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Biomedical subjects

M G Donovan

Publications and source records attributed to M G Donovan.

36 records · Page 2Linked to original sources
PubMed1991

A model of human small intestinal absorptive cells. 1. Transport barrier.

The Caco-2 cell culture model of human small intestinal absorptive cells was used to investigate transepithelial transport. Transport of permeability markers such as mannitol demonstrated that Caco-2 monolayers became less permeable with increasing age in culture. Cells were routinely used for transport studies between day 18 and day 32. A transport index was determined for each compound by calculating the ratio of transport of the molecules under investigation to transport of an internal standard such as the permeability marker mannitol. Comparison of transport rates at 4 and 37 degrees C was a simple approach for differentiating primary transport mechanisms (passive paracellular, passive transcellular, or transporter-mediated) but must be coupled with additional experimental manipulations for definitive determination of transport pathways. Compounds predicted to undergo predominantly paracellular transport (mannitol, FITC, PEG-900, and PEG-4000), transporter-mediated transcellular transport (glucose, biotin, spermidine, or alanine), or lipophilic transcellular transport (alprenolol, propranolol, clonidine, or diazepam) showed differential effects of temperature on rates of transport as well as the transport index.

Biological Transport↗
PubMed1990

Secretion of the Bordetella pertussis adenylate cyclase from Escherichia coli containing the hemolysin operon.

The extracellular calmodulin-sensitive adenylate cyclase produced by Bordetella pertussis is synthesized as a 215-kDa precursor. This polypeptide is transported to the outer membrane of the bacteria where it is proteolytically processed to a 45-kDa catalytic subunit which is released into the culture supernatant [Masure, H.R., & Storm, D.R. (1989) biochemistry 28, 438-442]. The gene encoding this enzyme, cyaA, is part of the cya operon that also includes the genes cyaB, cyaD, and cyaE. A comparison of the predicted amino acid sequences encoded by cyaA, cyaB, and cyaD with the amino acid sequences encoded by hlyA, hlyB, and hlyD genes from the hemolysin (hly) operon from Escherichia coli shows a large degree of sequence similarity [Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., & Danchin, A. (1988) EMBO J. 7, 3997-4004]. Complementation studies have shown that HlyB and HlyD are responsible for the secretion of HlyA (hemolysin) from E. coli. The signal sequence responsible for secretion of hemolysin has been shown to reside in its C-terminal 27 amino acids. Similarly, CyaB, CyaD, and CyaE are required for the secretion of CyaA from Bordetella pertussis. We placed the cyaA gene and a truncated cyaA gene that lacks the nucleotides that code for a putative C-terminal secretory signal sequence under the control of the lac promoter in the plasmid pUC-19. These plasmids were transformed into strains of E. coli which contained the hly operon. The truncated cyaA gene product, lacking the putative signal sequence, was not secreted but accumulated inside the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1990

Evidence that the adenylate cyclase secreted from Bordetella pertussis does not enter animal cells by receptor-mediated endocytosis.

Bordetella pertussis, the pathogen responsible for whooping cough, produces a calmodulin-sensitive adenylate cyclase. Several investigators have shown that the partially purified adenylate cyclase is capable of entering animal cells and elevating intracellular cAMP levels (Confer and Eaton: Science 217:948-950, 1982; Shattuck and Storm: Biochemistry 24:6323-6328, 1985). However, the mechanism for entry of the catalytic subunit of this adenylate cyclase into animal cells is unknown. It has been reported that the B. pertussis adenylate cyclase extracted from bacterial cells with urea does not enter animal cells by receptor-mediated endocytosis. There is, in addition to the cell associated form of the B. pertussis adenylate cyclase, a cell-invasive form of the enzyme secreted into the bacterial culture media. The properties of the cell-associated and secreted enzymes are significantly different (Masure and Storm: Biochemistry 28:438-442, 1989). In this study, we report evidence that the secreted form of the B. pertussis adenylate cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis.

Adenylyl Cyclases↗
PubMed1989

Isolation of a protein fraction from Bordetella pertussis that facilitates entry of the calmodulin-sensitive adenylate cyclase into animal cells.

Bordetella pertussis, the pathogen responsible for whooping cough, releases a soluble calmodulin-sensitive adenylate cyclase into its culture medium. Several investigators have shown that the partially purified adenylate cyclase is capable of entering animal cells and elevating intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24,6323-6328]. However, the mechanism for entry of the catalytic subunit of the adenylate cyclase into animal cells is unknown. Recently, it was determined that the purified catalytic subunit of the enzyme is unable to enter animal cells [Masure, H. R., Oldenburg, D. J., Donovan, M. G., Shattuck, R. L., & Storm, D. R. (1988) J. Biol. Chem. 263, 6933-6940]. On the basis of these data and other observations, we hypothesized that the culture medium of B. pertussis contains one or more additional polypeptides which facilitate entry of the adenylate cyclase catalytic subunit into animal cells. In this study, we report that a cell-invasive preparation of B. pertussis adenylate cyclase was rendered noninvasive after passage through a wheat germ lectin-agarose column. A fraction was eluted from the wheat germ lectin-agarose column with N-acetyl-D-glucosamine. This fraction, when combined with the noninvasive adenylate cyclase, was able to restore the ability of the adenylate cyclase preparation to enter neuroblastoma cells and increase intracellular cAMP levels. Furthermore, the fraction eluted from the wheat germ lectin-agarose column was found to be trypsin and chymotrypsin sensitive, suggesting that this material was proteinaceous.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1989

Light and electron microscopic evaluation of Proplast II TMJ disc implants.

Proplast II disc implants (Vitek Inc, Houston) were used in nine patients (14 joints) who underwent discectomy of the temporomandibular joint (TMJ). These patients subsequently underwent removal of the implants from 10 to 28 months postplacement due to pain and occlusal changes or radiographic changes in the joints. Light and electron microscopic evaluations were performed on the retrieved implants and surrounding tissue. Evidence of gross deterioration manifested by fracture of the implant was present in 10 of the 14 implants, and microscopic evidence of deterioration was present in all implants. Foreign-body giant cell reaction in surrounding tissues and microfragmentation of the implant were evident in all joints.

Adult↗
PubMed1988

The interaction of Ca2+ with the calmodulin-sensitive adenylate cyclase from Bordetella pertussis.

Bordetella pertussis, the etiologic agent of whooping cough, produces a calmodulin-sensitive adenylate cyclase which elevates intracellular cAMP in a variety of eucaryotic cells. Exogenous calmodulin added to the partially purified adenylate cyclase has been shown to inhibit invasion of animal cells by this enzyme (Shattuck, R. L., and Storm, D. R. (1985) Biochemistry 24, 6323-6328). In this study, several properties of the calmodulin-sensitive adenylate cyclase are shown to be influenced by Ca2+ in the absence of calmodulin. The presence or absence of Ca2+ during QAE-Sephadex ion exchange chromatography produced two distinct chromatographic patterns of adenylate cyclase activity. Two different forms of the enzyme (Pk1 and Pk2EGTA) were isolated by this procedure. Pk1 adenylate cyclase readily elevated intracellular cAMP levels in mouse neuroblastoma cells (N1E-115) while Pk2EGTA adenylate cyclase had no effect on cAMP levels in these cells. Gel exclusion chromatography of Pk1 adenylate cyclase gave apparent Stokes radii (RS) of 43.5 A (+/- 1.3) in the presence of 2 mM CaCl2 and 33.8 A (+/- 0.94) in the presence of 2 mM EGTA [( ethylenebis (oxyethylenenitrilo)]tetraacetic acid). These Stokes radii are consistent with molecular weights of 104,000 (+/- 6,400) and 61,000 (+/- 3,600), respectively. Pk2EGTA adenylate cyclase had an apparent RS of 33.0 (+/- 1.2) (Mr = 60,600 (+/- 2,800] in the presence of Ca2+ or excess EGTA. At 60 degrees C, Pk1 adenylate cyclase exhibited a Ca2+-dependent heat stability with a half-life for loss of enzyme activity of 10.3 min in 5 mM CaCl2 and a half-life of 2.8 min in the presence of 0.1 microM CaCl2. The stability of Pk2EGTA adenylate cyclase was not affected by changes in free Ca2+. The adenylate cyclase preparations described above were submitted to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and enzyme activity was recovered from gel slices by extraction with detergent containing buffers. The catalytic subunit isolated from SDS-polyacrylamide gels was activated 7-fold in the presence of Ca2+ with maximum activity observed at 1 microM free Ca2+. With both preparations, the apparent molecular weight of the catalytic subunit on SDS gels was 51,000 in the presence of 2 mM CaCl2 and 45,000 in the presence of 2 mM EGTA. The catalytic subunit of the enzyme was purified to apparent homogeneity by preparative SDS-polyacrylamide gel electrophoresis and resubmitted to SDS gel electrophoresis in the presence or absence of free Ca2+. The purified catalytic subunit also exhibited a Ca2+-dependent shift in its mobility on SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗
PubMed1987

Ureteral meatotomy by Sachse urethrotome in the management of lower ureteral stones.

From January 1981 to June 1985 inclusive 170 patients were hospitalized for transurethral ureteral stone manipulation. A meatotomy with the Sachse urethrotome was done in 46 of these patients because the stone was impacted in the intramural ureter or the basket containing the stone became impacted. This method resulted in successful endoscopic removal of the stones in 43 patients.

Adult↗
PubMed1985

Use of the artificial urinary sphincter in the management of severe incontinence in females.

AS 792 and AS 800 sphincters were implanted in 31 female patients with incontinence due to various causes including neurologic dysfunction, recurrent stress incontinence, spinal cord trauma and pelvic trauma. Continued continence has been achieved in 21 of these patients. Erosions required removal of the device in the ten other patients. The AS 800 model has significant modifications that allow activation and deactivation without additional operative procedures. Satisfactory continence can be achieved in properly selected patients. The integrity of the vesicovaginal surgical plane is the key to success. Patients with multiple operations are the most difficult to manage and delayed activation is mandatory. Intermittent catheterization can be safely done and mechanical reliability is good.

Adolescent↗
PubMedDate not supplied

Primary lymphoma of the bladder: a report of three cases.

BACKGROUND: Primary lymphoma of the bladder is rare and its management is an evolving field. AIMS: To highlight primary lymphoma of the bladder as a possible diagnosis in cases of bladder neoplasm and to illustrate the currently favoured management options. METHODS: Three cases of primary bladder lymphoma are reported and management is reviewed. RESULTS: Each of the three cases was managed differently with each management approach yielding a favourable outcome. CONCLUSION: Chemotherapy combined, if necessary, with surgery or radiation therapy, should be the standard of care, depending on the full histological diagnosis.

Aged↗
PubMedDate not supplied

Clinical and histological assessment of collagen-induced arthritis progression in the diabetes-resistant BB/Wor rat.

Collagen-induced arthritis in the diabetes-resistant BB (DR BB)/Wor rat is a severe, aggressive disease initiated by immunization with heterologous native Type II collagen. Onset of clinical symptoms reproducibly occurs in 100% of animals between days 10 and 12 following collagen immunization. Hypertrophy of the synovial lining is the first histological manifestation of the early inflammatory arthritis. A mild inflammatory infiltrate in the synovium rapidly becomes a fibrovascular pannus eroding articular cartilage and subchondral bone. Beginning at the joint margins, an active synovitis is present. Light microscopy and immunohistochemical staining show the infiltrate to be comprised of mononuclear (lymphocytes, macrophages) and polymorphonuclear inflammatory cells. In addition, there is histological evidence for chronic inflammatory nodules and necrotizing vasculitis in connective tissue from diseased joints, both morphologic features associated with rheumatoid arthritis in humans. Subchondral bone erosion appears to be mediated largely by the resorptive action of activated osteoclasts. These histological parameters of disease progression in the DR BB/Wor rat are similar to human rheumatoid arthritis. The extensive degree of similarity in the pathology of DR BB/Wor rat collagen-induced arthritis and human rheumatoid arthritis supports the role of this model as an in vivo disease model for human rheumatoid arthritis.

Animals↗