Biomedical subjects

A Martinez

Publications and source records attributed to A Martinez.

At least 235 records · Page 13Linked to original sources

PubMed1996

Efficacy of epiroprim (Ro11-8958), a new dihydrofolate reductase inhibitor, in the treatment of acute Toxoplasma infection in mice.

Toxoplasma gondii is a major cause of focal encephalitis in patients with acquired immunodeficiency syndrome. Epiroprim, an inhibitor of dihydrofolate reductase, was evaluated in vitro and in a mouse model of acute infection for activity against T. gondii. The 50% inhibitory concentration (IC50) of epiroprim for T. gondii dihydrofolate reductase was 0.9 micromole, similar to that of pyrimethamine, but epiroprim was 650-fold more selective than pyrimethamine for T. gondii compared with human dihydrofolate reductase. While intraperitoneally administered epiroprim (300 mg/kg/day for 14 days) alone was ineffective in mice acutely infected with the RH strain of T. gondii, 100% survival was seen when it was combined with orally administered sulfadiazine (375 mg/kg/day), which alone was also ineffective. Increases in survival were seen in combination with doses of sulfadiazine as low as 0.375 mg/kg/day. Orally administered epiroprim combined with dapsone also prolonged survival. Thus epiroprim is an active and potentially less toxic alternative pyrimethamine for the treatment of toxoplasmosis.

Acute Disease↗

PubMed1996

Effects of continuous low-dose-rate brachytherapy on the rectum of the rat.

The dose-limiting factor in essentially all curative radiotherapy is damage to normal tissue. In spite of this, our understanding of radiation-induced damage to normal tissue leaves much to be desired. Damage to the rectum is often observed after clinical brachytherapy of pelvic malignancies. A model in which the rectum of the rat is irradiated with a uniform and reproducible dose of brachytherapy irradiation was developed. This system consists of an intracavitary applicator, which is used in conjunction with methods that reduce fecal flow, a jacket that restrains rat movement and a lead-shielded chamber to contain rats during low-dose-rate brachytherapy. Irradiation was applied in 48-h intervals separated by 48-h breaks during which the applicator was removed. Methods to analyze the sequelae of brachytherapy irradiation of the rectum were also developed. The total dose of 0.75 Gy/h continuous low-dose-rate brachytherapy required to produce obstruction in the rat rectum has been measured. The ED50 was 70.6 +/- 4.2 Gy. The length of time between irradiation and obstruction was also correlated with total dose. Other effects on the rectum that were related to dose were diarrhea and rectovaginal fistula. This irradiation system can produce uniform and reliable brachytherapy doses to the rat rectum. The late effects in normal tissues observed in this model are similar to those observed in humans after irradiation of the rectum. The model should be appropriate for analyzing relative changes in the rectum resulting from different dose rates and fractionation schemes relevant to brachytherapy.

Animals↗

PubMed1996

[Results of a randomized study comparing combination of navelbine-cisplatin to combination of vindesine-cisplatin and to navelbine alone in 612 patients with inoperable non-small cell lung cancer].

The combination of vindesine and cisplatin is considered a reference regimen in advanced NSCLC which has yielded a significant improvement in the duration of survival. A phase II study of a new semi-synthetic vinca alkaloid, Navelbine, reported an unusually high 29% response rate in stage III-IV NSCLC and a phase I-II study established the feasibility of the combination of Navelbine and cisplatin. We, therefore, designed a prospective randomized trial to compare Navelbine and cisplatin (NVB-P) to vindesine and cisplatin (VDS-P) and to evaluate whether the best of these regimens affords a survival benefit compared to Navelbine alone (NVB), an outpatient regimen. Forty-five centers included 612 patients in this study: 206 in NVB-P, 200 in VDS-P and 206 in NVB. Navelbine was given at a dose of 30 mg/m2 weekly, cisplatin at 120 mg/m2 on day 1, day 29 and then every 6 weeks and vindesine at 3 mg/m2 weekly for 6 weeks and then every other week. Treatment was continued until progression or toxicity. Patients' characteristics were similar in the three groups with 59% of patients presenting with metastatic disease. An objective response rate was observed in 30% of patients in NVB-P versus 19% in VDS-P (P = .02) and 14% in NVB (P < .001). The median duration of survival was 40 weeks in NVB-P compared to 32 weeks in VDS-P and 31 weeks in NVB. The comparison of survival between the three groups demonstrated an advantage for NVB-P compared to VDS-P (P = .04) and NVB (P = .02). Neutropenia was significantly higher in the NVB-P group (P < .001) and neurotoxicity more frequent with VDS-P (P < .004). Since our results have demonstrated that NVB-P yields a longer survival duration and a higher response rate than VDS-P or NVB alone, with acceptable toxicity, this combination should be considered a reference regimen in advanced NSCLC.

Adult↗

PubMed1996

[Treatment of inoperable non-small cell bronchogenic carcinoma with a combination of cisplatin and vinorelbine--preliminary evaluation].

The optimal therapeutic approach to patients with inoperable non-small-cell carcinoma is still a matter of discussion. The reason is that chemotherapy improves the quality of life only in some patients. It prolongs their life only by several weeks. In recent years in this indication a new cytostatic is tested--vinorelbine which when used in monotherapy achieves a therapeutic response in 16--30%. In clinical trials of phase 2 a combination of vinorelbine and cisplatinum was most effective and therefore it was selected for the third phase of clinical trials. In the Czech Republic a clinical investigation was made with the objective to verify published data on the success of treatment, evaluate undesirable effects and consider whether it is suitable for routine use. Cisplatinum (Platidiam Lachema) was administered--80 mg/m2 on the first day, vinorelbine (Navelbine Pierre Fabre)--30 mg/m2 on the first and eighth day. The cycle was repeated on the 22nd day. All patients were treated for 12 weeks. Then followed the first evaluation and treatment was continued only in patients with regression or stabilization of the disease. In the group of 126 patients in 44 (35%) partial and in 3 (2.3%) complete therapeutic responses were obtained. In 35 (27%) the disease was evaluated during treatment as stabilized, in 38 (30%) of the patients the disease progressed despite treatment. The tolerance of treatment was, when effective antiemetic treatment was used (ondansetrone or granisetrone), relatively satisfactory.

Antineoplastic Agents↗

PubMed1996

Primary graft failure after heart transplantation. Successful recovery with pneumatic biventricular assistance.

A 41-year-old male underwent orthotopic heart transplantation complicated by intraoperative acute allograft failure. The patient required immediate placement of a pneumatic biventricular assist device which was kept for 49 days until graft recovery resulted in successful explantation of the device. The patient was discharged from hospital on postoperative day 112. Management of primary cardiac allograft failure with mechanical ventricular assistance is discussed.

Adult↗

PubMed1996

[Classification of operative laparoscopy].

We are proposing a new approach in the classification of Operative Laparoscopy, divided in 3 areas: Area 1: Reproductive laparoscopy, that is exclusively to techniques of assisted reproduction, Area 2: Reconstructive laparoscopy that includes all the pathology of the female's inner organs its treatment in sterility and Area 3: Ablative laparoscopy related to the extraction of tissues and organs with gynecological pathology.

Female↗

PubMed1995

Neuropeptide Y is a vasoconstrictor and adrenergic modulator in the hamster microcirculation by acting on neuropeptide Y1 and Y2 receptors.

The microvascular effects of neuropeptide Y, and two analogs with preferential affinity for different neuropeptide Y receptor subtypes, were assessed by intravital microscopy on the hamster cheek pouch. The interaction of neuropeptide Y and its analogs with noradrenaline was also studied. Superfusion with 0.1-300 nM neuropeptide Y caused a concentration-dependent reduction in microvascular conductance that was paralleled by reductions in arteriolar and venular diameters. These effects of neuropeptide Y were equipotent with noradrenaline, but slower to develop and longer-lasting than that of noradrenaline. Neuropeptide Y did not affect permeability to macromolecules, as measured by extravasation of fluorescent dextran. The neuropeptide Y Y1 receptor agonist, [Leu31,Pro34]neuropeptide Y, mimicked neuropeptide Y with similar potency but shorter duration, while neuropeptide Y-(13-36), a neuropeptide Y Y2 receptor agonist, was at least 10-fold less potent than neuropeptide Y to induce a delayed and prolonged reduction in microvascular conductance. The joint superfusion of 1 nM neuropeptide Y plus 0.1 mu M noradrenaline did not cause synergism, nor even summation of effects, but reduced the contractile effect of noradrenaline. No synergism was observed after a 10 min priming with 1 nM neuropeptide Y, followed by its joint application with 0.1 mu M noradrenaline, but a significant vasodilation and hyperemia ensued upon stopping noradrenaline application. Priming with 1 nM [Leu31,Pro34]neuropeptide Y prolonged noradrenaline vasoconstriction without evidence of hyperemia. In contrast, priming with 1 nM neuropeptide Y-(13-36) significantly antagonized noradrenaline vasoconstriction. These findings indicate that both neuropeptide Y receptor subtypes are present in arterioles and venules of the hamster, and suggest that their activation with neuropeptide Y induces a rapid (Y1 receptor subtype activation) and a delayed (Y2 receptor subtype activation) vasocontractile response. The interaction with noradrenaline is complex, without evidence for synergism, but neuropeptide Y Y2 receptor activation seems to antagonize noradrenaline and/or to facilitate auto-regulatory vasodilation after the catecholamine-induced vasoconstriction.

Animals↗

PubMed1995

Dosimetric evaluation of the conformation of the multileaf collimator to irregularly shaped fields.

PURPOSE: The goal of this study was to evaluate the dosimetric characteristics of geometric MLC prescription strategies and compare them to those of conventional shielding block. METHODS AND MATERIALS: Circular fields, square fields, and 12 irregular fields for patients with cancer of the head and neck, lung, and pelvis were included in this study. All fields were shaped using the MLC and conventional blocks. A geometric criterion was defined as the amount of area discrepancy between the MLC and the prescription outline. The "least area discrepancy" (LAD) of the MLC conformation was searched by selecting the collimator angle, meanwhile keeping a preselected position along the width of the leaf into the prescribed field. Five LAD conventions were studied. These included the LAD-0, LAD-1/3, LAD-1/2, and LAD-2/3 that inserted the leaves at the 0, 1/3, 1/2, and 2/3 of the leaf end into the prescription field, respectively. In addition, the LAD optimization was applied to the transecting (TRN) approach for leaf conformation that prescribed an equal area of overblocking and underblocking under each leaf. Film dosimetry was performed in a 20 cm polystyrene phantom at 10 cm depth 100 cm from source to axis distance (SAD) for both 6 and 18 MV photons with each of the above MLC conformations and conventional blocks. The field penumbra width, defined as the mean of the separation between the 20% and 80% isodose lines along the normal of the prescription field edge, was calculated using both the MLC and conventional block film dosimetry and compared. In a similar way, the d20 is defined as the mean separation between the 20% isodose line and the prescription field edge, and the d80 is defined as the mean separation between the 80% isodose line and the prescription field edge. RESULTS: The field penumbra width for all MLC conventions was approximately 2 mm larger than that of the conventional block. However, there was a larger variation of the separation distribution in the penumbra region of the irregular fields for the MLC, which had a standard deviation of 1 mm (a factor of 5 larger than the conventional block). The dosimetry for the circular fields showed that the LAD-TRN, LAD-1/2, and LAD-1/3 approximated the conventional blocking well in terms of d20 and d80; however, no single convention produced the best conformation for both measures. The dosimetric result of the patient treatment fields was similar for all sites. The LAD-1/3, LAD-1/2, and LAD-TRN strategies conformed to within 1 to 1.5 mm of the d80 of the conventional block for both 6 MV and 18 MV, respectively. The LAD-1/2 and LAD-TRN conformations were virtually identical, although it is proven analytically that the LAD-1/2 convention has the least overall area discrepancy of all conventions. CONCLUSIONS: The five MLC conformation conventions resulted in similar dosimetric penumbrae for all field shapes studied. The LAD-1/3, LAD-TRN, and LAD-1/2 produced the more favorable approximation to conventional block. The field penumbra width, although useful for evaluating irregular field shapes, could not describe the large local variations in the penumbra along the field edge for the MLC. These local variations could be of clinical concern when they appear near vital organs. However, the variation in a local region can potentially be reduced by minimizing the jaggedness of the leaf steps in that local region. The dosimetric results were useful as guidelines for the clinicians in the evaluation and adjustment of MLC leaf positions.

Head and Neck Neoplasms↗

PubMed1995

Conformal prostate brachytherapy: initial experience of a phase I/II dose-escalating trial.

PURPOSE: To improve treatment results on prostatic adenocarcinoma, conformal radiation therapy (CRT) has been used. Two major drawbacks of external CRT are: (a) internal organ motion/daily set-up variations, and (b) exclusion of several patients for CRT based on poor geometrical relationships as identified by three dimensional (3D) treatment planning. To overcome the above problems, we began the first prospective Phase I/II dose-escalating clinical trial of conformal brachytherapy (CB) and concurrent external beam irradiation. METHODS AND MATERIALS: Fifty-nine patients with T2b-T3c prostatic adenocarcinoma received 176 transperineal ultrasound-guided conformal high-dose rate (HDR) boost implants. All patients received concomitant external beam pelvic irradiation. Dose escalation of the three HDR-CB fractions proceeded as follows: 5.5 Gy (30 patients), 6 Gy (20 patients), and 6.5 Gy (9 patients). The CB dose was prescribed to the prostate contour as outlined using an online biplanar transrectal ultrasound probe. The urethra, anterior rectal wall, and prostate boundaries were identified individually and outlined at 5 mm intervals from the base to the apex of the gland. The CB using real-time ultrasound guidance with interactive online isodose distributions was performed on an outpatient basis. As needles were placed into the prostate, corrections for prostate displacement were recorded and the isodose distributions were recalculated to represent the new relationship between the needles, prostate, and normal structures. No computerized tomography (CT) planning or implant preplanning was required. RESULTS: No patient was rejected based on poor geometrical relation of pelvic structures. In every implant performed, prostate displacement was noted. Craniocaudal motion of the gland ranged from 0.5-2.0 cm (mean = 1.0 cm), whereas lateral displacement was 0.1-0.4 cm. With the interactive online planning system, organ motion was immediately detected, accounted for, and corrected prior to each HDR treatment. The rectal dose has ranged from 45 to 87%, and the urethral dose from 97 to 112% of the prostate dose. It is significant to note that operator dependence has been completely removed because the interactive online planning system uniformly guides the physicians. CONCLUSIONS: With ultrasound guidance and the interactive online dosimetry system, organ motion (as compared to external beam) is insignificant because it can be corrected during the procedure without increasing target volume margins. Common pitfalls of brachytherapy, including operator dependence and difficulty with reproducibility, have been eliminated with the intraoperative online planning system.

Adenocarcinoma↗

PubMed1995

Effects of treatment setup variation on beam's eye view dosimetry for radiation therapy using the multileaf collimator vs. the cerrobend block.

PURPOSE: The purpose of this study is to quantify and compare retrospectively the effects of treatment setup variation on beam's eye view (BEV) dosimetry for radiation therapy using a multileaf collimator (MLC) vs. cerrobend block. METHODS AND MATERIALS: A study was performed on a group of 18 patients with cancer of the head and neck, lung, and pelvis who were treated with irregularly shaped fields. The BEV dosimetry of the fields shaped with cerrobend blocks and the MLC was measured with films at the depth of dose prescription in a solid water phantom. A "one-half-leaf" insertion convention was used to shape the MLC. In addition, an average of 15 sequential daily port films was taken per patient during the course of radiotherapy. The port films were aligned with the prescription film for each patient. Systematic error and random error of treatment setup for each patient were calculated. The effects of setup variation were incorporated by convolving the patient portal imaging data with the corresponding BEV film dosimetry. Two parameters were used to quantify the BEV dosimetry. First, the field penumbra width was calculated, which represented the average of the normal separations between 20 and 80% isodose lines along the prescription outline. Second, the ratio of areas covered by the 90 and 20% isodose lines, A90/20, was determined. The BEV dosimetry was then characterized with and without the effects of treatment setup variation. In addition, the difference in BEV dosimetry between the cerrobend block and the MLC was used to estimate the corresponding changes in tumor control probability (TCP). These changes were also compared to the changes in TCP for the treatment with or without the effects of random setup variation. RESULTS: With or without daily setup variation, the use of cerrobend block was more favorable than the MLC in terms of the field penumbra width and A90/20 for all treatment sites. In the absence of daily variation, the MLC field penumbra width was on average 1.3 mm larger than that of the cerrobend block, and 0.9 mm larger in the presence of daily setup variation. Similarly, the ratio A90/20 of the cerrobend block was on average 0.03 larger than that of MLC without daily setup variation, and 0.02 with daily setup variation. The difference in field penumbra width and A90/20 between the MLC and the cerrobend block was slightly reduced due to the effects of daily setup variation. For both the cerrobend block and the MLC, daily setup variation produced a significant increase in the field penumbra width, 2.3 mm for the cerrobend block and 1.9 mm for the MLC, and a decrease in the A90/20, 0.06 for the former and 0.05 for the latter. The change due to the daily setup variation was about a factor of 2 larger than the changes due to replacing the cerrobend block with the MLC. Using the TCP model, the change in TCP due to the daily setup variation was more than a factor of 3 larger than the change in TCP due to replacing the cerrobend block with the MLC. It was noted that the average changes in the penumbra, the A90/20 and the TCP calculated for the patient population did not adequately describe the changes for the individual patient. CONCLUSION: Our results do not show significant dosimetric differences between the MLC and the cerrobend block in conventional radiation treatment, whether or not daily setup variation was taken into consideration. The effects of daily setup variation alone produced a larger dosimetric change. The same results were obtained when the data were applied to calculate changes in TCP. For optimal radiation therapy, efforts should be concentrated on reducing daily setup variation. Our results also demonstrate the importance of frequent evaluation of MLC treatment using electronic portal imaging devices.

Equipment Design↗

PubMed1995

Ultrasound-guided high dose rate conformal brachytherapy boost in prostate cancer: treatment description and preliminary results of a phase I/II clinical trial.

PURPOSE: To improve results for locally advanced prostate cancer, a prospective clinical trial of concurrent external beam irradiation and fractionated iridium-192 (Ir-192) high dose rate (HDR) conformal boost brachytherapy was initiated. METHODS AND MATERIALS: Between November 1991 and February 1994, 99 implants were performed on 33 patients with prostatic adenocarcinoma at William Beaumont Hospital. Using AJCC staging criteria, 9 patients had T2b tumors, 17 patients had T2c tumors, and 7 patients had T3 disease. Patients were treated with (a) 45.6 Gy whole pelvis external irradiation and (b) three HDR fractions of 5.5 Gy each (18 patients) or 6 Gy each (15 patients) to the prostate. Transperineal needle implants using real-time ultrasound guidance with interactive on-line isodose distributions were performed on an outpatient basis during weeks 1, 2, and 3 of external irradiation. Acute toxicity was scored using the Radiation Therapy Oncology Group (RTOG) morbidity grading system. RESULTS: This technique of concurrent external pelvic irradiation and conformal HDR brachytherapy was well tolerated. No significant intraoperative or perioperative complications occurred. Three patients (9%) experienced Grade 3 acute toxicity (two dysuria and one diarrhea). All toxicities were otherwise Grades 1 or 2 and were primarily as expected from pelvic external irradiation. Persistent implant-related toxicities included Grades 1-2 perineal pain (12%) and hematospermia (15%). Median follow-up time was 13 months. Serum prostatic-specific antigen (PSA) levels normalized in 91% of patients (29 out of 32) within 1-14 months (median 2.8 months) after irradiation. PSA levels were progressively decreasing in the other three patients at last measurement. Prospectively planned prostatic rebiopsies done at 18 months in the first 10 patients were negative in 9 out of 10 (90%). CONCLUSIONS: Acute toxicity has been acceptable with this unique approach using conformal high dose rate Ir-192 boost brachytherapy with concurrent external irradiation. The initial tumor response as assessed by serial PSA measurement and rebiopsy is extremely encouraging. Dose escalation will proceed in accordance with the protocol guidelines. Further patient accrual and longer follow-up will allow comparison to other techniques.

Adenocarcinoma↗

PubMed1995

A multileaf collimator field prescription preparation system for conventional radiotherapy.

PURPOSE: The purpose of this work is to develop a prescription preparation system for efficient field shaping using a multileaf collimator that can be used in community settings as well as research institutions. The efficiency advantage of the computer-controlled multileaf collimator, over cerrobend blocks, to shape radiation fields has been shown in conformal treatments, which typically require complete volumetric computerized tomographic data for three-dimensional radiation treatment planning--a utility not readily available to the general community. As a result, most patients today are treated with conventional radiation therapy. Therefore, we believe that it is very important to fully use the same efficiency advantage of multileaf collimator as a block replacement in conventional practice. METHODS AND MATERIAL: The multileaf collimator prescription preparation system developed by us acquires prescription images from different sources, including film scanner and radiation treatment planning systems. The multileaf collimator angle and leaf positions are set from the desired field contour defined on the prescription image, by minimizing the area discrepancies. Interactive graphical tools include manual adjustment of collimator angle and leaf positions, and definition of portions of the field edges that require maximal conformation. Data files of the final leaf positions are transferred to the multileaf collimator controller via a dedicated communication link. RESULTS: We have implemented the field prescription preparation system and a network model for integrating the multileaf collimator and other radiotherapy modalities for routine treatments. For routine plan evaluation, isodose contours measured with film in solid water phantom at prescription depth are overlaid on the prescription image. Preliminary study indicates that the efficiency advantage of the MLC over cerrobend blocks in conformal therapy also holds true for conventional treatments. CONCLUSION: Our model of computer-controlled prescription, evaluation, and treatment using multileaf collimators can be effectively implemented in both community settings and research institutions. The resultant increase in treatment efficiency and accuracy is now available for conventional radiotherapy.

Equipment Design↗

PubMed1995

Changes in TRH and its degrading enzyme pyroglutamyl peptidase II, during the development of amygdaloid kindling.

Pyroglutamyl peptidase II (PPII) is a neuronal ectoenzyme responsible for thyrotropin releasing hormone (TRH) degradation at the synaptic cleft. PPII, heterogeneously distributed in different brain regions and adenohypophysis, is regulated under various endocrine conditions where TRH is involved in thyrotropin or prolactin regulation but only at the adenohypophyseal level. TRH can downregulate PPII activity in cultured adenohypophyseal cells. TRH present in extrahypothalamic brain areas has been postulated to serve as a neuromodulator and levels of this peptide increase in amygdala, hippocampus and cortex after electrical stimulation (kindling or electroshock). To study whether brain PPII could be regulated in conditions that stimulate TRHergic neurons, TRH and PPII activity were determined during the development of amygdaloid kindling in the rat. TRH levels increased from stage II to V in amygdala and hippocampus in the ipsi- and contralateral side to stimulation. In n. accumbens a decrease, compared to sham was observed at stage II, but levels raised through stage V. In contrast, PPII activity was increased at stage II, in amygdala of both sides and in hipppocampus, frontal cortex, n. accumbens and hypothalamus of the contralateral side; levels decreased at stage V to sham values in most structures (except amygdala and hippocampus where the activity was 30% below controls). These results suggest that PPII activity in the central nervous system can be regulated in conditions known to affect TRHergic neurons.

Aminopeptidases↗

PubMed1995

A human (3.3 kb) haptoglobin-CAT transgene is modulated in lungs of transgenic mice by inflammation.

Four independent lines of transgenic mice were produced carrying integrated copies of a chimeric gene composed of 3.3 kb of the human haptoglobin 5' regulatory region fused to the CAT (chloramphenicol acetyl transferase) reporter gene. Although the endogenous mouse haptoglobin (Hp) and human haptoglobin (HP) genes express mainly in liver and lung, expression of the human 3.3-kb HP-CAT transgene was not detected until after induction of inflammation and then only in lungs. The results indicated that the transgene maintained the regulatory DNA elements required for lung specific responsiveness to inflammation in vivo but lacked the DNA sequence required for robust expression in liver. The DNA sequence(s) responsible for the normally high level of HP expression in liver either reside outside the 3.3-kb regulatory region of the HP chimeric gene or this region contains a suppressor sequence affecting tissue specific expression in the liver.

Animals↗

PubMed1995

Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme.

Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors. In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E. coli maltose-binding protein (MBP). The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield. By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin. Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c. After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography. The yield of highly purified hPAH was approx. 10 mg/l of culture. The specific activity of the isolated recombinant enzyme was high (i.e. 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver. The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.

ATP-Binding Cassette Transporters↗

PubMed1995

Outcome and prognostic factors for local recurrence in mammographically detected ductal carcinoma in situ of the breast treated with conservative surgery and radiation therapy.

PURPOSE: We have retrospectively reviewed our institution's experience treating a predominantly mammographically detected population of ductal carcinoma in situ (DCIS) patients with conservative surgery and radiation therapy (CSRT) to determine outcome and prognostic factors for local recurrence. METHODS AND MATERIALS: Between January 1, 1982 and December 31, 1988, 52 consecutive cases of DCIS of the breast were treated with CSRT at William Beaumont Hospital. Forty-six (88%) were mammographically detected nonpalpable lesions. All patients underwent at least an excisional biopsy and 28 (54%) were reexcised. The axilla was surgically staged in 41 (79%) and all were N0. The entire breast was irradiated to 45-50 Gy over 5-6 weeks. The tumor bed was boosted in 49 (94%) so that the minimum dose was 60 Gy. The three patients not boosted received a minimum dose of 50 Gy to the entire breast. Pathologic materials were reviewed by one of the authors. RESULTS: The predominant DCIS pattern was comedo in 40%, cribriform in 28%, solid in 17%, and micropapillary in 15%. The predominant nuclear grade was Grade I in 51%, Grade II in 49%, and Grade III in 0%. The median follow-up is 68 months. There have been three recurrences in the treated breast at a median time to failure of 30 months. The 5- and 8-year actuarial local recurrence rate is 6%. One patient recurred with invasive ductal cancer at 28 months, and the other two recurrences were DCIS at 30 and 50 months. All three patients were treated with salvage mastectomy. The patient who recurred locally with an invasive cancer developed metastasis and died at 64 months. The 5- and 8-year actuarial cause-specific survival rates are 100% and 97%, respectively. The following pathologic factors were analyzed for an association with local recurrence: predominant DCIS histology, predominant nuclear grade, and highest nuclear grade. Of these, the predominant nuclear grade was the best predictor of local recurrence (p = 0.070). No clinical or treatment related factor analyzed was associated with local recurrence. CONCLUSION: Our results indicate that excellent local control (94%) at 8 years is obtainable after CSRT in a mammographically detected population of patients with DCIS. The predominant nuclear grade was the only factor found that may be associated with local recurrence.

Adult↗

PubMed1995

A new model for "accept or reject" strategies in off-line and on-line megavoltage treatment evaluation.

PURPOSE: With megavoltage on-line imaging, a temporal record of the treatment setup variation is made available during the course of radiotherapy. This work describes the theoretical framework where the imaging data is incorporated as feedback for deriving off-line and on-line "accept or reject" decisions to improve the quality of radiotherapy on an individual patient basis. METHODS AND MATERIALS: A dynamic model of treatment setup variation is developed to describe: (a) the variations of multifraction treatment position and (b) the variation of measurement. Based on this model, a confidence region of subsequent treatment position is estimated from measurements of previous treatment position. The confidence region is compared with an allowance region of the treatment position, representing the acceptable margin of setup variation. The difference of the two regions are then evaluated to derive off-line and on-line "accept or reject" treatment decisions. RESULTS: The model was simulated retrospectively using sequential daily images from different treatment sites. "Accept or reject" decisions were produced for on-line and off-line applications. The results also demonstrate the ability of the model to include the effects of setup variation that drifted during the course of treatment. CONCLUSION: The preliminary result demonstrates that "accept or reject" treatment decisions can be derived quantitatively from on-line imaging data. The model shows potential to guide the physician and therapist in implementing efficient and accurate treatment adjustments. Further work is required to test the model in the clinical setting.

Humans↗

PubMed1995

Upstream stimulatory factor proteins are major components of the glucose response complex of the L-type pyruvate kinase gene promoter.

L-type pyruvate kinase (L-PK) gene transcription is induced by glucose through its glucose response element (GlRE) composed of two degenerated E boxes able to bind in vitro ubiquitous upstream stimulator factor (USF) proteins. Here we demonstrate in vivo, by transient transfections in hepatoma cells, that (i) native USF proteins synthesized from expression vectors can act as transactivators of the L-PK promoter via the GlRE, stimulating transcription without glucose and, therefore, decreasing the glucose responsiveness of the promoter; (ii) expression of the truncated USF proteins, able to bind the GlRE but devoid of the NH2-terminal activation domain, represses the activation of the L-PK promoter by glucose; and (iii) a similar repression of the glucose effect is observed upon expression of mutant USF proteins devoid of the basic DNA binding domain, able to dimerize with endogenous USF but not to bind the GlRE. We conclude that USF proteins are components of the transcriptional glucose response complex assembled on the L-PK gene promoter.

Base Sequence↗