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Biomedical subjects

R F Ryan

Publications and source records attributed to R F Ryan.

At least 19 recordsLinked to original sources
PubMed1999

KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Krüppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing.

Krüppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1alpha and HP1gamma) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1alpha, which are normally found in centromeric heterochromatin, as well as M32 and hHP1gamma, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP-KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments.

3T3 Cells↗
PubMed1998

The role of zinc finger linkers in p43 and TFIIIA binding to 5S rRNA and DNA.

Transcription factor IIIA (TFIIIA) and p43 zinc finger protein form distinct complexes with 5S ribosomal RNA in Xenopus oocytes. Additionally, TFIIIA binds the internal promoter of the 5S RNA gene and supports assembly of a transcription initiation complex. Both proteins have nine tandemly repeated zinc fingers with almost identical linker lengths between corresponding fingers, yet p43 has no detectable affinity for the 5S RNA gene. TFIIIA zinc fingers 1-3 are connected by highly conserved linkers, first identified in the Drosophila protein Krüppel, that are found in many DNA binding zinc finger proteins. To understand the role of these linkers in RNA and DNA binding we exchanged three TFIIIA linker amino acids with the equivalent amino acids from p43. The major effect of linker substitution is a 50-fold reduction in DNA specificity, concomitant with an 8-fold reduction in affinity. N-Terminal zinc fingers from either TFIIIA or p43 bind to multiple specific sites on 5S RNA that are resistant to competition by tRNA or poly(rA). This mode of RNA binding is unaffected by linker substitution. These data suggest that zinc finger linkers significantly facilitate the specificity of DNA binding.

Amino Acid Sequence↗
PubMed1994

Regional chemotherapy for melanoma. A 35-year experience.

OBJECTIVE: The authors present their 35-year experience with intra-arterial chemotherapeutic regional perfusion of 1139 patients with melanomas, using an extracorporeal oxygenated circuit and heart-lung apparatus. SUMMARY BACKGROUND DATA: Intra-arterial chemotherapy produces improved responses in many tumors. By isolating and sustaining the area with extracorporeal oxygenated circulation, high doses can be delivered to the tumor area, limited only by local toxicity. Drug levels up to 10 times those achieved by systemic administration are obtained. METHODS: Techniques for hyperthermic perfusion were developed for limbs, pelvis, head, neck, and skin of the breast. Melphalan (Burroughs Wellcome, Research Triangle Park, NC) was used in 753 patients. Combinations with melphalan or other drugs were used in remaining cases at temperature of 38 to 40 C for 30 to 60 minutes. RESULTS: Chemotherapy perfusion followed by tumor excision or node dissection, was performed where indicated. The cumulative 10-year survival for patients with localized melanomas was 70%. For patients with local recurrences or satellites within 3 cm, survival was 61%. For those with regionally confined intransit tumors, survival was 30%; for those with regional node involvement, 38%; for those with intransit and nodal metastases, 16%; for those with distant metastases and perfusion--mainly to save functional limbs--survival was 7%. Multiple perfusions were performed in 158 patients with recurrent disease on 366 occasions. Patients with indolent regionally confined melanomas were benefited by prolongation of useful life. CONCLUSIONS: Safe perfusion techniques are available for most anatomic regions. Increased chemotherapeutic doses are delivered to isolated areas limited only by local toxicity. Adjunct perfusion in poor prognosis stage I cases is useful in reducing local recurrence, and intransit or lymph node metastases. Regional perfusion reduces the need for major amputation. Multiple perfusion can be useful in treating recurrent chronic melanoma.

Adult↗
PubMed1989

Cellular senescence involves stochastic processes causing loss of expression of differentiated function genes: visualization by in situ hybridization for steroid 17 alpha-hydroxylase in bovine adrenocortical cells.

When grown for long periods in culture, bovine adrenocortical cells lose the expression of a differentiated function gene, steroid 17 alpha-hydroxylase. Previously, we documented a decline in 17 alpha-hydroxylase mRNA with increasing culture passage level after induction with cyclic AMP (P. J. Hornsby et al., 1987, Proc. Natl. Acad. Sci. USA 84, 1580). We used in situ hybridization to investigate the loss of expression of this gene during cellular senescence at an individual cell level. In primary cultures, cells were uniformly positive for hybridization with cDNA for 17 alpha-hydroxylase after cyclic AMP induction. After two passages, cultures comprised a mixture of hybridizing and nonhybridizing cells. Cells appeared either to hybridize at a level comparable to that in primary cultures or to be nonhybridizing. When in situ hybridization was combined with immunofluorescence, cells positive for immunofluorescence were also positive for hybridization. Senescing mass cultures showed decreasing numbers of positive cells, and after 30 passages cultures comprised entirely nonhybridizing cells. Thus, the previously observed decline in overall 17 alpha-hydroxylase mRNA levels results from a decline in the fraction of expressing cells in the culture, and the rate of loss of expressing cells is in agreement with the rate of loss of total 17 alpha-hydroxylase mRNA. Primary clones, even when isolated at an early stage of clonal expansion, had mixtures of subclones of hybridizing and nonhybridizing cells. On recloning, hybridizing subclones usually produced uniformly nonhybridizing sub-subclones. Some subclones within primary clones had a morphology associated with replicative senescence (flattened cells with sparse intercellular contacts), yet had high numbers of hybridizing cells. We conclude that, in both mass and clonal populations, cells initially expressing 17 alpha-hydroxylase rapidly give rise to clones of nonexpressing cells. Such cells are continually derived by a stochastic process from cells originally expressing the gene.

Adrenal Cortex↗
PubMed1989

Cellular senescence involves stochastic processes causing loss of expression of differentiated function genes: transfection with SV40 as a means for dissociating effects of senescence on growth and on differentiated function gene expression.

In the accompanying work we demonstrated that the decline in expression of steroid 17 alpha-hydroxylase in mass cultures and clones of adrenocortical cells is the result of a stochastic switching process which yields mixtures of expressing and nonexpressing cells. There is an apparent positive correlation between the replicative potential of adrenocortical cell cultures and the number of cells in the culture that can express 17 alpha-hydroxylase. We investigated this by extending the cells' replicative potential by transfecting them with cloned SV40 virus. Cells from a senescent subclone, with very limited remaining replicative potential, were transfected. The cell population showed a progressive increase in growth rate and gave rise to a line of cells that expressed T antigen and which was apparently immortalized. Induction of mRNA for 17 alpha-hydroxylase by cyclic AMP was absent in this line of cells, as it was in the senescent cells prior to transfection. The cells remained responsive to gene induction by cyclic AMP as evidenced by increases in mRNA and activity for cholesterol side-chain cleavage. The absence of 17 alpha-hydroxylase expression in this line was not the result of interference by SV40 T antigen. When early passage cells were transfected with pSV3neo, which contains the early region of SV40 and neo, and were selected with G418, SV40 T antigen-expressing lines were derived which showed high levels of expression of 17 alpha-hydroxylase after induction with cyclic AMP. These cells maintained high levels of expression of 17 alpha-hydroxylase through four successive recloning events, over a period of replication much longer than that achievable by nontransfected cells. Thus, transfection by SV40 can be used to dissociate effects of senescence on growth and differentiated gene expression. T antigen expression selectively affects growth, but preserves the state of expression of a differentiated function gene as it was prior to transfection.

Adrenal Cortex↗
PubMed1989

Replicative senescence and differentiated gene expression in cultured adrenocortical cells.

We have used a differentiated endocrine cell type, the adrenocortical cell, to investigate the interrelationship of senescence and differentiation, the effects of the environment on differentiated gene expression, and the interrelationship of differentiated gene expression and proliferation. In bovine adrenocortical cells, expression of some differentiated functions is maintained to very late points in the replicative life span, whereas expression of others is lost at various times prior to senescence. There is clonal variation in the rate and extent of loss of functions. For steroid 17 alpha-hydroxylase, in situ hybridization shows that the observed decline in induction of 17 alpha-hydroxylase mRNA during senescence results from a decline in the fraction of cells expression the gene. Descendants of expressing cells in the primary cell population randomly become nonexpressing. Among clones there is a correlation between the fraction of cells expressing the gene and remaining replicative potential, although several experiments show no direct mechanism linking replicative senescence and 17 alpha-hydroxylase expression. Transfection with SV40 early region genes also dissociates the decline in growth and the change in 17 alpha-hydroxylase expression. SV40 T antigen selectively affects growth; expression of 17 alpha-hydroxylase is stabilized either in the on state, when cells are transfected early in the culture life span, or in the off state, when senescent cells are transfected. Thus, although the switching off of 17 alpha-hydroxylase expression and the loss of replicative potential are independent events, the switching process requires DNA replication. Because the switch is irreversible, changes in replicative potential occurring after the switch-off event do not affect the state of expression of the switched-off gene. Changes in differentiated cell properties and changes in replicative potential may be two facets of a general phenomenon of stochastic changes in gene expression in normal cells during senescence.

Adrenal Cortex↗
PubMed1988

A role for topical 5-fluorouracil therapy in melanoma.

Topical 5-fluorouracil (Efudex) has been shown to be of great value in the treatment of skin cancers, Marjolin ulcers, and advanced squamous cell carcinoma of the mouth and esophagus. It has been advocated for use on patients with melanocytic dysplasia and lentigo maligna melanoma. This report confirms the use of topical 5-FU for lentigo maligna melanoma and melanocytic dysplasia. In melanomas with poor prognosis, preoperative treatment with topical 5-FU converts the cellular infiltrate from acute inflammatory cells to round cells which have been shown to be T-cells. We believe this preoperative stimulation of the patients' own immune reaction to the tumor may be of great significance in improving the surgical results for melanomas with a poor prognosis.

Administration, Topical↗
PubMed1988

Topical 5-fluorouracil in treatment of carcinoma of nasal floor and nasal alae.

Squamous cell carcinomas of the nasal floor present problems for surgical therapy. Similarly, basal cell carcinoma of the nasal floor and alae requires extensive reconstruction when cures are obtained. To improve the results, we have treated the squamous cell carcinomas with topical 5-fluorouracil to obtain sensitization and shrinkage of the tumor before resection. The same has been done with basal cell carcinomas, but in selected patients the topical 5-fluorouracil has been continued until biopsies are negative. Thus, the costs of resection and reconstruction have been avoided and excellent cosmetic results are possible. This treatment is lengthy and requires weekly supervision. It offers a different approach that gives surgeons further options in treating these difficult carcinomas in carefully selected and cooperative patients.

Administration, Topical↗
PubMed1987

Loss of expression of a differentiated function gene, steroid 17 alpha-hydroxylase, as adrenocortical cells senescence in culture.

Senescence in cultured adrenocortical cells involves changes in expression of differentiated functions as well as changes in responses to mitogenic stimulation. Steroid 17 alpha-hydroxylase (steroid 17 alpha-monooxygenase, EC 1.14.99.9) is an adrenal-specific enzyme, the expression of which is dependent on the presence of stimulators of cyclic AMP production, such as cholera toxin. Dot-blot hybridization of RNA from bovine adrenocortical cells that had been incubated with cholera toxin showed a marked decline in 17 alpha-hydroxylase mRNA levels as a function of population doubling level, closely paralleling the decline in induction of 17 alpha-hydroxylase enzyme activity. The lower levels of 17 alpha-hydroxylase induction did not result from a requirement for a longer time period for induction or from a specific defect in response to cholera toxin and were not caused by a general failure of enzyme induction in response to cyclic AMP. The decreased growth rate in older cells results from a general decline in response to several growth factors. However, the decline in 17 alpha-hydroxylase induction did not result from a loss of response of the cells to mitogens, since quiescent cells at a low population doubling level showed stimulation of 17 alpha-hydroxylase mRNA by cholera toxin to levels similar to those in nonquiescent cultures and added mitogens either had no effect on 17 alpha-hydroxylase mRNA levels or decreased them. There was, however, a specific posttranscriptional effect of insulin on 17 alpha-hydroxylase. The loss of 17 alpha-hydroxylase induction is unlikely to result from overgrowth of a minority cell type lacking the ability to induce 17 alpha-hydroxylase, because adrenocortical cell clones that had high levels of 17 alpha-hydroxylase induction gave rise to cells with lower levels of induction on subcloning. Thus, loss of 17 alpha-hydroxylase activity in adrenocortical cellular senescence results from a primary failure of accumulation of 17 alpha-hydroxylase mRNA after incubation with the inducing agent.

Adrenal Cortex↗
PubMed1987

Previously undescribed treatment for persistent peritoneal-perineal fistula following proctectomy.

Persistent perineal sinus is a common complication following proctectomy for inflammatory bowel disease. Complete excision of the sinus tract and obliteration of dead space have been stressed as important aspects of treatment. A patient is described in whom the perineal sinus persisted despite three surgical attempts at cure. Combined perineal exploration and laparotomy revealed a communicating peritoneal-perineal fistula. A combined perineal and peritoneal approach may be indicated in treating the perineal sinus that persists despite perineal excision.

Abscess↗
PubMed1987

Squamous cell carcinoma of the parotid gland.

Squamous cell carcinoma involving the parotid gland is an aggressive and rapidly advancing lesion which if not recognized and treated early will result in a high morbidity and mortality. We reviewed 30 patients with squamous cell carcinoma involving the parotid gland. Twenty-four patients had had previous epidermoid skin lesions in an area known to drain to the parotid gland and three resulted from direct extension into the gland from an overlying skin carcinoma, whereas only three were primary lesions of the gland. Patients who presented with involvement of the gland more than 4 months after excision of the skin lesion had a poor prognosis. Patients with epidermoid skin cancer in areas with a propensity to secondarily involve the parotid gland must be closely followed after treatment of the primary skin lesion.

Adult↗
PubMed1986

Salvage of stage IV intraoral squamous cell carcinomas with preoperative 5-fluorouracil.

A regimen for improving the salvage rate for Stage IV squamous cell carcinoma of the tongue, alveolar ridge and floor of mouth is presented. This method utilizes pre-operative sensitization of the tumor and regional lymph nodes by the topical application of 5-fluorouracil (5-FU) in the form of Efudex (Roche). The drug must be used topically at the tumor skin or tumor-mucous membrane interface to utilize the sensitizing properties of skin or mucous membrane. Further response is obtained by direct injections of 5-FU into the tumor. Later intravenous (IV) drip of 5-FU can be used particularly at the time of surgical resection. During the period of preparation until sensitized to 5-FU, patients must be restored to positive nitrogen balance and concurrent infections are controlled. Because of the importance of nutrition in restoring immunity, a feeding gastrostomy for these patients is recommended. The definitive surgery must include all bone that is involved, as 5-FU alone will not sterilize the bone. Of 15 patients who underwent the regimen outlined in this study, 12 of the patients with Stage IV intra-oral squamous cell carcinoma have had their primary tumor controlled for 17 months to 5 years at the time of this report.

Administration, Topical↗
PubMed1986

Osteogenic sarcoma of the mandible: a plea for radical initial surgery.

A total of 24 cases of osteogenic sarcoma of the mandible have been reported to the SEER Program of the National Cancer Institute from 1973 to 1983. Out of 14 patients whose disease was staged as regional, only 9 were eligible for 5-year survival figures and only 2 are alive, for a 23 percent 5-year cure. For those patients staged as localized, only four of six eligible for 5-year cure survived, for a 66 percent 5-year survival. Considering all patients reported to SEER who were eligible for 5-year cure, the rate is 40 percent. We believe this reflects the practice of localized resection of these tumors. At Charity Hospital of Louisiana at New Orleans there have been 10 cases of osteogenic sarcoma of the mandible since 1948. There have been no 5-year survivors for those operated on by other services, usually by local resection. On the Tulane Plastic Surgery Service, a total of six cases of osteogenic sarcoma of the mandible were treated by radical surgery with 100 percent survival from 11 to 22 years.

Adolescent↗
PubMed1985

Regeneration of upper femur after subperiosteal resection.

A 16-year-old patient had total thigh fillet for decubitus ulcer with an infected hip joint. The patient had extraosseous calcification and infection and was hospitalized again one year later for operation. Approximately four years after the initial operation the patient regenerated a femoral head, neck, and approximately 20 cm of the femoral shaft. This was resected. How often does this occur and should we take steps to obliterate the periosteum when doing a subperiosteal thigh fillet procedure?

Adolescent↗
PubMed1985

Virginal hypertrophy.

A patient with extensive juvenile hypertrophy of the breasts has been presented. Several interesting facts in the case history are as follows: After pregnancy, the breasts did not regress with "hormone shots" to stop lactation. The patient took high-dosage estrogen birth control pills for 3 years before the breasts started to grow rapidly. Within 1 month after reduction mammaplasty and despite 20 mg dydrogesterone per day, the breasts started to enlarge. A total of 60 mg b.i.d. of dydrogesterone did not stop breast regrowth. Tamoxifen citrate did cause regression of the breasts. After two reductions, the breasts regrew with a subsequent pregnancy. The breast tissue regrew in the axilla with a subsequent pregnancy after simple mastectomy-subcutaneous mastectomy and free nipple transplants. Chronic marijuana use may have an effect on the breast tissue in certain susceptible females as well as in some males. Much needs to be learned about the control of growth of female breast tissue.

Adult↗