Biomedical subjects

T J Fitzgerald

Publications and source records attributed to T J Fitzgerald.

At least 19 recordsLinked to original sources

PubMed2006

Radiotherapy in pediatric medulloblastoma: quality assessment of Pediatric Oncology Group Trial 9031.

PURPOSE: To evaluate the potential influence of radiotherapy quality on survival in high-risk pediatric medulloblastoma patients. METHODS AND MATERIALS: Trial 9031 of the Pediatric Oncology Group (POG) aimed to study the relative benefit of cisplatin and etoposide randomization of high-risk patients with medulloblastoma to preradiotherapy vs. postradiotherapy treatment. Two-hundred and ten patients were treated according to protocol guidelines and were eligible for the present analysis. Treatment volume (whole brain, spine, posterior fossa, and primary tumor bed) and dose prescription deviations were assessed for each patient. An analysis of first site of failure was undertaken. Event-free and overall survival rates were calculated. A log-rank test was used to determine the significance of potential survival differences between patients with and without major deviations in the radiotherapy procedure. RESULTS: Of 160 patients who were fully evaluable for all treatment quality parameters, 91 (57%) had 1 or more major deviations in their treatment schedule. Major deviations by treatment site were brain (26%), spinal (7%), posterior fossa (40%), and primary tumor bed (17%). Major treatment volume or total dose deviations did not significantly influence overall and event-free survival. CONCLUSIONS: Despite major treatment deviations in more than half of fully evaluable patients, underdosage or treatment volume misses were not associated with a worse event-free or overall survival.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2005

High dose fractionated ionizing radiation inhibits prostate cancer cell adhesion and beta(1) integrin expression.

BACKGROUND: The effect of ionizing radiation on extracellular matrix (ECM)-mediated cellular functions is an important area of research for translational science. Mechanisms of tumor cell ability to proliferate, migrate, and survive appear dependent on integrin-mediated adhesion to the ECM; however, the exact role therapeutic radiation plays in altering signaling pathways and promoting cell death within remains less well established. METHODS: To examine these effects on prostate carcinoma cell lines, cells were irradiated at sub-lethal doses. We have studied two human prostate cancer cell lines (PC3 and DU-145) irradiated with different fractionated radiation schedules. Three groups were compared to non-irradiated controls. Group A was given a single dose of 5 Gy. Group B was given 5 Gy the first week and then 10 Gy the second week for a total of 15 Gy. Group C was given 5 Gy the first week, and then 10 Gy the second and third week for a total of 25 Gy. Cells were analyzed at their prescribed total dose. At 48 hr post irradiation, cells were detached from culture dishes and were subsequently used for adhesion assays and immunoblotting analysis. RESULTS: Our findings revealed that two prostate carcinoma cell lines, PC3 and DU-145, had a reduced cellular adhesion to fibronectin (FN) compared to the non-irradiated control groups. Both prostate cancer cell lines showed decreased adhesion to FN and reduced beta(1) integrin protein levels at a total dose of 25 Gy, but not at the doses of 15 and 5 Gy. In a parallel analysis, at the maximum total dose of 25 Gy, both PC3 and DU-145 demonstrated a significant decrease in cell proliferation. CONCLUSIONS: High dose radiation treatment of prostate cancer cell lines inhibits integrin expression. Our study suggests that promoting a synergistic decrease in adhesion could bring additional therapeutic benefit to patients treated with radiation therapy.

Apoptosis↗

PubMed2005

Analysis of axillary coverage during tangential radiation therapy to the breast.

PURPOSE: To evaluate the percent of the prescribed radiation dose to the breast delivered to the axillary tissue and to evaluate the volume of the axilla receiving 95% of the prescribed dose with normal and with high tangential fields. METHODS AND MATERIALS: Computed tomographic scan images with 5-mm sections were retrospectively analyzed for 35 patients who had undergone three-dimensional (3D) planning for whole-breast radiation. The axillary nodal region was identified and divided into Levels I to III and Rotter's nodes (RN). Digitally reconstructed radiographs were created, and two plans were developed: (a) the standard clinical opposed tangential irradiation fields and (b) the high-tangential irradiation fields. Axillary coverage was examined by use of dose-volume histograms (DVH), and the average coverage for the four nodal groups was obtained. RESULTS: The data show that with the standard tangential irradiation fields, the average dose delivered to Levels I, II, III, and RN is 66% (standard deviation, or SD = 13%), 44% (SD = 18%), 31% (SD = 20%), and 70% (SD = 19%) of the prescribed dose, respectively. The coverage increases to 86% (SD = 9%), 71% (SD = 19%), 73% (SD = 17%), and 94% (SD = 8%) of the prescribed dose, respectively, for Levels I, II, III, and RN when the high tangential irradiation fields are used. 51% of Level I, 26% of Level II, and 15% of Level III receive 95% of the prescribed dose with normal tangents. The volume increases to 79%, 51%, and 49% of Levels I, II, and III, respectively, with high tangents. CONCLUSION: The tangential fields designed to treat only the breast do not adequately cover the axillary region and, therefore, cannot be relied upon for prophylactic therapy of the axilla. The high tangential irradiation fields increase the dosages received by the axillary region, but the average dosages received by the lower axillary regions are still less than 90% of the prescribed dose.

Adult↗

PubMed2002

An evaluation of the relationship between the quality of prophylactic cranial radiotherapy in childhood acute leukemia and institutional experience: a Quality Assurance Review Center-Pediatric Oncology Group study.

PURPOSE: The Pediatric Oncology Group Protocol 9404 was a prospective clinical trial of two forms of chemotherapy in childhood T-cell acute lymphoblastic leukemia and advanced stage T-cell lymphoblastic non-Hodgkin's lymphoma. The protocol called for prophylactic C1 whole brain external beam irradiation, 18 Gy in 2 Gy/fraction for 9 fractions. We hypothesized that a correlation would be found between the number of children irradiated on protocol by an institution and the compliance rate of that institution with radiotherapy quality assurance (QA) guidelines. We also hypothesized that QA compliance would improve as the study progressed. METHODS AND MATERIALS: We scored the radiation dose as a minor deviation from protocol guidelines if the dose to the prescription point differed from the protocol by 6-10%, and a major deviation if it differed from protocol by > 10%. Treatment volumes were scored as a minor deviation if the margins were less than specified or the fields were excessively large. A major deviation was defined as the transection of a potential leukemia-bearing volume such as would be caused by blocking the cribriform plate, optic nerve, or temporal lobe. When the treating physician submitted a treatment plan and simulator film at the initiation of therapy to the Quality Assurance Review Center (QARC), a rapid turn-around review of the plan and suggestions for improvement was provided. At the end of therapy, all simulator and port films were reviewed at the QARC. RESULTS: We reviewed the data from 353 patients treated at 73 institutions in the United States, Canada, and Europe. Of these patients, 2% (n = 7) were not assessable for QA because of incomplete information. Minor quality deviations were found in 27.7% of patients (n = 98) and major deviations in 7.9% (n = 28). The frequency of major deviations for institutions placing 1-4 patients on study was 11% vs. 5.5% for institutions placing > or =5 patients (p < 0.09). The frequency of minor deviations was 28.6% for institutions placing 1-4 patients on study vs. 27.1% for institutions placing >5 patients (p not significant). The frequency of major deviations fell over time (1996-1997, 15.5% vs. 1998-2001, 4.7%, p < 0.001). The frequency of minor deviations did not (1996-1997, 29.9% vs. 1998-2001, 26.9%, p not significant). CONCLUSION: For a relatively simple radiotherapy field, with clearly written protocol guidelines and rapid turn-around corrections from QARC, the rate of minor deviations was no different between institutions placing 1-4 patients on study and those placing > or =5. A trend (p < 0.09) was noted, however, for major deviations to decrease as a function of institutional experience, as well as over time (p < 0.001), supporting the validity of the hypothesis that pediatric clinical experience matters in QA for C1 whole brain leukemia radiotherapy.

Brain↗

PubMed2000

Quantitative analysis of thymosin alpha1 in human serum by LC-MS/MS.

A high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to measure the thymosin alpha 1 (Talpha1) concentration in human serum. Tá1 in human serum was determined by solid phase extraction and reverse phase LC-MS/MS. The high-performance liquid chromatography (HPLC) system interfaced with the MS/MS system with a Turbo Ion spray interface. Positive ion detection and multiple reaction monitoring (MRM) mode were used for this human serum quantitation. Eight different concentration standards were used to establish the detection range. Six quality control (QC) and 2 matrix blanks were checked by calibration curves performed on the same day. The lower quantitation limit was 0.5 ng/mL Talpha1 in human serum. Calibration curves were established between 0.5 to 100 ng/mL by weighted linear regression. The correlation coefficients for different days were 0.9955 or greater. Quantitation of Talpha1 by the LC-MS/MS method is fast, accurate, and precise.

Calibration↗

PubMed2000

Intensity modulation for breast treatment using static multi-leaf collimators.

PURPOSE: To achieve more uniform dose distributions in breast cancer treatment using multiple sets of multi-leaf collimator (MLC) defined fields. Dose uniformity for many breast cancer patients can be significantly improved by using two or more sets of portals and the "hot" regions of a traditional treatment can be significantly reduced. METHODS AND MATERIALS: Patients for breast cancer treatment are immobilized with alpha cradle in the traditional arm-up position and have a CT scan in the treatment position. The target volume is delineated on the 5-mm thick CT slices that are obtained from the lower neck to well below the breast target volume. Medial and lateral tangential fields at conventional gantry angles are designed with the aid of digitally reconstructed radiographs (DRRs). The MLC, without collimator rotation, is used to shape the field to spare as much lung as possible. The wedges and relative weights of the beams are optimized to provide the best dose uniformity. For the patients with large dose inhomogeneity, a second set of fields is designed. The weight of the original set of fields is reduced (usually to approximately 90%) so that the "original hot" regions receive the prescription dose; the second set of fields delivers a supplemental dose to the "cold" region, typically approximately 10% of the total dose. The second set of fields has the same beam parameters but "treat" only the part of breast tissue that is "cool." Presently, the design of the reduced field is an iterative process. The process can be extended to more than two sets of portals to obtain the desired dose uniformity. RESULTS: With 3D planning and multiple MLC fields, dose uniformity in the treatment of breast patients was improved from 7%-22% to approximately 7%-15%. The volume receiving these high doses decreased significantly and shifted from the lung to the target. By keeping the gantry angles and wedges the same for the multiple fields, treatments can be delivered quickly and reliably. The internal mammary nodes (IM) can also be treated without including significant amount of lung or heart in the field. CONCLUSION: Dose uniformity can be significantly improved by using this intensity modulation technique to treat certain breast patients. With these static MLC fields creating the intensity modulation, the dose uniformity to the breast can be significantly improved and the hot region in lung reduced. There is no increase in setup complexity. The small increase in treatment time is insignificant.

Breast Neoplasms↗

PubMed1999

Self-association of the alpha subunit of phosphorylase kinase as determined by two-hybrid screening.

The structural organization of the (alphabetagammadelta)(4) phosphorylase kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of alpha subunit interactions. By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the alpha subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the alpha subunit containing its C-terminal region. Domain mapping of binary interactions between alpha constructs revealed two regions involved in the self-association of the alpha subunit: residues 833-854, a previously unrecognized leucine zipper, and an unspecified region within residues 1015-1237. The cognate binding partner for the latter domain has been inferred to lie within the stretch from residues 864-1059. Indirect evidence from the literature suggests that the interacting domains contained within the latter two, overlapping regions may be further narrowed to the stretches from 1057 to 1237 and from 864 to 971. Cross-linking of the nonactivated holoenzyme with N-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced intramolecularly cross-linked alpha-alpha dimers, consistent with portions of two alpha subunits in the holoenyzme being in sufficient proximity to associate. This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase. Additionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed.

Amino Acid Sequence↗

PubMed1999

Mg2+ induces conformational changes in the catalytic subunit of phosphorylase kinase, whether by itself or as part of the holoenzyme complex.

Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition (alpha beta gamma delta)4. Previous studies have revealed that the activity of its catalytic gamma subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb gamma79) has been generated against isolated gamma subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the gamma subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the gamma-calmodulin binary complex, and the free gamma subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb gamma79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the gamma subunit containing the epitope for mAb gamma79. The activating ligand Mg2+ also stimulated the binding of the PhK holoenzyme to immobilized mAb gamma79, as well as the binding of mAb gamma79 to immobilized gamma subunit. Thus, Mg2+ increases the accessibility of the mAb gamma79 epitope in both the isolated gamma subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg2+ on the quaternary structure of the PhK holoenzyme are directly mediated by the gamma subunit.

Animals↗

PubMed1998

Induction chemotherapy with docetaxel, cisplatin, fluorouracil, and leucovorin for squamous cell carcinoma of the head and neck: a phase I/II trial.

PURPOSE: A phase I/II trial of docetaxel, cisplatin, fluorouracil (5-FU), and leucovorin (TPFL5) induction chemotherapy for patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN). PATIENTS AND METHODS: Twenty-three previously untreated patients with stage III or IV SCCHN and Eastern Cooperative Oncology Group functional status less than or equal to 2 were treated with TPFL5. Postchemotherapy home support included intravenous fluids, prophylactic antibiotics, and granulocyte colony-stimulating factor (G-CSF). Docetaxel dose was escalated to determine the maximum-tolerated dose (MTD). Fifteen patients were treated with three cycles of TPFL5 at MTD. Patients who achieved either a partial response (PR) or complete response (CR) to three cycles of TPFL5 then received definitive twice-daily radiation therapy. Toxicity and clinical and pathologic response to TPFL5 were assessed. RESULTS: Twenty-three patients received a total of 69 cycles of TPFL5. The MTD was determined to be docetaxel 60 mg/m2. Dose-limiting toxicity (DLT) was neutropenia. Additional significant toxicities at MTD were nausea, mucositis, diarrhea, peripheral neuropathy, and sodium-wasting nephropathy. The overall response rate to TPFL5 was 100%, which included 14 of 23 (61%) clinical CRs and nine of 23 (39%) clinical PRs. Primary-site clinical and pathologic CR rates were 19 of 22 (86%) CRs and 20 of 22 (91%) CRs, respectively. Eight patients had less than a CR in the neck to chemotherapy and, therefore, had postradiation neck dissections, four of which were positive for residual tumor. CONCLUSION: TPFL5 is a tolerable induction regimen in patients with good performance status. The DLT is neutropenia with significant mucositis, diarrhea, peripheral neuropathy, and sodium-wasting nephropathy. The high response rates to TPFL5 justify further evaluation of this combination of agents in the context of formal clinical trials.

Adult↗

PubMed1997

Interactions of a bicyclic analog of colchicine with beta-tubulin isoforms alphabeta(II), alphabeta(III) and alphabeta(IV).

Tubulin exists as various isoforms, which differ in their assembly, drug-binding properties, and the dynamic properties of the microtubules they compose. One of the most striking differences in drug binding among the isoforms is observed with colchicine, which binds much better to the alphabeta(II) and alphabeta(IV) isoforms than to the alphabeta(III) isoform. Here we have studied the interaction of these isoforms with 2-methoxy-5-(2',3',4'-trimethoxyphenyl) tropone (MTPT), an analog of colchicine that lacks the B-ring. The kinetics of association and dissociation were studied fluorometrically, and the kinetic parameters for the two-step binding were determined for different beta-tubulin isoforms. The apparent on-rate constants for alphabeta(II), alphabeta(III) and alphabeta(IV) were 13358, 4558 and 10828 M(-1) s(-1), the off-rate constants (k(-2)) were 0.04, 0.03 and 0.02 s(-1), and the affinity constants are 3.33 x 10(5), 1.56 x 10(5) and 5.44 x 10(5) M(-1), respectively. The differences in kinetic parameters among different beta-tubulin isoforms are greatly reduced when the B-ring is removed. Our results indicate that the B-ring plays a major role in determining the isoform differences, and the results might be of importance for designing tissue-specific analogs of colchicine for cancer chemotherapy.

Colchicine↗

PubMed1993

Primary radiation therapy for endometrial carcinoma: a case controlled study.

PURPOSE: Primary radiation therapy is generally considered inferior to a surgical approach for patients with endometrial carcinoma and is reserved for patients with a high operative risk. These patients are usually elderly, have multiple medical problems and frequently die of intercurrent disease. To evaluate the efficacy of primary radiation therapy a case controlled analysis comparing corrected survival of patients treated with primary radiation to patients treated with surgical therapy with or without radiation therapy was performed. METHODS AND MATERIALS: Sixty-four patients treated with primary radiation therapy were retrospectively studied. A Kaplan-Meier product limit survival analysis was used to estimate survival among patients treated with primary radiation therapy. A case control study matched by clinical stage, tumor grade, and time of diagnosis was performed. The Mantel-Cox statistic was used to evaluated the equality of the survival curves. RESULTS: Primary radiation therapy was used to treat 9.0% of the patients with endometrial carcinoma during the study period. Cardiovascular disease, diabetes, age greater than 80 and morbid obesity were the most common indications. Ninety percent of patients had either Stage I or II disease. Forty-eight of the 64 patients (75%) completed treatment which included both teletherapy and brachytherapy. Ten patients received brachytherapy only. Twelve complications, both acute and chronic, occurred in eleven patients (17%). Intercurrent disease accounted for 13 of the 36 (36%) of the deaths. Clinical stage of disease and histologic grade of the tumor were significant predictors of survival, p = 0.0001 and p = 0.013, respectively. The case controlled study of Stage I and II patients treated by primary radiation therapy matched to surgically treated controls showed no statistical difference in survival. Dilatation and curettage after the completion of radiation therapy was predictive of local control, p = 0.003. CONCLUSION: Although surgery followed by tailored radiation therapy has become widely accepted therapy for Stage I and II endometrial carcinoma, even in patients who are a poor operative risk, the survival with primary radiation therapy is not statistically different.

Adult↗

PubMed1993

Tolerances in setup and dosimetric errors in the radiation treatment of breast cancer.

PURPOSE: Treatment failure in radiation therapy, as well as unexpected complications, can be associated with set up changes or variations that can cause deviations from the prescribed radiation dose distribution both inside and outside the target volume. The effect of various deviations from the planned setup on the delivery of the prescribed radiation dose to the desired treatment volume was studied. METHODS AND MATERIALS: Adding a second simulation was investigated as means of minimizing setup changes on treatment. The first simulation was used for planning the treatment and the second simulation was essentially a mock treatment. Dosimetric evaluations based on dose volume histograms were analyzed for each deviation in the setup. RESULTS: In 95% of the patients, the frequency of the changes in the setup parameters between the second simulation and the treatment setup were reduced significantly from the changes that occurred between the first simulation and the second simulation. The changes in isocenter coordinates up to +/- 1.0 cm have minimal effects (+/- 2%) on the dose distributions. Gantry angle variations up to +/- 4 degrees produce a change of less than +/- 5% in the dose distribution within the target volume. However, this angular variation resulted in additional tissue irradiation outside of the desired treatment field (about 10 cm3 for a large patient). A gantry angle variation of +/- 6 degrees can change the volume of tissue that receives the prescribed dose by at least +/- 10%. In addition, such a change can increase the volume of tissue outside the desired treatment field that is irradiated. CONCLUSION: It is concluded that individually, deviations in one of the parameters from the planned setup of +/- 1.0 cm in isocenter position and +/- 4 degrees in gantry angle do not produce significant deviations from the planned dose distribution. However, a significant change in dose distribution is observed if the setup parameters are concurrently changed. A second simulation may minimize the deviations of the treatment setup from the planned setup and maximize the precision in dose delivery to the target volume.

Breast Neoplasms↗

PubMed1993

Evidence for an alternative pathway for colchicine binding to tubulin, based on the binding kinetics of the constituent rings.

The kinetics of tropolone methyl ether binding to tubulin were measured by following the loss of colchicine binding capacity upon preincubation of tubulin with tropolone methyl ether. At 25 degrees C a bimolecular association rate constant of 2.7 (+/- 0.2) M-1 min-1 was determined, and from the temperature dependence an activation energy of 37 (+/- 8) kJ.mol-1 was calculated. By displacement experiments a dissociation rate constant of 2.9 (+/- 0.6) x 10(-2) min-1 was determined at 25 degrees C. The effect of 3',4',5'-trimethoxyacetophenone (TMA) is 2-fold. TMA reduces the apparent association rate constant of colchicine, indicating that it equilibrates very rapidly and reversibly with the colchicine binding site. From this reduction the binding constant for TMA can be obtained. At 25 degrees C a value of 112 (+/- 13) M-1 is estimated. The binding of TMA is practically thermoneutral. Preincubation of tubulin with TMA over 30 min not only reduces the subsequent binding rate constant of colchicine but also the amplitude. This indicates that TMA also binds slowly in a second mode or site. Stopped-flow kinetic studies reveal that fast TMA binding competes for the initial binding of colchicine. From these results it is concluded that colchicine binds initially with its trimethoxybenzene ring and in a subsequent step with the tropolone ring.

Acetophenones↗

PubMed1993

Pathology of congenital syphilis in rabbits.

We have developed a model for congenital syphilis in the rabbit. This report provides additional information on newborn tissue pathology in animals that were infected in utero. A total of 35 pregnancies were evaluated, each containing 6 to 12 newborns. In the infected group, the mortality was approximately 50%; of the live newborns, half appeared normal and half were hyperreflexic, weak, and runty. Gross pathology in the sickly newborns was quite prevalent and involved enlarged spleens with isolated spots of necrosis; enlarged livers that were overtly congested and hemorrhagic and had numerous granular, white spots; and brains with hemorrhage in the occipital area. Histopathology was apparent in different tissues. Lymphocytes, plasma cells, and vacuolated macrophages were prominent in livers, spleens, brains, and bones. A few actively motile treponemes were visualized by dark-field microscopy within extracts of spleen and within cerebrospinal fluid. Low numbers of treponemes were also demonstrated in sections of brain and liver by using the Warthin-Starry silver stain technique. Blood hematocrits were decreased, and extramedullary hematopoiesis was prominent within spleens and livers; this is consistent with anemia. This rabbit model exhibits many of the same pathologic features commonly found in human congenital syphilis.

Animals↗

PubMed1992

Rhom-2 expression does not always correlate with abnormalities on chromosome 11 at band p13 in T-cell acute lymphoblastic leukemia.

A frequent site for nonrandom recombination in T-cell acute lymphoblastic leukemia (T-ALL) is chromosome 11 at p13. The molecular characterization of a (7;11)(q35;p13) translocation showed that the translocation breakpoint was 2 kb 5' to the T-ALLbcr locus resulting in the juxtaposition of the T-cell receptor (TCR) beta gene to the rhom-2 gene locus. Northern blot analysis did not detect expression of the rhom-2 gene in the leukemic blasts of the (7;11) translocation. However, using a sensitive polymerase chain reaction (PCR)-based assay, the (7;11) translocation showed a trace expression of rhom-2 at a level of 0.01% of TCR-beta message. Because rhom-2 is considered a proto-oncogene, the significance of the trace expression of rhom-2 in the (7;11) translocation was investigated by comparing the level of rhom-2 expression in 7 additional T-ALLs, normal thymocytes, and CEM (pre-T) and HPB (mature-T) cell lines using the PCR assay. The CEM cells, normal thymocytes, and one patient, whose blasts had no cytogenetic abnormality of chromosome 11, did not express rhom-2 indicating that rhom-2 is not normally expressed in T cells. The other six T-ALLs fell into three categories: (1) two T-ALLs overexpressed rhom-2 in the presence of a translocation; (2) two T-ALLs had trace expression in the presence of a translocation; and (3) two T-ALLs had trace expression with no observable abnormalities on chromosome 11 at p13. Therefore, the data indicate that not all translocations at the T-ALLbcr locus result in overexpression of rhom-2. To account for the sharp contrast in rhom-2 expression seen in these T-ALLs, a model is proposed with a negative regulatory element in the T-ALLbcr locus that is disrupted in some of the cases leading to overexpression of rhom-2.

Base Sequence↗

PubMed1992

Brain and egg tubulins from antarctic fishes are functionally and structurally distinct.

The multitubulin hypothesis proposes that chemically distinct tubulins may possess different polymerization properties or may form functionally different microtubules. To test this hypothesis, we have examined the functional properties and the structures of singlet-specific nonneural and neural tubulins from Antarctic fishes. Tubulins were purified from eggs of Notothenia coriiceps neglecta, and from brain tissues of N. coriiceps neglecta or N. gibberifrons, by DEAE ion-exchange chromatography and cycles of microtubule assembly/disassembly. At temperatures between 0 and 20 degrees C, each of these tubulins polymerized efficiently in vitro to yield microtubules of normal morphology. Critical concentrations for polymerization of egg tubulin ranged from 0.057 mg/ml at 3 degrees C to 0.002 mg/ml at 18 degrees C, whereas those for brain tubulin at like temperatures were 4-10-fold larger. Polymerization of both tubulins was entropically driven, but the apparent standard enthalpy and entropy changes for microtubule elongation by egg tubulin (delta Happ0 = +33.9 kcal/mol, delta Sapp0 = +151 entropy units) were significantly greater than values observed for brain tubulin (delta Happ0 = +26.5 kcal/mol, delta Sapp0 = +121 entropy units). Egg tubulin was composed of approximately six alpha and two beta chains and lacked the beta III isotype, whereas brain tubulin was more complex (greater than or equal to 10 of each chain type). Furthermore, egg alpha tubulins were more basic, and their carboxyl termini more resistant to cleavage by subtilisin, than were the alpha chains of brain. We conclude that brain and egg tubulins from the Antarctic fishes are functionally distinct in vitro, due either to qualitative or quantitative differences in isotypic composition, to differential posttranslational modification of shared isotypes, or to both.

Animals↗

PubMed1992

Macrophage and lymphocyte functions are down-regulated in newborn rabbits.

We have developed an animal model for congenital syphilis. Treponema pallidum is injected intravenously into pregnant rabbits and fetuses are infected in utero. As a prelude to characterizing the immunologic consequences of fetal infection, it was necessary to expand on the baseline information about newborn rabbit immune capabilities. Studies were undertaken to determine splenic macrophage and T lymphocyte functions with emphasis on newer immunologic parameters. Newborns aged 2 weeks were compared to adults. Macrophage capabilities in newborn rabbits differed from those of their adult counterparts. These cells produced similar basal levels of interleukin 1 (IL-1) but failed to respond to the IL-1 stimulants of lipopolysaccharide (LPS) or T. pallidum. Macrophages also exhibited diminished levels of la expression and increased levels of prostaglandin E2 (PGE2) secretion. T lymphocyte functions were altered in newborn spleen preparations. Following concanavalin A (Con A) stimulation, interferon gamma production was half that of adults; in direct contrast, IL-2 production was twice that of adults. Con A-induced lymphocyte proliferation was markedly decreased in newborn preparations. This diminished response resulted from down-regulation rather than immaturity. When newborn splenic cells were stimulated with Con A in the presence of indomethacin, anti-transforming growth factor (anti-TGF), or exogenous IL-1/IL-2, better proliferation resulted. PGE2, which is well established as a down-regulator of newborn immune functions in human and mouse systems, also appears to play a role in suppressing newborn rabbit functions. TGF is a potent suppressor of a number of adult immunologic reactions. This is the first documentation of the potential role of this factor in down-regulating newborn immune capabilities. These findings provide a framework for future investigations of our congenital syphilis model.

Animals↗

PubMed1992

Expression of the V(D)J recombinase gene RAG-1 is tightly regulated and involves both transcriptional and post-transcriptional controls.

The V(D)J recombinase activating genes, RAG-1 and RAG-2, are coexpressed only in immature lymphocytes, and are sufficient and necessary for V(D)J recombination to occur in non-lymphoid cells. In order to examine control mechanisms operative in the regulation of RAG-1 and RAG-2, we have studied the pattern of expression of these genes in human pre-T cells, pre-B cells, and thymocytes treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA); an agent which mimics some of the lymphocyte maturation changes seen in vivo. The expression of RAG-1 and RAG-2 was tightly controlled in a rapid, yet very complex, manner with both positive and negative control elements operating. Treatment of immature lymphocytes with TPA caused the specific and rapid elimination of steady-state RAG-1 and RAG-2 RNA. Nuclear run-on assays showed that TPA completely repressed the transcription of RAG-1 within 30 min. In addition to repressing the transcription of RAG-1, TPA treatment caused the rapid and specific degradation of RAG-1 transcripts by decreasing the apparent half-life of RAG-1 mRNA more than two-fold. As judged by cycloheximide treatment of cells, the effects of TPA were not dependent on new protein synthesis. A labile transcriptional repressor, separate from the TPA-associated repression of transcription, was also active in cells transcribing RAG-1 and RAG-2 RNA. After depletion of this labile repressor by cycloheximide treatment, steady-state RAG-1 and RAG-2 RNA levels, and their transcription rates, were elevated four- to six-fold; but were still susceptible to elimination by TPA treatment. Treatment of pre-T CEM cells with interleukin-2, or theophylline (an agent that increases intracellular cAMP) resulted in a two-fold increase in RAG-1 RNA suggesting that lymphokines, either independently or through second messengers, may modulate RAG-1 and RAG-2 expression. The complex, rapid and precise regulation of RAG-1 and RAG-2 expression is consistent with the view that it is necessary for the cell to tightly regulate V(D)J recombinase levels; lower expression may result in inefficient recombination of Ig/TCR genes, whereas increased expression may lead to recombination errors that are deleterious to the cell.

Animals↗