strengthening of the terminal actin-actin bond.

Acrosome↗
PubMed1991

Localization of the calmodulin- and the actin-binding sites of caldesmon.

Expression of the C-terminal third of chicken gizzard caldesmon in Escherichia coli, using the Nagai vector (Nagai, K., and Thøgersen, H.V. (1987) Methods Enzmol. 153, 461-481), produces a cII-caldesmon fusion protein (27 kDa) with caldesmon sequence beginning at Lys579. Degradation during purification yields five peptides with molecular masses of 24, 22, 19 (two peptides), and 15 kDa. The 24-kDa peptide begins at Phe581; the 22-kDa peptide begins at Leu597, the two 19-kDa peptides begin at Phe581 and Val629, respectively; the 15-kDa peptide also begins at Val629. We estimate that the 15-kDa and one of the 19-kDa peptides end near Leu710. Site-directed mutagenesis was used to produce truncated peptides with known C termini; one peptide (17 kDa) terminates at Asn675. Digestion of the fragments with chymotrypsin generates a second 15-kDa fragment that begins at Ser666 (15K'). All of the peptides, with the exception of 15K', bind Ca(2+)-calmodulin-Sepharose and share a common 37-amino acid peptide between Val629 and Ser666, suggesting this contains the calmodulin binding site. Comparison with published sequences (Takagi, T., Yazawa, M., Ueno, T., Suzuki, S., and Yagi, K. (1989) J. Biochem. (Tokyo) 106, 778-783 and Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238) for other calmodulin-binding fragments further restricts the binding site to 7 residues, Trp-Glu-Lys-Gly-Asn-Val-Phe, between Trp659 and Ser666. All of the fragments, except the two 15-kDa peptides, co-sediment with F-actin, indicating that there are two segments in the C-terminal third of caldesmon that can interact with F-actin: one between Leu597 and Val629, the other between Arg711 and Pro756. Although separated in the primary sequence, these domains may interact with the calmodulin-binding region in the folded structure.

Actins↗
PubMed1991

Detection of human papillomavirus types 6 and 11 E4 gene products in condylomata acuminatum.

Polyclonal antiserum to an Escherichia coli-produced beta-galactosidase/E4 fusion protein of human papillomavirus type 6b (antiserum 256), and affinity purified HPV 11 anti-E4 antibodies were tested for reactivity in Western blots with bacterially expressed trpE/E4 fusion proteins of HPV types 6b, 11, 16, and 18. To further characterize the affinity purified anti-E4 antibodies, a dot-immunobinding assay was performed using overlapping synthetic HPV 11 E1E4 peptides as antigens. Protein extracts of condylomata acuminatum from 18 patients containing HPV type 6 or 11 DNA sequences were tested in Western blots using antiserum 256 or affinity purified HPV 11 anti-E4 antibodies. In the Western blots of the trpE proteins, antiserum 256 identified the HPV types 6b and 11 fusion proteins; the affinity purified HPV 11 anti-E4 antibodies identified only the HPV 11 fusion protein. In the dot-immunobinding assay, three HPV 11 peptides were recognized, each containing a shared 8 amino acid sequence that differs significantly from the corresponding sequences of HPV types 6b, 16, or 18. In the Western blots of protein extracts from 18 condylomata acuminatum samples shown to contain HPV types 6 or 11 DNA, putative E4 gene products were identified in six samples by antiserum 256. The affinity purified HPV 11 anti-E4 antibodies identified putative E4 gene products in one of these same six lesions, which was shown to contain HPV 11 sequences by the Southern blot method. All six samples containing E4 gene products were from women. Three of these women were pregnant, one had serum antibodies to the human immunodeficiency virus, and one was a renal transplant recipient receiving glucocorticoids.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Sequence of an avian non-muscle caldesmon.

We have sequenced a cloned cDNA from a chicken gizzard library which encodes a short version of caldesmon with the properties expected for the non-muscle protein. The predicted protein is 524 amino acids long with a molecular mass of 60,174 daltons. Expression in bacteria produces a protein with an apparent molecular weight on SDS gels of 75-77,000, that comigrates with human platelet caldesmon. The expressed protein binds to F-actin and is retained on calmodulin-Sepharose in the presence, but not absence of Ca2+. This non-muscle caldesmon isoform is identical to the smooth muscle protein at the N- and C-terminal ends, but is missing 232 amino acids from the centre. This central segment is thought to be helical, suggesting that non-muscle caldesmon is approximately 35 nm shorter, about 40 nm overall length, than smooth muscle caldesmon.

Actins↗
PubMed1991

Sulfonylurea signal transduction.

In the pancreatic beta cells the proximal step in sulfonylurea signal transduction is the binding of these clinically important drugs to high-affinity receptors in the beta cell membrane. Using HIT cells as a model system, we have established an extremely close correlation between the affinity of binding of glyburide and its analog, iodoglyburide, and the activation of various steps in stimulus-secretion coupling--inhibition of 86Rb+ efflux, increase in [Ca2+]i resulting from gating of voltage-gated calcium channels by cell depolarization, and the exocytosis of insulin. Two different L-type channel cDNAs have been identified in an HIT cell library, one neuroendocrine in type and one more cardiac-like. A HIT cell membrane protein of Mr 140,000, which we believe to be the high-affinity sulfonylurea receptor, can be covalently linked to 5(125)-iodo-2-hydroxyglyburide by ultraviolet irradiation. The receptor has been solubilized and retains binding activity and the same rank order of displacement of the 5(125)-iodo-2-hydroxyglyburide as observed with the native receptor. The Mr 140,000 protein has been partially purified and the amino acid sequences of three proteolytic fragments have been used to design oligonucleotides to screen HIT cell cDNA libraries. Since the binding constant of glyburide or iodoglyburide is closely correlated with the ability of these compounds to inhibit the ATP-sensitive K+ channel, increase [Ca2+]i, and elicit insulin secretion, we have identified the Mr 140,000 protein as the sulfonylurea receptor. Expression of the cloned cDNA should allow us to test this hypothesis directly.

ATP-Binding Cassette Transporters↗
PubMed1991

Positive mood and math performance.

Two studies are reported that assessed the impact of positive moods on students' feelings of self-efficacy and math performance. In the first study, 32 black and Hispanic girls and boys in the third to fifth grades, who had been identified by teachers as at risk for school failure, and a control sample of normally achieving classmates served as subjects. In the second study, 15 males and 3 females in junior high and high school attending a private school for students with learning disabilities served as subjects. In both studies, students were randomly assigned to a positive-mood induction condition or a no-treatment control condition. Following this, they completed a measure of self-efficacy for math and were then given 5 minutes to do 50 math problems. In both studies, analyses of covariance using standardized math scores as the adjustor variable found that children in the positive-mood condition completed significantly more problems accurately than children in the no-treatment control condition. In addition, in the junior high and high school sample, students in the positive-mood induction condition expressed greater self-efficacy for math than students in the control condition. The results of these two studies raise many questions about the role of affect, positive and negative, in the learning and performance of students with learning disabilities that researchers and teachers might explore.

Adolescent↗
PubMed1990

The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli.

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca2(+)-calmodulin and binding to actin, actin-tropomyosin, Ca2(+)-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca2(+)-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.

Animals↗
PubMed1990

An efficient deletion mutant packaging system for defective herpes simplex virus vectors: potential applications to human gene therapy and neuronal physiology.

We have previously described a defective herpes simplex virus (HSV-1) vector system that permits the introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli beta-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. We now report an efficient packaging system for HSV-1 vectors using a deletion mutant, D30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses beta-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.

Animals↗