In vitro to in vivo extrapolation from three-dimensional hiPSC-derived cardiac microtissues and physiologically based pharmacokinetic modeling to inform next-generation arrhythmia risk assessment.

Proarrhythmic cardiotoxicity remains a substantial barrier to drug development as well as a major global health challenge. In vitro human pluripotent stem cell-based new approach methodologies have been increasingly proposed and employed as alternatives to existing in vitro and in vivo models that do not accurately recapitulate human cardiac electrophysiology or cardiotoxicity risk. In this study, we expanded the capacity of our previously established three-dimensional human cardiac microtissue model to perform quantitative risk assessment by combining it with a physiologically based pharmacokinetic model, allowing a direct comparison of potentially harmful concentrations predicted in vitro to in vivo therapeutic levels. This approach enabled the measurement of concentration responses and margins of exposure for two physiologically relevant metrics of proarrhythmic risk (ie, action potential duration and triangulation assessed by optical mapping) across concentrations spanning three orders of magnitude. The combination of both metrics enabled accurate proarrhythmic risk assessment of four compounds with a range of known proarrhythmic risk profiles (ie, quinidine, cisapride, ranolazine, and verapamil) and demonstrated close agreement with their known clinical effects. Action potential triangulation was found to be a more sensitive metric for predicting proarrhythmic risk associated with the primary mechanism of concern for pharmaceutical-induced fatal ventricular arrhythmias, delayed cardiac repolarization due to inhibition of the rapid delayed rectifier potassium channel, or hERG channel. This study advances human induced pluripotent stem cell-based three-dimensional cardiac tissue models as new approach methodologies that enable in vitro proarrhythmic risk assessment with high precision of quantitative metrics for understanding clinically relevant cardiotoxicity.

Gq-activated fibroblasts induce cardiomyocyte action potential prolongation and automaticity in a three-dimensional microtissue environment.

Cardiac fibroblasts (CFs) are known to regulate cardiomyocyte (CM) function in vivo and in two-dimensional in vitro cultures. This study examined the effect of CF activation on the regulation of CM electrical activity in a three-dimensional (3-D) microtissue environment. Using a scaffold-free 3-D platform with interspersed neonatal rat ventricular CMs and CFs, Gq-mediated signaling was selectively enhanced in CFs by Gαq adenoviral infection before coseeding with CMs in nonadhesive hydrogels. After 3 days, the microtissues were analyzed by signaling assay, histological staining, quantitative PCR, Western blots, optical mapping with voltage- or Ca2+-sensitive dyes, and microelectrode recordings of CF resting membrane potential (RMPCF). Enhanced Gq signaling in CFs increased microtissue size and profibrotic and prohypertrophic markers. Expression of constitutively active Gαq in CFs prolonged CM action potential duration (by 33%) and rise time (by 31%), prolonged Ca2+ transient duration (by 98%) and rise time (by 65%), and caused abnormal electrical activity based on depolarization-induced automaticity. Constitutive Gq activation in CFs also depolarized RMPCF from -33 to -20 mV and increased connexin 43 and connexin 45 expression. Computational modeling confers that elevated RMPCF and increased cell-cell coupling between CMs and CFs in a 3-D environment could lead to automaticity. In conclusion, our data demonstrate that CF activation alone is capable of altering action potential and Ca2+ transient characteristics of CMs, leading to proarrhythmic electrical activity. Our results also emphasize the importance of a 3-D environment where cell-cell interactions are prevalent, underscoring that CF activation in 3-D tissue plays a significant role in modulating CM electrophysiology and arrhythmias.NEW & NOTEWORTHY In a three-dimensional microtissue model, which lowers baseline activation of cardiac fibroblasts but enables cell-cell, paracrine, and cell-extracellular matrix interactions, we demonstrate that selective cardiac fibroblast activation by enhanced Gq signaling, a pathophysiological trigger in the diseased heart, modulates cardiomyocyte electrical activity, leading to proarrhythmogenic automaticity.

Cross talk between cardiac myocytes and fibroblasts: from multiscale investigative approaches to mechanisms and functional consequences.

The heart is comprised of a syncytium of cardiac myocytes (CM) and surrounding nonmyocytes, the majority of which are cardiac fibroblasts (CF). CM and CF are highly interspersed in the myocardium with one CM being surrounded by one or more CF. Bidirectional cross talk between CM and CF plays important roles in determining cardiac mechanical and electrical function in both normal and diseased hearts. Genetically engineered animal models and in vitro studies have provided evidence that CM and CF can regulate each other's function. Their cross talk contributes to structural and electrical remodeling in both atria and ventricles and appears to be involved in the pathogenesis of various heart diseases that lead to heart failure and arrhythmia disorders. Mechanisms of CM-CF cross talk, which are not yet fully understood, include release of paracrine factors, direct cell-cell interactions via gap junctions and potentially adherens junctions and nanotubes, and cell interactions with the extracellular matrix. In this article, we provide an overview of the existing multiscale experimental and computational approaches for the investigation of cross talk between CM and CF and review recent progress in our understanding of the functional consequences and underlying mechanisms. Targeting cross talk between CM and CF could potentially be used therapeutically for the modulation of the cardiac remodeling response in the diseased heart and may lead to new strategies for the treatment of heart failure or rhythm disturbances.

Functional scaffold-free 3-D cardiac microtissues: a novel model for the investigation of heart cells.

To bridge the gap between two-dimensional cell culture and tissue, various three-dimensional (3-D) cell culture approaches have been developed for the investigation of cardiac myocytes (CMs) and cardiac fibroblasts (CFs). However, several limitations still exist. This study was designed to develop a cardiac 3-D culture model with a scaffold-free technology that can easily and inexpensively generate large numbers of microtissues with cellular distribution and functional behavior similar to cardiac tissue. Using micromolded nonadhesive agarose hydrogels containing 822 concave recesses (800 µm deep × 400 µm wide), we demonstrated that neonatal rat ventricular CMs and CFs alone or in combination self-assembled into viable (Live/Dead stain) spherical-shaped microtissues. Importantly, when seeded simultaneously or sequentially, CMs and CFs self-sorted to be interspersed, reminiscent of their myocardial distribution, as shown by cell type-specific CellTracker or antibody labeling. Microelectrode recordings and optical mapping revealed characteristic triangular action potentials (APs) with a resting membrane potential of -66 ± 7 mV (n = 4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100 ± 30 ms and 18.0 ± 1.9 cm/s, respectively (n = 5 each). The presence of CFs led to a twofold AP prolongation in heterogenous microtissues (CM-to-CF ratio of 1:1). Importantly, Ba(2+)-sensitive inward rectifier K(+) currents and Ca(2+)-handling proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, were detected in CM-containing microtissues. Furthermore, cell type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we developed a novel scaffold-free cardiac 3-D culture model with several advancements for the investigation of CM and CF function and cross-regulation.

Diffusion tensor imaging screening of radiation-induced changes in the white matter after prophylactic cranial irradiation of patients with small cell lung cancer: first results of a prospective study.

BACKGROUND AND PURPOSE: Diffusion tensor imaging (DTI) will show abnormal fractional anisotropy (FA) in the normal-appearing brain after prophylactic cranial irradiation (PCI). These abnormalities will be more accentuated in patients with underlying vascular risk factors. MATERIALS AND METHODS: A prospective study by use of DTI and conventional T2-weighted MR images was performed with a 1.5T unit with 16 patients with small cell lung cancer and undergoing PCI. All of the T2-weighted images were evaluated with respect to abnormalities in signal intensity of white matter as markers of radiation damage. Measurements of FA were performed before, at the end of, and 6 weeks after radiation therapy. On the FA maps, the bifrontal white matter, the corona radiata, the cerebellum, and the brain stem were evaluated. FA values were compared with respect to age, demographic, and vascular risk factors. Statistical analyses (Friedman test, Wilcoxon test, and Mann-Whitney U test) were performed. RESULTS: Fractional anisotropy decreased significantly in supratentorial and infratentorial normal-appearing white matter from the beginning to the end of PCI (P < .01). A further decline in FA occurred 6 weeks after irradiation (P < .05). A stronger reduction in FA was observed in patients with more than 1 vascular risk factor. There was an age-related reduction of white matter FA. Patients 65 years and older showed a trend toward a stronger reduction in FA. CONCLUSION: During the acute phase, after PCI, patients with many vascular risk factors showed stronger damage in the white matter compared with patients with only 1 risk factor.

Osteoradionecrosis of the jaws as a side effect of radiotherapy of head and neck tumour patients--a report of a thirty year retrospective review.

This retrospective study aimed to demonstrate the incidence and the aetiological factors involved in osteoradionecrosis (ORN) in a group of 830 head and neck tumour patients who received radiotherapy between 1969 and 1999. The data showed an over all incidence of 8.2% and a 3-fold higher incidence for men than for women. Osteoradionecrosis was most commonly located in the body of the mandible. Concerning the risk factors, a negative influence was shown for advanced tumours, segmental resections of the mandible and pre-/post-radiation tooth extractions. Tooth extractions were found to be responsible for 50% of all cases. The osteoradionecroses were observed significantly earlier in patients who received pre-surgical radiotherapy than those who received post-surgical radiotherapy. Combined pre-surgical radio- and chemotherapy significantly hastened the appearance of osteoradionecrosis compared to pre-surgical radiotherapy alone. Only 40% of patients with osteoradionecrosis could be healed completely by means of surgery and antibiotic medication. Hyperbaric oxygenation (HBO) therapy was performed only in individual cases. The data suggest that osteoradionecrosis has a multifactorial aetiology. Therefore, a very close follow-up of tumour patients and a strict prophylactic management are required.

Comparative analysis of locally adaptive image enhancement for 3D ultrasound images.

In this article we compare methods for locally adaptive preprocessing of 3D ultrasound (US) images and their different capabilities of enhancing diagnostically important anatomical structures. We show that suitability and optimization of methods depend on the objective of the preprocessing task, e.g., slice representation or volume rendering. This article analyzes which features should be used to control the enhancement algorithm For slice representations optimization of local statistics is appropriate to maintain diagnostically important structures. Features with good region separation properties are preferable for volume rendering tasks. We demonstrate our results with US images from clinical examinations.

[3D-ultrasound: a valuable adjunct for therapy planning and follow-up of head and neck tumours]. / 3D-Ultraschall: Eine wertvolle Ergänzung bei Therapie-planung und Verlaufskontrolle von Kopf-Hals-Tumoren.

AIM: The three-dimensional visualization of orofacial tumours and adjacent organs at risk of infiltration is an important requirement for staging, therapy planning and follow-up. Artifacts from the mandible or metal implants often reduce the diagnostic power of computed tomography (CT). The value of 3D-ultrasound (3D-US) in respect to the standard methods CT and magnetic resonance imaging (MRI) was therefore analyzed. METHOD: Between 9/97 and 10/99 the visualization of orofacial tumours in 243 patients by 3D-US was examined, classified on a five-point scale and compared to the corresponding CT and MRI scans. RESULTS: Complete visualization of the tumours was possible in 85.6 % by 3D-US, whereas the rates of MRI and CT were lower with 77.4 % and 61.3 % respectively, mainly because of artifacts. The best combinations of methods, 3D-US + CT (96.7 %) and 3D-US + MRI (95.1 %) gave almost equivalent results, whereas CT + MRI (83.5 %) was inferior. In 2.5 % of the cases none of the methods produced adequate results. CONCLUSION: By free selection of sectional planes and direct correlation to the clinical findings 3D-ultrasound can improve staging, therapy planning and follow-up of orofacial tumours, especially in the case of small tumour size, dental crowns/inlays, metal implants or contraindications to MRI.

[Ultrasonography of tumors of the locomotor system]. / Sonographie von Tumoren des Bewegungsapparats.

Sonography is an integral part of primary tumor diagnosis and follow-up for the great majority of organ systems. However, its value in the field of space-occupying processes of the locomotor system, especially of malignant bone tumors, has mostly been underestimated. The sonographic examination has to investigate the whole tumor region and the corresponding lymph nodes statically and dynamically. The examination procedure should be standardized and the documentation reliable. Evaluation criteria are the localization, dimensions, and volume of the tumors, echogenicity and homogeneity, peri- and intratumoral vascularization (vessel density and architecture), borders of the tumor, and neighboring structures. Pathologic changes not only of the soft tissues but also of the bones can be evaluated sonographically. Even subtle analysis does not permit definitive assessment of tumor status. Taking its physical limitations into consideration, high-resolution sonography, enhanced by color/PowerDoppler and three-dimensional techniques, is a valuable adjunct to improve diagnosis, therapy planning, monitoring, and posttherapeutic care of tumors of the locomotor system.

Dilated cardiomyopathy in two transgenic mouse lines expressing activated G protein alpha(q): lack of correlation between phospholipase C activation and the phenotype.

We previously described a transgenic mouse line (alpha(q)*52) in which cardiac-specific expression of activated G alpha(q)protein (HA alpha(q)*) leads to activation of phospholipase C beta (PLC beta), the immediate downstream target of HA alpha(q)*, with subsequent development of cardiac hypertrophy and dilation. We now describe a second, independent line in the same genetic background (alpha(q)*44h) with lower expression of HA alpha(q)* protein that ultimately results in the same phenotype: dilated cardiomyopathy (DCM) with severely impaired left ventricular systolic function (assessed by M-mode and 2D echocardiography), but with a much delayed disease onset. We asked if PLC activation correlates with the development of the phenotype. At 12-14 months, 65% of alpha(q)*44h mice still had normal cardiac function and ventricular weight/body weight ratios (VW/BW). However, their basal PLC activity, which began to increase in ventricles at 6 months, was threefold higher than in wild-type by 12 months. This increase was even more pronounced than in 2.5-month-old alpha(q)*52 mice, in which a twofold increase was accompanied by a 25% increase in VW/BW. Furthermore, at 12-14 months the increase in PLC activity in alpha(q)*44h mice with and without DCM was comparable. Thus, the delayed time course in alpha(q)*44h mice unmasked a lack of correlation between PLC activation and development of DCM in response to HA alpha(q)* expression, suggesting a role for additional pathways and/or mechanisms. It also revealed a differential temporal regulation of protein kinase C isoform expression. The markedly different ages of disease onset in these two mouse lines provide a model for studying both genetic modifying factors and potential environmental influences in DCM.

[Computer-assisted oral, maxillary and facial surgery]. / Computerunterstützte Mund-, Kiefer- und Gesichtschirurgie.

BACKGROUND: Methods from the area of virtual reality are used in oral and maxillofacial surgery for the planning and three-dimensional individual simulation of surgeries. SIMULATION: In order to simulate complex surgeries with the aid of a computer, the diagnostic image data and especially various imaging modalities (CT, MRT, US) must be arranged in relation to each other, thus enabling rapid switching between the various modalities as well as the viewing of mixed images. Segmenting techniques for the reconstruction of three-dimensional representations of soft-tissue and osseous areas are required. We must develop ergonomic and intuitively useable interaction methods for the surgeon, thus allowing for precise and fast entry of the planned surgical intervention in the planning and simulation phase. SURGERY: During the surgical phase, instrument navigation tools offer the surgeon interactive support through operation guidance and control of potential dangers. This feature is already available today. Future intraoperative assistance will take the form of such passive tools for the support of intraoperative orientation as well as so-called tracking systems (semi-active systems) which accompany and support the surgeons' work. The final form are robots which execute specific steps completely autonomously. DISCUSSION: The techniques of virtual reality keep gaining in importance for medical applications. Many applications are still being developed or are still in the form of a prototype. However, it is already clear that developments in this area will have a considerable effect on the surgeon's routine work.

Altered regulation of potassium and calcium channels by GABA(B) and adenosine receptors in hippocampal neurons from mice lacking Galpha(o).

To examine the role of G(o) in modulation of ion channels by neurotransmitter receptors, we characterized modulation of ionic currents in hippocampal CA3 neurons from mice lacking both isoforms of Galpha(o). In CA3 neurons from Galpha(o)(-/-) mice, 2-chloro-adenosine and the GABA(B)-receptor agonist baclofen activated inwardly rectifying K(+) currents and inhibited voltage-dependent Ca(2+) currents just as effectively as in Galpha(o)(+/+) littermates. However, the kinetics of transmitter action were dramatically altered in Galpha(o)(-/-) mice in that recovery on washout of agonist was much slower. For example, recovery from 2-chloro-adenosine inhibition of calcium current was more than fourfold slower in neurons from Galpha(o)(-/-) mice [time constant of 12.0 +/- 0.8 (SE) s] than in neurons from Galpha(o)(+/+) mice (time constant of 2.6 +/- 0.2 s). Recovery from baclofen effects was affected similarly. In neurons from control mice, effects of both baclofen and 2-chloro-adenosine on Ca(2+) currents and K(+) currents were abolished by brief exposure to external N-ethyl-maleimide (NEM). In neurons lacking Galpha(o), some inhibition of Ca(2+) currents by baclofen remained after NEM treatment, whereas baclofen activation of K(+) currents and both effects of 2-chloro-adenosine were abolished. These results show that modulation of Ca(2+) and K(+) currents by G protein-coupled receptors in hippocampal neurons does not have an absolute requirement for Galpha(o). However, modulation is changed in the absence of Galpha(o) in having much slower recovery kinetics. A likely possibility is that the very abundant Galpha(o) is normally used but, when absent, can readily be replaced by G proteins with different properties.

Signal transduction in atria and ventricles of mice with transient cardiac expression of activated G protein alpha(q).

We recently showed that the transient expression of a hemagglutinin (HA) epitope-tagged, constitutively active mutant of the G protein alpha(q) subunit (HAalpha(q)*) in the hearts of transgenic mice is sufficient to induce cardiac hypertrophy and dilatation that continue to progress after HAalpha(q)* protein becomes undetectable. We demonstrated that the activity of phospholipase Cbeta, the immediate downstream target of activated Galpha(q), is increased at 2 weeks, when HAalpha(q)* is expressed, but also at 10 weeks, when HAalpha(q)* is no longer detectable. This observation suggested that the transient HAalpha(q)* expression causes multiple, persistent changes in cellular signaling pathways. We now demonstrate changes in the level, activity, or both of several signaling components, including changes in the amount and hormone responsiveness of phospholipase Cbeta enzymes, in the basal level of diacylglycerol (which predominantly reflects activation of phospholipase D), in the amount or distribution of protein kinase C (PKC) isoforms (PKCalpha, PKCdelta, and PKCepsilon), and in the amount of several endogenous G proteins. These changes vary depending on the isoform of the signaling molecule, the chamber in which it is expressed, and the presence or absence of HAalpha(q)*. Our results suggest that a network of linked signaling functions determines the development of hypertrophy. They also suggest that atria and ventricles represent different signaling domains. It is likely that such changes occur in other model systems in which the activity of a single signaling component is increased, either due to an activating mutation or due to overexpression of the wild type.

[Uncommon sonographic findings of an epidermoid cyst in the head and neck]. / Ungewöhnlicher sonographischer Befund einer Epidermoidzyste.

Epidermoid cysts are rare lesions in the head and neck. Most often the are located in the submental region, although they sometimes can impose certain diagnostic problems. We report the case of a 19-year-old girl who presented with a fluctuant, painless, midline swelling of the floor of her mouth that had persisted for three months. Ultrasonographic examination showed a 5 x 5 x 4 cm cystic tumor with the unusual aspect of multiple smaller spherical formations. The differential diagnosis of the lesion included dermoid cysts, epidermoid cysts, ranula and parasitic lesions. Computed tomography and magnetic resonance tomography were not diagnostic. The patient underwent excision of the lesion, during which the cyst was completely removed. Histopathological diagnosis was consistent with an epidermoid cyst. The striking sonographic aspect of multiple spherical formations in the lesion was caused by multiple spherical keratin formations.

Transient cardiac expression of constitutively active Galphaq leads to hypertrophy and dilated cardiomyopathy by calcineurin-dependent and independent pathways.

Cardiac hypertrophy and dilatation can result from stimulation of signal transduction pathways mediated by heterotrimeric G proteins, especially Gq, whose alpha subunit activates phospholipase Cbeta (PLCbeta). We now report that transient, modest expression of a hemagglutinin (HA) epitope-tagged, constitutively active mutant of the Gq alpha subunit (HAalpha*q) in hearts of transgenic mice is sufficient to induce cardiac hypertrophy and dilatation that continue to progress after the initiating stimulus becomes undetectable. At 2 weeks, HAalpha*q protein is expressed at less than 50% of endogenous alphaq/11, and the transgenic hearts are essentially normal morphologically. Although HAalpha*q protein declines at 4 weeks and is undetectable by 10 weeks, the animals develop cardiac hypertrophy and dilatation and die between 8 and 30 weeks in heart failure. As the pathology develops, endogenous alphaq/11 rises (2.9-fold in atria; 1.8-fold in ventricles). At 2 weeks, basal PLC activity is increased 9- to 10-fold in atria but not ventricles. By 10 weeks, it is elevated in both, presumably because of the rise in endogenous alphaq/11. We conclude that the pathological changes initiated by early, transient HAalpha*q expression are maintained in part by compensatory changes in signal transduction and other pathways. Cyclosporin A (CsA) prevents hypertrophy caused by activation of calcineurin [Molkentin, J. D., Lu, J.-R., Antos, C. L., Markham, B., Richardson, J., Robbins, J., Grant, S. R. & Olson, E. N. (1998) Cell 93, 215-228]. Because HAalpha*q acts upstream of calcineurin, we hypothesized that HAalpha*q might initiate additional pathways leading to hypertrophy and dilatation. Treating HAalpha*q mice with CsA diminished some, but not all, aspects of the hypertrophic phenotype, suggesting that multiple pathways are involved.

Effect of deletion of the major brain G-protein alpha subunit (alpha(o)) on coordination of G-protein subunits and on adenylyl cyclase activity.

Heterotrimeric G-proteins, composed of alpha and betagamma subunits, transmit signals from cell-surface receptors to cellular effectors and ion channels. Cellular responses to receptor agonists depend on not only the type and amount of G-protein subunits expressed but also the ratio of alpha and betagamma subunits. Thus far, little is known about how the amounts of alpha and betagamma subunits are coordinated. Targeted disruption of the alpha(o) gene leads to loss of both isoforms of alpha(o), the most abundant alpha subunit in the brain. We demonstrate that loss of alpha(o) protein in the brain is accompanied by a reduction of beta protein to 32+/-2% (n = 4) of wild type. Sucrose density gradient experiments show that all of the betagamma remaining in the brains of alpha(o)-/- mice sediments as a heterotrimer (s20,w = 4.4 S, n = 2), with no detectable free alpha or betagamma subunits. Thus, the level of the remaining betagamma subunits matches that of the remaining alpha subunits. Protein levels of alpha subunits other than alpha(o) are unchanged, suggesting that they are controlled independently. Coordination of betagamma to alpha occurs posttranscriptionally because the mRNA level of the predominant beta1 subtype in the brains of alpha(o)-/- mice was unchanged. Adenylyl cyclase can be positively or negatively regulated by betagamma. Because the level of other alpha subunits is unchanged and alpha(o) itself has little or no effect on adenylyl cyclase, we could examine how a large change in the level of betagamma affects this enzyme. Surprisingly, we could not detect any difference in the adenylyl cyclase activity between brain membranes from wild-type and alpha(o)-/- mice. We propose that alpha(o) and its associated betagamma are sequestered in a distinct pool of membranes that does not contribute to the regulation of adenylyl cyclase.

G alpha(o) is necessary for muscarinic regulation of Ca2+ channels in mouse heart.

Heterotrimeric G proteins, composed of G alpha and G betagamma subunits, transmit signals from cell surface receptors to cellular effector enzymes and ion channels. The G alpha(o) protein is the most abundant G alpha subtype in the nervous system, but it is also found in the heart. Its function is not completely known, although it is required for regulation of N-type Ca2+ channels in GH3 cells and also interacts with GAP43, a major protein in growth cones, suggesting a role in neuronal pathfinding. To analyze the function of G alpha(o), we have generated mice lacking both isoforms of G alpha(o) by homologous recombination. Surprisingly, the nervous system is grossly intact, despite the fact that G alpha(o) makes up 0.2-0.5% of brain particulate protein and 10% of the growth cone membrane. The G alpha(o)-/- mice do suffer tremors and occasional seizures, but there is no obvious histologic abnormality in the nervous system. In contrast, G alpha(o)-/- mice have a clear and specific defect in ion channel regulation in the heart. Normal muscarinic regulation of L-type calcium channels in ventricular myocytes is absent in the mutant mice. The L-type calcium channel responds normally to isoproterenol, but there is no evident muscarinic inhibition. Muscarinic regulation of atrial K+ channels is normal, as is the electrocardiogram. The levels of other G alpha subunits (G alpha(s), G alpha(q), and G alpha(i)) are unchanged in the hearts of G alpha(o)-/- mice, but the amount of G betagamma is decreased. Whichever subunit, G alpha(o) or G betagamma, carries the signal forward, these studies show that muscarinic inhibition of L-type Ca2+ channels requires coupling of the muscarinic receptor to G alpha(o). Other cardiac G alpha subunits cannot substitute.

[Ultrasound criteria for staging and follow-up of malignant lymphoma]. / Sonographische Kriterien für Staging und Verlaufskontrolle bei malignen Lymphomen.

Ultrasonography is an integral part of the radiological imaging of malignant lymphomas for staging, therapy planning, monitoring, follow-up, post-therapeutic care, and detection of recurrences, as well as the diagnostic differentiation of benign lesions and metastasis of solid tumors. None of the imaging methods, including sonography, however, can safely determine the stage (UICC certainty level C4) in agreement with the pathohistological examination. Also considering the clinical appearance the sonographic criteria in the synoptic evaluation will nevertheless provide important indications for diagnosis and therapy planning. Not only changes in size and shape, but also the echogenicity (gray-scale histogram) and vascularization (color Doppler) of nodal and extranodal processes are decisive criteria for estimating the therapeutic response rates and detecting relapses.

[99m-Tc-MIBI for recurrent and metastatic differentiated thyroid carcinoma]. / 99mTc-MIBI zur Rezidiv- und Metastasensuche bei differenzierten Schilddrüsenkarzinomen.

AIM: The aim of the study has been the examination of the diagnostic value of 99mTc-MIBI scintigraphy for the detection of local tumor or metastases following total thyroidectomy and 131I ablation therapy in differentiated thyroid carcinoma. METHODS: MIBI-scintigraphy has been indicated in 85 patients because of ascending thyroglobulin values or suspected local recurrencies by ultrasonography. The results have been compared to cytology or histology or ultrasonography, computed tomography, X-ray and radioiodine scanning. RESULTS: MIBI scintigraphy was found positive in 32 of 40 metastases. Only 18 metastases have been seen by radioiodine. MIBI scintigraphy was most effective in detecting local tumor recurrencies and lymph node metastases (94%). The specificity of MIBI and radioiodine was similar (100%). In inflammatory enlarged lymph nodes no MIBI uptake was found, so it is possible to differentiate reactive lymph node enlargement from metastatic disease. CONCLUSION: In conclusion scintigraphy with 99mTc-MIBI is advisable in suspected local recurrencies and negative radioiodine scan. It is favourable that withdrawing TSH-suppressive hormone medication is not necessary.

Maintenance of cellular levels of G-proteins: different efficiencies of alpha s and alpha o synthesis in GH3 cells.

G-proteins couple membrane-bound receptors to intracellular effectors. Each cell has a characteristic complement of G-protein alpha, beta and gamma subunits that partly determines the cell's response to external signals. Very little is known about the mechanisms that set and maintain cellular levels of G-proteins or about potential points of regulation. We have assayed the steady-state levels of mRNA and protein for two types of G-protein subunits, alpha s and alpha o, in rat brain, heart and GH3 cells, and found that in all these cases, it takes 9- to 20-fold more mRNA to produce a given amount of alpha s protein than to produce the same amount of alpha o protein. Such a situation could arise from a relatively rapid rate of alpha s protein degradation, requiring rapid protein synthesis to compensate, or from relatively inefficient translation of alpha s mRNA compared with alpha o mRNA. The latter appears to be the case in GH3 cells. These cells contain 94 times more mRNA for alpha s than for alpha o, yet the rate of alpha s protein synthesis is only 9 times greater than alpha o protein synthesis. The degradation rates of the two proteins are similar (13 h for alpha s and 18 h for alpha o). To begin to define the mechanism that accounts for the fact that it takes more mRNA to synthesize a given amount of alpha s than alpha o, we asked whether there is a pool of alpha s mRNA that does not participate in protein synthesis. We found that virtually all alpha s and alpha o mRNA is associated with ribosomes. Therefore, all the mRNA is likely to be capable of directing protein synthesis. Since the rate-limiting step in protein synthesis is usually binding of the ribosome to mRNA at initiation, our results suggest that the relatively slow rate of alpha s protein synthesis is regulated by a mechanism that acts beyond initiation at peptide elongation and/or termination.