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Biomedical subjects

T J Fitzgerald

Publications and source records attributed to T J Fitzgerald.

At least 55 records · Page 3Linked to original sources
PubMed1988

Allosteric nucleotide specificity of phosphorylase kinase: correlation of binding, conformational transitions, and activation. Utilization of lin-benzo-ADP to measure the binding of other nucleoside diphosphates, including the phosphorothioates of ADP.

Recent work has shown that ADP is an allosteric activator of nonphosphorylated phosphorylase kinase from rabbit skeletal muscle (Cheng, A., Fitzgerald, T. J., and Carlson, G. M. (1985) J. Biol. Chem. 260, 2535-2542). The specificity of the allosteric site for nucleoside diphosphates is further investigated in this study. Only purine nucleoside diphosphates are capable of causing allosteric activation, and an amino group at position 2 or 6 of the purine ring is required. Comparisons are made of the abilities of 5'-diphosphate analogs of ADP, including phosphorothioates, to activate, to bind, and to induce in the enzyme's beta subunits conformational changes associated with activation. Binding is measured by competition titrations utilizing fluorescence polarization of lin-benzo-ADP, itself an allosteric activator; and conformational changes are measured by partial proteolysis and chemical cross-linking. When measured at an identical percentage of saturation at the allosteric site, the abilities of ADP analogs to induce conformational changes in the beta subunits parallel their abilities to activate the holoenzyme. An unmodified beta-phosphate of ADP, although not necessary for binding at the allosteric site, is needed to fully drive the activating conformational transition. The activating nucleoside diphosphate appears to be the free species, as opposed to its Mg2+ complex.

Adenosine Diphosphate↗
PubMed1987

Autophosphorylation of the alpha subunit of phosphorylase kinase from rabbit skeletal muscle.

The autophosphorylation of the alpha subunit of phosphorylase kinase occurs simultaneously at multiple sites during incorporation of the first mol of phosphate. The predominant and initial autophosphorylation site on this subunit is different than the major site phosphorylated by cAMP-dependent protein kinase, which also phosphorylates multiple sites, as evidenced by two-dimensional phosphopeptide maps. All of the sites on the alpha subunit phosphorylated by cAMP-dependent protein kinase comigrate on peptide maps with autophosphorylation phosphopeptides; however, several phosphopeptides observed after autophosphorylation are not evident following phosphorylation by cAMP-dependent protein kinase. The phosphopeptide maps of the alpha subunit are the same whether autophosphorylation is carried out at pH 6.8 or 8.2 or whether MnATP is used instead of MgATP; there is only a slight difference in the maps brought about by EGTA-insensitive autophosphorylation. The autophosphorylation is shown to be an intrinsic activity of the phosphorylase kinase molecule; this conclusion is based on the observed copurification of the autophosphorylation activity with activities toward phosphorylase b and kappa-casein and the unaltered influence of various effectors on these activities throughout different sequential adsorption chromatography purification steps. Additional support to that already in the literature that the initial autophosphorylation events are predominantly intramolecular is gained by showing that previously autophosphorylated enzyme has little ability to catalyze the phosphorylation of nonphosphorylated enzyme.

Adenosine Triphosphate↗
PubMed1987

A fluorescence stopped flow study of the competition and displacement kinetics of podophyllotoxin and the colchicine analog 2-methoxy-5-(2',3',4'-trimethoxyphenyl) tropone on tubulin.

The colchicine analog 2-methoxy-5-(2',3',4'-trimethoxyphenol) tropone (AC) was used as a fluorescent probe to study the binding kinetics of podophyllotoxin at high concentrations. The observed pseudo-first order rate constant showed a linear concentration dependence up to 1 mM. The bimolecular rate constant (195 M-1 s-1 at 15 degrees C) and the activation energy (57 kJ/mol) correspond perfectly with those previously determined in the submicromolar range (Cortese, F., Bhattacharyya, B., and Wolf, J. (1977) J. Biol. Chem. 252, 1134-1140). Displacement kinetics of bound AC by podophyllotoxin, allow the determination of the dissociation rate constants for AC. By studying the temperature dependence, and combining with the binding rate constants previously determined (Engelborghs, Y., and Fitzgerald, T.J. (1986) Ann. N.Y. Acad. Sci. 466, 709-717) a full characterization of the kinetic pathway is possible. This is shown to differ considerably from the pathway of colchicine binding.

Animals↗
PubMed1987

The hyaluronidase associated with Treponema pallidum facilitates treponemal dissemination.

Treponema pallidum contains hyaluronidase (Hase) associated with its surface. Experiments were performed to determine the functional role of this enzyme in syphilitic infection. The effects of incubating organisms with rabbit anti-bovine Hase or normal or immune sera were compared. Preincubation of treponemes with anti-Hase resulted in inhibition of treponemal degradation of hyaluronic acid, indicating that these antisera did in fact retard enzyme activity. Anti-Hase did not immobilize or neutralize T. pallidum. In addition, rabbits were immunized with bovine Hase and then challenged intradermally with organisms; subsequent lesion development was not affected. Anti-Hase did not block treponemal attachment to cultured testicular fibroblasts but did inhibit attachment to isolated capillaries. Rabbit amnions were used as an in vitro model for dissemination of T. pallidum. Anti-Hase retarded the penetration of organisms through the amnions. This inhibitory effect was dependent on the presence of amniotic hyaluronic acid. When this glycosaminoglycan was selectively removed, the anti-Hase lost its ability to inhibit treponemal penetration. When exogenous hyaluronic acid was added back to treated amnions, the inhibitory effect of anti-Hase was restored. Evans blue experiments were used to characterize treponeme-induced vascular leakage following intradermal inoculation of T. pallidum. Prior treatment of organisms with anti-Hase reduced dermal leakage of the dye, indicating the involvement of the treponemal Hase in causing vessel leakage. Finally, rabbit testicular infections were used as an in vivo model for dissemination; one testis was infected, and after 10 to 13 days, treponemes in the opposite testis were quantitated. The anti-Hase restricted dissemination of organisms. These findings point to the functional role of the treponemal Hase in facilitating disseminated syphilis.

Amnion↗
PubMed1987

Activation of the classical and alternative pathways of complement by Treponema pallidum subsp. pallidum and Treponema vincentii.

Both in vivo and in vitro studies have indicated that complement plays an important role in the syphilitic immune responses. Few quantitative data are available concerning activation of the classical pathway by Treponema pallidum subsp. pallidum, and no information is available on treponemal activation of the alternative pathway. Activation of both pathways was compared by using T. pallidum subsp. pallidum and the nonpathogen T. vincentii. With rabbit and human sources of complement, both organisms rapidly activated the classical pathway, as shown by hemolysis of sensitized sheep erythrocytes and by the generation of soluble C4a. With human sources of complement, both organisms also activated the alternative pathway, as shown by hemolysis of rabbit erythrocytes and by the generation of soluble C3a in the presence of magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). During incubation, organisms remained actively mobile and did not lyse, indicating that activation was a function of complement reactivity with the intact outer treponemal surface. In addition, freshly harvested T. pallidum subsp. pallidum immediately activated both pathways of complement; preincubation of organisms did not enhance complement reactivity. T. vincentii was a more potent activator of this pathway. T. pallidum subsp. pallidum contained almost four times as much surface sialic acid as T. vincentii did. When sialic acid was enzymatically removed from T. pallidum subsp. pallidum, enhanced activation of the alternative pathway was detected. It is proposed that T. pallidum subsp. pallidum retards complement-mediated damage by the alternative pathway through surface-associated sialic acid. This may be an important virulence determinant that enables these organisms to readily disseminate through the bloodstream to infect other tissues.

Animals↗
PubMed1986

Solubilization of the vasopressin receptor from rat liver plasma membranes. Evidence for a receptor X GTP-binding protein complex.

The GTPase activity of plasma membranes isolated from rat livers was stimulated 20% over basal by vasopressin. A concentration dependency curve showed that maximal stimulation was obtained with 10(-8) M vasopressin. The vasopressin-stimulated GTPase activity was not inhibited in plasma membranes that had been ADP-ribosylated with either cholera toxin or pertussis toxin. Identical results were obtained from plasma membranes that had been solubilized with 1% digitonin. When membranes that had been solubilized after preincubation with [3H]vasopressin were subjected to sucrose gradient centrifugation, the majority of protein-bound [3H]vasopressin migrated as a single band with a sedimentation constant of 16.8 S. Moreover, there was a GTPase activity that migrated with the bound [3H]vasopressin. This peak of bound [3H]vasopressin was decreased by 90% when the sucrose gradient centrifugation was run in the presence of 10 microM guanosine 5'-O-(thiotriphosphate). When the 16.8 S peak of bound [3H]vasopressin was further purified over a wheat germ lectin-Sepharose column, a GTPase activity co-eluted from the column with the protein-bound [3H]vasopressin. Direct evidence that a GTP-binding protein was present in the 16.8 S peak was obtained by the immunodetection of a 35-kDa beta subunit of a GTP-binding protein. These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.

Adenosine Diphosphate Ribose↗
PubMed1985

Adenosine 5'-diphosphate as an allosteric effector of phosphorylase kinase from rabbit skeletal muscle.

Equilibrium binding and activity studies indicate that adenosine 5'-diphosphate binds to phosphorylase kinase with high affinity at a site, or sites, distinct from the catalytic site. Equilibrium dialysis at pH 6.8 and 8.2, with and without Mg2+, and with phosphorylated and nonphosphorylated enzyme preparations revealed approximately 8 ADP binding sites per alpha 4 beta 4 gamma 4 delta 4 hexadecamer, with Kd values ranging from 0.26 to 17 microM. Decreasing the pH from 8.2 to 6.8 or removing the Mg2+ enhanced the affinity for ADP. At pH 6.8, ADP stimulated the phosphorylase conversion and autophosphorylation activities of the nonactivated enzyme. Analogs of ADP with modifications at the 2'-, 3'-, and 5'-positions allowed determination of structural requirements for the stimulation of activity. ADP seems to alter the conformation of the beta subunit because addition of the nucleotide inhibits its dephosphorylation by phosphoprotein phosphatase and its chemical cross-linking by 1,5-difluoro-2,4-dinitrobenzene. The binding affinities and effects of ADP suggest that it may function physiologically as an allosteric effector of phosphorylase kinase.

Adenosine Diphosphate↗
PubMed1985

Treatment complications after sequential combination chemotherapy and radiotherapy with or without surgery in previously untreated squamous cell carcinoma of the head and neck.

One hundred consecutive patients with previously untreated advanced squamous cell carcinoma of the head and neck were treated with induction combination chemotherapy followed by definitive surgery and/or radiotherapy, and were evaluated for radiotherapy related toxicity. The induction regimen consisted of cisplatin, bleomycin and methotrexate/leucovorin. Acute toxicity consisted predominantly of mucositis and weight loss, and was mild or moderate by degree in 94% of patients. Six percent of patients experienced severe or life threatening acute toxicities. Two acute toxic deaths were noted in this series, one from a combination of mucositis, weight loss and infection and one from hypoglycemia of unknown origin. Thirty-five percent of patients had radiation treatment interrupted briefly because of acute toxicity. Toxicity was greatest in patients who were nonresponders to induction chemotherapy and such may have been related to the continued presence of advanced tumor. Radiotherapy dose, surgical intervention and age did not have an impact on the presence or degree of acute toxicity. Late toxicities included: hypothyroidism in 32% of patients tested: osteoradionecrosis in 5% of patients, associated primarily with a composite resection (4 of 5 cases); and soft tissue ulcerations in 3%. Taken together, these data indicate that induction combination chemotherapy did not significantly increase the toxicity of subsequent radiotherapy with or without surgery.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1985

Binding of glycosaminoglycans to the surface of Treponema pallidum and subsequent effects on complement interactions between antigen and antibody.

Acidified bovine serum albumin (acid BSA) reacts with glycosaminoglycans to form a precipitate. This reaction was adapted to Treponema pallidum to show glycosaminoglycans associated with the surface of the micro-organism. As testicular infection progressed from days 4 to 18, treponemes showed increasing amounts of these surface components. High speed centrifuging effectively removed the glycosaminoglycans, thus indicating that they were loosely bound. The subsequent addition of commercial preparations of hyaluronic acid or chondroitin sulphate resulted in their immediate adherence to the surface of the pathogens T pallidum and T pertenue, but not to the non-pathogens T vincenti, T denticola, or T phagedenis. The amount adhering to the treponemal surface varied depending on the concentration added. Intradermal inoculation showed that the virulence of T pallidum was not altered by the glycosaminoglycans associated with its surface. The coating of treponemes with hyaluronic acid or chondroitin sulphate did not interfere with neutralising antibodies or antibodies found by radioimmunoassay using whole organisms. In contrast, hyaluronic acid or chondroitin sulphate on the treponemal surface did interfere with immobilising antibodies. Results are discussed in terms of the potential role of the treponemal glycosaminoglycans in the infectious process.

Animals↗
PubMed1985

Interactions of fibronectin with Treponema pallidum.

The adhesive or opsonic glycoprotein, fibronectin, is associated with the surface of Treponema pallidum as shown by immunofluorescence. A quantitative assay using iodine-125 (125I) showed that T pallidum harvested seven days after infection bound more fibronectin than T pallidum harvested 14 days after infection. This increased binding by "younger" organisms was confirmed by radioimmunoassay techniques. Fibronectin appears to have a role in treponemal attachment. Preincubation of T pallidum with goat or rabbit antibody to fibronectin blocked treponemal attachment to cultured cells and to isolated capillaries and inhibited treponemal virulence. Treponemes were incubated in glass wool columns pretreated with fibronectin and were then eluted from the columns. This technique yielded a population of T pallidum that failed to bind to fibronectin. Compared with treponemes eluted from control ovalbumin columns, organisms eluted from fibronectin columns attached to cultured cells in larger numbers but did not survive as long and were not as virulent. Findings are discussed in terms of the relevance of interaction between treponemes and fibronectin in the pathogenesis of T pallidum.

Animals↗
PubMed1985

Detection and functional characterization of early appearing antibodies in rabbits with experimental syphilis.

Following testicular infection of rabbits with Treponema pallidum, different antibodies become detectable initially at the time of healing. Experiments were performed to determine a functional role for these antibodies. Rabbits were sacrificed after 4-8 days. Treponemal numbers steadily increased for 10-12 days. Thereafter, host defenses were sufficiently stimulated to begin clearing the organisms. Antibodies in serum and antibodies localized at the site of infection were quantitated using radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) techniques. Anti-treponemal IgG was detected as early as day 4. Quantities of antibody correspondingly increased with time following infection. Treponema pallidum was harvested 7 and 14 days postinfection and tested for surface antibodies. With increasing days postinfection, more antibody was found on the organisms. Two functional properties of these antibodies were shown. Sera from 24 of 45 rabbits infected for 14 days immobilized T. pallidum in the presence of complement and 14-day sera blocked the attachment of T. pallidum to tissue culture cells. We suggest that antibody-mediated, complement-dependent immobilization of T. pallidum and blockage of attachment are at least partially responsible for healing of testicular lesions.

Animals↗
PubMed1985

Experimental congenital syphilis in rabbits.

A model for congenital syphilis in rabbits was developed based on multiple intravenous injections of pregnant does with high concentrations of Treponema pallidum. A total of 48 pregnant does and 394 newborns were evaluated. Indications of in utero infection included a 7- to 10-fold increase in fetal mortality and a 49% reduction in birth weight. The size of the stillborns varied. Some developed to normal size, whereas others were poorly developed, hemorrhagic, and 1/10th normal size. Fetuses were surgically removed after 25 to 28 days of gestation and extracts of the fetal tissues were injected into adult rabbits. Syphilitic lesions resulted demonstrating the presence of T. pallidum within the fetal tissues. Treponemes were also demonstrated within splenic tissue from a 1-week-old newborn. Isolated amniotic membranes were placed in Franz Biologic chambers. Viable organisms readily penetrated through the amnion but heat-inactivated organisms did not. Further evidence for in utero transmission of organisms was provided by intradermal injection of 6- to 7-week-old newborns. In control newborns in which the does were not infected during pregnancy, lesions occurred at 90% of the sites injected and developed in typical fashion. In newborns from does infected during pregnancy, lesions occurred in 18% of the sites injected and developed in atypical fashion (flat, nonulcerating, rapid healing). Finally, overt congenital syphilis did not occur if the does were immune prior to pregnancy, then infected with T. pallidum during pregnancy. The percent mortalities and birth weights of newborns were equivalent to control newborns from noninfected does.

Animals↗
PubMed1985

Immune immobilization of Treponema pallidum: antibody and complement interactions revisited.

The Treponema pallidum immobilization test was designed for serodiagnosis of syphilis and is dependent upon specific antibody and a heat labile component of normal serum. Investigators have shown the component to be dependent upon divalent cations and it is presumed to be complement. Experiments were performed to reevaluate the interactions of antibody and complement and the mechanism of immobilization. The loss of treponemal motility was correlated to the loss of complement activity in the reaction mixture. When motility of treponemes incubated with immune serum IgG and complement had dropped to 50% (3.4 h), 72% of the available complement had been consumed. At the same time, treponemes incubated with normal serum IgG and complement were 82% motile and only 51% of the complement had been consumed. C6 deficient rabbit serum and C4 deficient guinea pig serum were used in conjunction with immune serum IgG to determine which components of the complement cascade were necessary for immobilization. Treponemes were not immobilized by either sera. Results suggest that the heat labile factor in normal sera is complement, that both early and late components of the complement cascade are necessary, and that the reaction proceeds via the classical complement pathway. Although T. pallidum is susceptible to the actions of antibody and complement, the organisms must interact with these components for at least 2 h before immobilization will result.

Animals↗
PubMed1985

Sphincter preservation in patients with low rectal cancer treated with radiation therapy with or without local excision or fulguration.

Twenty-six patients with small cancers limited to the lower two-thirds of the rectum were treated with conservative surgery and radiation therapy (XRT). The selection factors for this approach were age, refusal of a permanent colostomy, or the high risk of local recurrence because of inadequate surgical margin or palpable residual tumor after local surgical treatment alone. In patients treated with local excision or fulguration followed by XRT, there was a 6% local failure rate (one in 17); in 16 of 17 patients receiving radiation doses above 4,500 cGy, the local control was complete for follow-up periods of 6 months to 7 years. In nine patients treated with XRT for residual tumor, local failure occurred in five (56%). The disease-free survival for those without residual tumor versus those with residual tumor was 88% and 44%, with median follow-up periods of 20 and 23 months, respectively. Serious late complications occurred only if total doses were greater than 6,300 cGy. Local excision combined with XRT proved to be a safe alternative to radical surgery in selected patients and resulted in excellent local control while allowing preservation of anal sphincter function.

Adenoma↗
PubMed1984

Activated states of phosphorylase kinase as detected by the chemical cross-linker 1,5-difluoro-2,4-dinitrobenzene.

When phosphorylase kinase from rabbit skeletal muscle was activated by phosphorylation and then cross-linked with 1,5-difluoro-2,4-dinitrobenzene at pH 6.8, dimers of beta subunits were formed that were not observed during cross-linking of nonphosphorylated enzyme under the same conditions. The ability to form these dimers was due to phosphorylation of the beta subunit because when enzyme phosphorylated in the alpha and beta subunits was incubated with a protein phosphatase relatively specific for the beta subunit (Ganapathi, M.K., Silberman, S.R., Paris, H., and Lee, E.Y.C. (1981) J. Biol. Chem. 256, 3213-3217), the ability to form the cross-linked beta dimers was lost. Significant amounts of two complexes also judged to be dimers of beta subunits were observed when nonphosphorylated phosphorylase kinase was cross-linked after preincubation with Ca2+ plus Mg2+ ions, after proteolysis by chymotrypsin, or when it was cross-linked at pH 8.2, three conditions known to stimulate the activity of the nonphosphorylated enzyme. From these results, we conclude that 1,5-difluoro-2,4-dinitrobenzene can serve as a structural probe for activated states of phosphorylase kinase. The activation is associated with a conformational change in which two beta subunits either move closer together or have a reactive group on one, or both, of them unmasked. Our results suggest that the diverse mechanisms listed above for stimulating phosphorylase kinase activity cause a common conformational change to occur.

Animals↗
PubMed1984

Attachment of Treponema pallidum to fibronectin, laminin, collagen IV, and collagen I, and blockage of attachment by immune rabbit IgG.

As shown by scanning electron and phase contrast microscopy, Treponema pallidum attached in vitro to basement membranes purified from kidney cortex tissues or from retinal vessels. This organism also attached to the extracellular matrix remaining after cultured cells had been solubilised with Triton X. Fibronectin, laminin, collagen, IV, collagen I, and hyaluronic acid are structural components of basement membranes and extracellular matrices. Experiments were performed to investigate the in vitro attachment of T pallidum to each of these components. Viable or heat inactivated treponemes were added to glass coverslips precoated with different concentrations of each component. After various times of incubation, coverslips were washed and the attached organisms were counted. Large numbers of viable organisms attached to each of these five components. In contrast, heat inactivation sharply reduced numbers of attached organisms. The IgG fractions of immune and non-immune rabbit serum samples were affinity purified using protein A. T pallidum was preincubated with both fractions, then incubated with either intact cultured cells or with coverslips coated with the five tissue components. The IgG from immune serum blocked treponemal attachment to the cultured cells and to fibronectin, laminin, collagen IV, and collagen I, but not to hyaluronic acid. These results are discussed in terms of attachment mechanisms of T pallidum and potential applications to in vivo infection.

Adhesiveness↗
PubMed1983

Characterization of initial autophosphorylation events in rabbit skeletal muscle phosphorylase kinase.

Initial autophosphorylation of nonactivated rabbit skeletal muscle phosphorylase kinase at pH 8.0 caused an increase in enzymatic activity that closely paralleled phosphorylation of the beta subunit. Peptide maps revealed that the first phosphate incorporated into the beta subunit during autophosphorylation was on the same tryptic peptide previously isolated from phosphorylase kinase that had been phosphorylated by cAMP-dependent protein kinase (Cohen P., Watson, D.C., and Dixon, G.H. (1975) Eur. J. Biochem. 51, 79-92). When preincubated with phosphorylase kinase for one min, Ca2+ and Mg2+ synergistically stimulated subsequent autophosphorylation at pH 6.8. After this treatment phosphorylation of both the alpha and beta subunits became linear, and the first site phosphorylated on the beta subunit at pH 6.8 corresponded to the first site phosphorylated at pH 8.0. Removal of the lag as a consequence of the synergistic action of the metal ions allowed determination of a Km for MgATP of approximately 20 microM during initial autophosphorylation at either pH 6.8 or 8.2. With phosphorylase b as the substrate the Km values for MgATP under identical conditions were determined to be approximately 30 and 60 microM at pH 6.8 and 8.2, respectively. Initial rates of autophosphorylation over a 30-fold range of phosphorylase kinase concentrations suggest that incorporation of the first 1 to 2 mol of phosphate per alpha beta gamma delta tetramer occurs through an intramolecular mechanism.

Adenosine Triphosphate↗