Tumor-induced hypophosphatemic rickets in an adolescent boy--clinical presentation, diagnosis, and histological findings in growth plate and muscle tissue.

CONTEXT: The mechanism behind disabling muscle weakness in tumor-induced hypophosphatemic rickets is obscure. Histological investigation of growth plate tissue of patients with tumor-induced osteomalacia has so far not been reported. PATIENT: A mesenchymal tumor was detected in the left distal fibula by (68)Ga-DOTATOC in a 17-yr-old boy with adolescent onset of severe hypophosphatemic rickets. Disabling muscle weakness improved within days after surgery, and normal mobility was restored within months. METHODS AND RESULTS: The resected tissue included part of the growth plate allowing immunohistochemical investigation. Positive staining of FGF23 was found in the tumor cells and in hypertrophic chondrocytes, osteoblasts, and osteoclasts of the adjacent growth plate. This distribution matched that found in growth plate tissue of a healthy control. We found positive staining for the somatostatin receptor not only in the tumor but also within the growth plate and adjacent bony tissue in the patient and the healthy control. Muscle tissue provided evidence for a partial defect in respiratory chain complexes I-IV. Biochemical markers were nearly or completely restored to normal 12 months after surgery. CONCLUSIONS: Hypertrophic growth plate chondrocytes are a target or source of FGF23 in tumor-induced osteomalacia. Low serum phosphate, FGF23, or other factors produced by the tumor may interfere with mitochondrial function.

If you can't see it, you can miss it: the role of biomedical imaging in radiation oncology.

During the last three decades, 3D imaging as X-ray computerised tomography and magnetic resonance imaging was introduced to characterise tumour morphology for an improved delineation of the gross target volume and the clinical target volume. At present, the time has come to also start the assessment and correction of the temporal changes of the target volume. This is the basis of 'image-guided radiotherapy' (IGRT), which is characterised by the integration of 2D and 3D imaging modalities into the radiotherapy workflow. The vision is to detect deformations and motion between radiotherapy fractions (inter-fractional IGRT) and during beam delivery (intra-fractional IGRT). Another challenge in radiotherapy is to develop concepts to include and integrate biological imaging into radiotherapy, first by extending the morphological towards a biological planning target volume and second by delivering appropriate inhomogeneous dose distributions, e.g. with the new tools of photon- and particle- Intensity Modulated Radiotherapy (IMRT) techniques ('dose painting').

The design, physical properties and clinical utility of an iris collimator for robotic radiosurgery.

Robotic radiosurgery using more than one circular collimator can improve treatment plan quality and reduce total monitor units (MU). The rationale for an iris collimator that allows the field size to be varied during treatment delivery is to enable the benefits of multiple-field-size treatments to be realized with no increase in treatment time due to collimator exchange or multiple traversals of the robotic manipulator by allowing each beam to be delivered with any desired field size during a single traversal. This paper describes the Iris variable aperture collimator (Accuray Incorporated, Sunnyvale, CA, USA), which incorporates 12 tungsten-copper alloy segments in two banks of six. The banks are rotated by 30 degrees with respect to each other, which limits the radiation leakage between the collimator segments and produces a 12-sided polygonal treatment beam. The beam is approximately circular, with a root-mean-square (rms) deviation in the 50% dose radius of <0.8% (corresponding to <0.25 mm at the 60 mm field size) and an rms variation in the 20-80% penumbra width of about 0.1 mm at the 5 mm field size increasing to about 0.5 mm at 60 mm. The maximum measured collimator leakage dose rate was 0.07%. A commissioning method is described by which the average dose profile can be obtained from four profile measurements at each depth based on the periodicity of the isodose line variations with azimuthal angle. The penumbra of averaged profiles increased with field size and was typically 0.2-0.6 mm larger than that of an equivalent fixed circular collimator. The aperture reproducibility is < or =0.1 mm at the lower bank, diverging to < or =0.2 mm at a nominal treatment distance of 800 mm from the beam focus. Output factors (OFs) and tissue-phantom-ratio data are identical to those used for fixed collimators, except the OFs for the two smallest field sizes (5 and 7.5 mm) are considerably lower for the Iris Collimator. If average collimator profiles are used, the assumption of circular symmetry results in dose calculation errors that are <1 mm or <1% for single beams across the full range of field sizes; errors for multiple non-coplanar beam treatment plans are expected to be smaller. Treatment plans were generated for 19 cases using the Iris Collimator (12 field sizes) and also using one and three fixed collimators. The results of the treatment planning study demonstrate that the use of multiple field sizes achieves multiple plan quality improvements, including reduction of total MU, increase of target volume coverage and improvements in conformality and homogeneity compared with using a single field size for a large proportion of the cases studied. The Iris Collimator offers the potential to greatly increase the clinical application of multiple field sizes for robotic radiosurgery.

Proteomics in cancer.

Proteomic studies have generated numerous datasets of potential diagnostic, prognostic, and therapeutic significance in human cancer. Two key technologies underpinning these studies in cancer tissue are two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Although surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF)-MS is the mainstay for serum or plasma analysis, other methods including isotope-coded affinity tag technology, reverse-phase protein arrays, and antibody microarrays are emerging as alternative proteomic technologies. Because there is little overlap between studies conducted with these approaches, confirmation of these advanced technologies remains an elusive goal. This problem is further exacerbated by lack of uniform patient inclusion and exclusion criteria, low patient numbers, poor supporting clinical data, absence of standardized sample preparation, and limited analytical reproducibility (in particular of 2D-PAGE). Despite these problems, there is little doubt that the proteomic approach has the potential to identify novel diagnostic biomarkers in cancer. In therapeutic proteomics, the challenge is significant due to the complexity systems under investigation (i.e., cells generate over 10(5) different polypeptides). However, the most significant contribution of therapeutic proteomics research is expected to derive not from single experiments, but from the synthesis and comparison of large datasets obtained under different conditions (e.g., normal, inflammation, cancer) and in different tissues and organs. Thus, standardized processes for storing and retrieving data obtained with different technologies by different research groups will have to be developed. Shifting the emphasis of cancer proteomics from technology development and data generation to careful study design, data organization, formatting, and mining is crucial to answer clinical questions in cancer research.

Kilovoltage CT using a linac-CT scanner combination.

Modern radiotherapy techniques such as intensity modulation are capable of generating complex dose distributions whose high dose areas tightly conform to the tumour target volume, sparing critical organs even when they are located in close proximity. This potential can only be exploited to its full extent when the accumulated dose actually delivered over the complete treatment course is sufficiently close to the dose computed on the initial CT scan used for treatment planning. Exact patient repositioning is mandatory, but also other sources of error, e.g. changes of the patient's anatomy under therapy, should be taken into account. At the German Cancer Research Center, we use a combination of a linear accelerator and a CT scanner installed in one room and sharing the same couch. It allows the quantification and correction of interfractional variations between planning and treatment delivery. In this paper, we describe treatments of prostate, paraspinal and head and neck tumours. All patients were immobilized by customized fixation devices and treated in a stereotactic setup. For each patient, frequent CT scans were taken during the treatment course. Each scan was compared with the original planning CT using manual checks and automatic rigid matching algorithms. Depending on the individual case, the adaptation to variations was carried out offline after several fractions or in real-time between the CT scan and linac irradiation. We discuss the techniques for detecting and correcting interfractional errors and outline the procedural steps of a linac-CT scanner-supported radiation treatment course.

Comparative efficiency of the multi-leaf collimator and variable-aperture collimator in intensity-modulated radiotherapy.

The potential of the variable-aperture collimator (VAC) in intensity-modulated radiation therapy (IMRT) has been evaluated by comparing its performance with that of the multi-leaf collimator (MLC). This comparison used a decomposition algorithm to find the series of collimator segments that would treat a given intensity-modulated beam (IMB). Collimator performance was measured using both the number of segments required to complete the IMB and the monitor-unit efficiency of the treatment. The VAC was modelled with aperture sizes from 4 x 4 cm to 20 x 20 cm, and these apertures were allowed to be located anywhere within the IMB. To enable a direct comparison, a similar scanning MLC was modelled at the same range of aperture sizes. Using both collimators, decompositions were run on 10 x 10 and 20 x 20 random IMBs with integer bixel values ranging from 1 to 10. Clinical IMBs from lung, head and neck, and pelvic patients were taken from a Pinnacle treatment-planning system and tested in the same manner. It was found that for all treatment sites, a small, scanning MLC performs as well or better than an equivalent sized VAC in both number of segments and monitor-unit efficiency, and would be an efficient choice for centres looking for a simple collimator for IMRT.

Changes in the ratio of type-I and type-II collagen expression during monolayer culture of human chondrocytes.

We compared the changes in the ratio of type-I and type-II collagen in monolayer cultures of human articular chondrocytes (HAC). HAC were isolated from samples of cartilage from normal joints and cultivated in monolayer for up to 46 days. Expression of collagen type-I and type-II was determined by immunocytochemistry, Western blotting, and the nested reverse transcription polymerase chain reaction (RT-PCR), and quantified by real-time PCR. The transition from a spherical morphology to the flattened morphology of an anchorage-dependent culture was accompanied by a rapid change in the collagen phenotype with the replacement of collagen type II by collagen type I. This was confirmed by immunocytochemistry and Western blotting between days 21 and 28. Using techniques for the analysis of gene transcription (nested RT-PCR and real-time PCR), a complete switch of collagen gene expression was not observed. Expression of collagen type I increased 100-fold during the culture time. That of collagen type II was found during the entire period and decreased more than 100-fold. The main finding was that expression of the genes encoding collagen type I and II was highly time-dependent and the ratio of collagen type II to I (CII/CI), defined as an index of cell differentiation, was significantly higher (215- to 480-fold) at the beginning of the culture. At the end of the experimental culture time, ratios between 0.1 and 1 were reached.

An application framework for computer-aided patient positioning in radiation therapy.

The importance of exact patient positioning in radiation therapy increases with the ongoing improvements in irradiation planning and treatment. Therefore, new ways to overcome precision limitations of current positioning methods in fractionated treatment have to be found. The Department of Medical Physics at the German Cancer Research Centre (DKFZ) follows different video-based approaches to increase repositioning precision. In this context, the modular software framework FIVE (Fast Integrated Video-based Environment) has been designed and implemented. It is both hardware- and platform-independent and supports merging position data by integrating various computer-aided patient positioning methods. A highly precise optical tracking system and several subtraction imaging techniques have been realized as modules to supply basic video-based repositioning techniques. This paper describes the common framework architecture, the main software modules and their interfaces. An object-oriented software engineering process has been applied using the UML, C + + and the Qt library. The significance of the current framework prototype for the application in patient positioning as well as the extension to further application areas will be discussed. Particularly in experimental research, where special system adjustments are often necessary, the open design of the software allows problem-oriented extensions and adaptations.

Intensity-modulated radiation therapy using a variable-aperture collimator.

This paper extends some earlier concepts of using a tertiary mask plus jaws for delivering IMRT without a multileaf collimator. The new concept is to sweep a variable-aperture collimator (VAC) across the space of the intensity-modulated beam (IMB) to be delivered and to strip this IMB down into multiple-static-field components, each deliverable with the VAC. The stripping algorithm is described and it is shown, for several designs of VAC, that the mean number of field components and mean number of monitor units is less using the VAC than would be required for a jaws-only (JO) decomposition. The VAC would be simpler to construct than several previously suggested jaws-plus-mask (J+M) combinations. As well as describing a simple VAC for the use with jaws, we propose a design concept of a hybrid VAC. We also show that adding the potential to rotate the simple or hybrid VAC for some components relative to the field to be modulated is advantageous.

Collagen expression in tissue engineered cartilage of aged human articular chondrocytes in a rotating bioreactor.

This study describes the culture and three-dimensional assembly of aged human articular chondrocytes under controlled oxygenation and low shear stress in a rotating-wall vessel. Chondrocytes cultured in monolayer were released and placed without any scaffold as a single cell suspension in a rotating bioreactor for 12 weeks. Samples were analyzed with immunohistochemistry, molecular biology and electron microscopy. During serial monolayer cultures chondrocytes dedifferentiated to a "fibroblast-like" structure and produced predominantly collagen type I. When these dedifferentiated cells were transferred to the rotating bioreactor, the cells showed a spontaneous aggregation and formation of solid tissue during the culture time. Expression of collagen type II and other components critical for the extracellular cartilage matrix could be detected. Transmission electron microscopy revealed a fine network of randomly distributed collagen fibrils. This rotating bioreactor proves to be a useful tool for providing an environment that enables dedifferentiated chondrocytes to redifferentiate and produce a cartilage-specific extracellular matrix.

Intensity-modulated radiotherapy - technology and clinical applications.

Intensity-modulated radiotherapy (IMRT) is one of the most important developments in radiooncology of the last years. As an extension of 3D conformal radiotherapy, it provides the possibility of delivering a high radiation dose to the tumor tissue, protecting radiosensible critical organs nearby or even surrounded by the target. This is realized by the overlaying of beams from different directions, which are not homogeneous like in conventional radiotherapy, but inhomogeneous themselves. The sum of the beams from all directions forms finally the desired complex-shaped dose distribution. Because choosing the right intensity modulation of each beam is a nontrivial problem, the best treatment plan could only hardly be found in a trial-and-error process, but has to be computer- optimized. Therefore, IMRT has always to be spoken together with the term of 'inverse planning'. This describes the approach of telling the computerized planning system the desired radiation doses to the target and the allowed maximum doses to organs at risk (OAR), then letting the system compute the optimal modulated beams, called 'fluence profiles', by an iterative algorithm. Because the ideal dose distribution, giving 100% dose to the tumor and 0% dose to the healthy tissue, is never realizable, inverse planning has to be understood as an optimization problem. The present article shows the technical bases of IMRT and inverse planning and then describes important clinical applications.

Comparison of forward planned conformal radiation therapy and inverse planned intensity modulated radiation therapy for esthesioneuroblastoma.

The purpose of this study was to compare dose distribution of inverse planned intensity modulated radiation therapy (IMRT) with that of conformal radiation therapy (SCRT) in the treatment of esthesioneuroblastoma, and to report initial clinical results. 13 patients with esthesioneuroblastoma were planned both with IMRT and SCRT using complete three-dimensional data sets. A target dose of 60 Gy was prescribed. We performed a detailed dose volume histogram analysis. Dose coverage was equal in both plans while dose distribution was more conformal to the target volume with IMRT. Mean and maximum dose of the brain stem, chiasm, optic nerves and orbits were lower using IMRT than SCRT. The reduction was significant regarding orbit and optic nerve (p<0.05). IMRT was superior in sparing of organs at risk compared with SCRT. The additional sparing by IMRT was positively correlated to the size of the target volume, which was evident with target volumes above 200 cm3. Treatment time was approximately 20 minutes per fraction using IMRT compared with 15 minutes per fraction using SCRT. We conclude that IMRT is both feasible and a valuable tool for more conformal dose distribution in the treatment of esthesioneuroblastoma and to spare organs at risk that are in critical relationship to the tumour. This advantage could be seen especially well in complex shaped target volumes above 200 cm3. Thus, using IMRT, risk of complications may be minimized and local tumour control may be increased.

Management of benign cranial nonacoustic schwannomas by fractionated stereotactic radiotherapy.

Schwannomas are the most common tumors of cranial nerves. Nonacoustic schwannomas are very rare tumors, accounting for approximately 10% of intracranial schwannomas. Standard treatment is complete surgical resection if possible. The role of fractionated stereotactic radiotherapy remains to be defined. Thirteen patients with cranial nonacoustic schwannomas underwent fractionated stereotactic radiotherapy. Seven patients had trigeminal schwannomas, three schwannomas of the lower cranial nerves, and three located in the cerebellopontine angle without involvement of the acoustic nerve. Treatment included primary or adjuvant radiotherapy in progressive disease. Tumor volume ranged from 4.5 to 76.0 cc (median 19.8 cc). Median dose was 57.6 Gy with 1.8 Gy/fraction. Median follow-up was 33 months (range 13-70 months). Local tumor control rate was 100% (13/13). Tumor size remained stable in nine patients and decreased in four. Improvement of preexisting neurological deficits was seen in four cases. No patient developed new cranial nerve or brain stem deficits. No patient showed clinically significant complications of irradiation. Fractionated stereotactic radiotherapy is an effective and well-tolerated noninvasive treatment for cranial nonacoustic schwannomas with excellent tumor control rates. It is an option for patients at higher risk for microsurgical resection or in residual and recurrent tumors.

Fitting tumor control probability models to biopsy outcome after three-dimensional conformal radiation therapy of prostate cancer: pitfalls in deducing radiobiologic parameters for tumors from clinical data.

PURPOSE: The goal of tumor control probability (TCP) models is to predict local control for inhomogeneous dose distributions. All existing fits of TCP models to clinical data have utilized summaries of dose distributions (e.g., prescription dose). Ideally, model fits should be based on dose distributions in the tumor, but usually only dose-volume histograms (DVH) of the planning target volume (PTV) are available. We fit TCP models to biopsy outcome after three-dimensional conformal radiation therapy of prostate cancer using either a dose distribution summary or the full DVH in the PTV. We discuss differences in the radiobiologic parameters and dose-response curves and demonstrate pitfalls in interpreting the results. METHODS AND MATERIAL: Two mechanistic TCP models were fit with a maximum likelihood technique to biopsy outcome from 103 prostate patients treated at Memorial Sloan-Kettering Cancer Center. Fits were performed separately for different patient subgroups defined by tumor-related prognostic factors. Fits were based both on full DVHs, denoted TCP(DVH(calc)), and, alternatively, assuming a homogeneous PTV dose given by the mean dose (Dmean) of each DVH, denoted TCP(Dmean(calc)). Dose distributions for these patients were very homogeneous with any cold spots located on the periphery of the PTV. These cold spots were uncorrelated with biopsy outcome, likely because the low-dose regions may not contain tumor cells. Therefore, fits of TCP models that are potentially sensitive to cold spots (e.g., TCP(DVH(calc))) likely give biologic parameters that diminish this sensitivity. In light of this, we examined differences in fitted clonogenic cell number, N(C), or density, rho(C), surviving fraction after 2 Gy, SF(2), or radiosensitivity, alpha, and their standard deviations in the population, sigma(SF(2)) and sigma(alpha), resulting from fits based on TCP(DVH(calc)) and TCP(Dmean(calc)). Dose-response curves for homogeneous irradiation (characterized by TCD(50), the dose for a TCP of 50%) and differences in TCP predictions calculated from the DVH using alternatively derived parameters were evaluated. RESULTS: Fits of TCP(Dmean(calc)) are better (i.e., have larger likelihood) than fits of TCP(DVH(calc)). For TCP(Dmean(calc)) fits, matching values of SF(2) and sigma(SF(2)) (or alpha and sigma(alpha)) exist for all N(C) (rho(C)) above a threshold that give fits of equal quality, with no maximum in likelihood. In contrast, TCP(DVH(calc)) fits have maximum likelihood for high SF(2) (low alpha) values that minimize effects of cold spots. Consequently, small N(C) (rho(C)) values are obtained to match the observed control rate. For example, for patients in low-, intermediate-, and high-risk groups, optimum values of SF(2) and N(C) are 0.771 and 3.3 x 10(3), 0.736 and 2.2 x 10(4), and 0.776 and 1.0 x 10(4), respectively. The TCD(50) of dose-response curves for intermediate-risk patients is 2.6 Gy lower using TCP(DVH(calc)) parameters (TCD(50) = 67.8 Gy) than for TCP(Dmean(calc)) parameters (TCD(50) = 70.4 Gy). TCP predictions calculated from the DVH using risk group-dependent TCP(Dmean(calc)) parameters are up to 53% lower than corresponding calculations with TCP(DVH(calc)) parameters. CONCLUSION: For our data, TCP parameters derived from DVHs likely do not reflect true radiobiologic parameters in the tumor, but are a consequence of the reduced importance of low-dose regions at the periphery of the PTV. Deriving radiobiologic parameters from TCP(Dmean(calc)) fits is not possible unless one parameter is already known. TCP predictions using TCP(DVH(calc)) and TCP(Dmean(calc)) parameters may differ substantially, requiring consistency in the derivation and application of model parameters. The proper derivation of radiobiologic parameters from clinical data requires both substantial dose inhomogeneities and understanding of how these coincide with tumor location.

High efficacy of fractionated stereotactic radiotherapy of large base-of-skull meningiomas: long-term results.

PURPOSE: Large skull-base meningiomas are difficult to treat due to their proximity or adherence to critical structures. We analyzed the long-term results of patients with skull-base meningiomas treated by a new approach with high-precision fractionated stereotactic radiotherapy. PATIENTS AND METHODS: One hundred eighty-nine patients with benign meningiomas were treated with conformal fractionated stereotactic radiotherapy between 1985 and 1998. Patients were undergoing a course of radiotherapy either as primary treatment, following subtotal resection, or for recurrent disease. The median target volume was 52.5 mL (range, 5.2 to 370 mL). The mean radiation dose was 56.8 Gy (+/- 4.4 Gy). Follow-up examinations, including magnetic resonance imaging, were performed at 6-month intervals thereafter. RESULTS: The median follow-up period was 35 months (range, 3 months to 12 years). Overall actuarial survival for patients with World Health Organization (WHO) grade I meningiomas was 97% after 5 years and 96% after 10 years. Local tumor failure was observed in three of 180 patients with WHO grade I tumors and was significantly higher in two of nine patients with WHO grade II tumors. A volume reduction of more than 50% was observed in 26 patients (14%). Preexisting cranial nerve symptoms resolved completely in 28% of the patients. Clinically significant treatment-induced toxicity was seen in 1.6% of the patients. No treatment-related deaths occurred. CONCLUSION: The results of this study demonstrate that fractionated stereotactic radiotherapy is safe and effective in the therapy of subtotally resected or unresectable meningiomas. The overall morbidity and incidence subacute and late side effects of this conformal radiotherapy approach were low.

Fractionated stereotactically guided radiotherapy and radiosurgery in the treatment of functional and nonfunctional adenomas of the pituitary gland.

PURPOSE: We evaluated survival rates and side effects after fractionated stereotactically guided radiotherapy (SCRT) and radiosurgery in patients with pituitary adenoma. METHODS AND MATERIALS: Between 1989 and 1998, 68 patients were treated with FSRT (n = 63) or radiosurgery (n = 5) for pituitary adenomas. Twenty-six had functional and 42 had nonfunctional adenomas. Follow-up included CT/MRI, endocrinologic, and ophthalmologic examinations. Mean follow-up was 38.7 months. Seven patients received radiotherapy as primary treatment and 39 patients received it postoperatively for residual disease. Twenty-two patients were treated for recurrent disease after surgery. Mean total dose was 52.2 Gy for SCRT, and 15 Gy for radiosurgery. RESULTS: Overall local tumor control was 93% (60/65 patients). Forty-three patients had stable disease based on CT/MRI, while 15 had a reduction of tumor volume. After FSRT, 26% with a functional adenoma had a complete remission and 19% had a reduction of hormonal overproduction after 34 months' mean. Two patients with STH-secreting adenomas had an endocrinologic recurrence, one with an ACTH-secreting adenoma radiologic recurrence, within 54 months. Reduction of visual acuity was seen in 4 patients and partial hypopituitarism in 3 patients. None of the patients developed brain radionecrosis or radiation-induced gliomas. CONCLUSION: Stereotactically guided radiotherapy is effective and safe in the treatment of pituitary adenomas to improve local control and reduce hormonal overproduction.

Myocardial adrenergic dysfunction in rats with transgenic, human renin-dependent hypertension.

OBJECTIVES: We investigated cardiac function in rats transgenic for the human renin and angiotensinogen genes (TGR) to test the hypothesis that elevated local angiotensin II precipitates adrenergic dysfunction and abnormal contractile function. METHODS: Hearts from TGR and Sprague-Dawley control rats, aged 6 weeks, were studied using the Langendorff model and papillary muscle preparations (n = 6-10 per group). Incremental isoproterenol (1 - 1000 nmol/l) and external Ca2+-concentrations (0.75-6.0 mmol/l) were tested. Cardiac protein and mRNA expression levels were determined by Western blot and RNAase protection assay. RESULTS: TGR rats showed left ventricular hypertrophy (54%), higher blood pressures (76 mmHg), and elevated plasma renin activity (seven-fold) compared to controls (P < 0.01). The effect of isoproterenol on TGR rat systolic and diastolic left ventricular performance was decreased in both in-vitro models compared to controls (two- to threefold, P < 0.01). TGR rat papillary muscles showed impaired force generation with abnormal basal and Ca2+-dependent relaxation. Gialpha2 and Gialpha3 protein levels were increased (20-30%) and SERCA2a and adenylyl cyclase protein levels were decreased (23 and 37%, respectively) in TGR hearts compared to controls, while Gsalpha or beta1 and beta2-receptor levels were unchanged. Cardiac angiotensin converting enzyme and atrial natriuretic peptide mRNA levels were increased more than four-fold in TGR with no differences for the angiotensin type1 receptor, beta1-receptor, SERCA2a, phospholamban, adenylyl cyclase V and angiotensinogen genes. CONCLUSIONS: TGR rat hearts develop severe adrenergic dysfunction with decreased adenylyl cyclase and abnormal intracellular Ca2+-homeostasis. Our findings emphasize angiotensin II as a major risk factor promoting early functional decline in cardiac hypertrophy. The data may have implications for patients with activating polymorphisms of the renin-angiotensin system and support the need for an early therapeutic intervention.

Heme histidine ligands within gp91(phox) modulate proton conduction by the phagocyte NADPH oxidase.

The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H(+) channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91(phox) generated voltage-activated, pH-dependent, H(+)-selective currents that were reversibly blocked by Zn(2+). The gp91(phox) currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91(phox) proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.

MAP kinase phosphatase-1 gene transcription in rat neuroendocrine cells is modulated by a calcium-sensitive block to elongation in the first exon.

Transcriptional elongation of many eukaryotic, prokaryotic, and viral genes is tightly controlled, which contributes to gene regulation. Here we describe this phenomenon for the MAP kinase phosphatase 1 (MKP-1) immediate early gene. In rat GH4C1 pituitary cells, MKP-1 mRNA is rapidly and transiently induced by the thyrotropin-releasing hormone (TRH) and the epidermal growth factor EGF via transcriptional activation of the gene. Ca(2+) signals are necessary for the induction of MKP-1 in response to TRH but not to EGF. Reporter gene analysis with the newly cloned rat promoter sequence shows only limited induction in response to various stimuli, including TRH or EGF. By nuclear run-on assays we demonstrate that in basal conditions, a strong block to elongation in the first exon regulates the MKP-1 gene and that stimulation with either TRH or EGF overcomes the block. Ca(2+) signals are important to release the MKP-1 elongation block in a manner similar to the c-fos oncogene. These results suggest that a common mechanism of intragenic regulation may be conserved between MKP-1 and c-fos in mammalian cells.