Impact of coronary vasa vasorum functional structure on coronary vessel wall perfusion distribution.

Noncoronary vasa vasorum have been described as networks of microvessels in the wall of arteries and veins. However, we have shown, using microcomputerized tomography (micro-CT) imaging methods, that porcine coronary vasa vasorum have a tree-like branching structure similar to the vasculature in general. In this study, we elucidate functional aspects of coronary vasa vasorum perfusion territories. Three pig hearts were injected with radiopaque Microfil via the coronary sinus to fill the left anterior descending coronary arteries (LADs) retrogradely at atmospheric pressure. In three other hearts, LADs were injected antegradely at 100-mmHg pressure via the left main carotid artery. Additionally, six LADs were injected in vivo with a suspension of 100- or 300-microm-diameter microspheres before harvesting of the hearts and injection of the LADs with Microfil. All harvested LADs were scanned intact with micro-CT (20 microm cubic voxels). The spatial density of vasa vasorum (no. of vasa/mm2) was measured in 20-microm-thick cross sections (at 0.4-mm intervals). Retrogradely injected LADs showed high and uniformly distributed vasa vasorum densities in the adventitia (means +/- SE; 5.38 +/- 0.09 vs. 3.58 +/- 0.1 vasa/mm2 in antegradely prepared LADs; P < 0.001). Antegradely prepared LADs showed patchy distributed, low-vasa-vasorum-density territories especially on the myocardial side of the coronary artery wall (epicardial density: 4.29 +/- 0.13 vasa/mm2 vs. myocardial density: 2.80 +/- 0.1 vasa/mm2, P < 0.001). Microembolization reduced vasa vasorum densities significantly (100-mum-diameter microspheres: 3.26 +/- 0.07 vasa/mm2, P < 0.05; 300-microm-diameter microspheres: 2.66 +/- 0.07 vasa/mm2, P < 0.001 vs. antegrade controls) and increased the size of low-vasa-vasorum-density territories. We conclude that coronary vasa vasorum are functional endarteries not connected via a plexus. This characteristic may have a significant impact on the spatial distribution of perfusion and drainage of the coronary vessel wall.

Time course of bioluminescent signal in orthotopic and heterotopic brain tumors in nude mice.

In vivo bioluminescence imaging is becoming increasingly popular. Quantification of bioluminescence signals requires knowledge of the variability and reproducibility of this technique. The objective of this study was to analyze the time course of luminescent signal emitted from firefly luciferase-expressing tumors in two locations, following luciferin injection and at different times after tumor cell implantation. Knowledge of the kinetics of the bioluminescent signals is required for the reliable quantification and comparison of signal during longitudinal studies. The kinetics of bioluminescence was evaluated in orthotopic and heterotopic brain tumors in mice using a human brain tumor cell line constitutively expressing luciferase. Tumor cells were implanted in the brains and flanks of the animals, and whole-body images revealing tumor location were obtained. Tumor burden was monitored over time by the quantitation of photon emission. The magnitude of bioluminescence measured in vivo varied with time after the injection of luciferin, as well as with dose, which necessitated that the comparison of the quantitative results take into consideration the time after injection. Heterotopic and orthotopic tumors exhibited significantly different time courses; however, time after implantation as characterized by kinetic studies performed on days 4 and 14 after cell implantation revealed no significant differences in orthotopic tumors. Future quantitative longitudinal studies must take into account the differences in the kinetics of different models.

Troponin organization on relaxed and activated thin filaments revealed by electron microscopy and three-dimensional reconstruction.

The steric model of muscle regulation holds that at low Ca(2+) concentration, tropomyosin strands, running along thin filaments, are constrained by troponin in an inhibitory position that blocks myosin-binding sites on actin. Ca(2+) activation, releasing this constraint, allows tropomyosin movement, initiating actin-myosin interaction and contraction. Although the different positions of tropomyosin on the thin filament are well documented, corresponding information on troponin has been lacking and it has therefore not been possible to test the model structurally. Here, we show that troponin can be detected on thin filaments and demonstrate how its changing association with actin can control tropomyosin position in response to Ca(2+). To accomplish this, thin filaments were reconstituted with an engineered short tropomyosin, creating a favorable troponin stoichiometry and symmetry for three-dimensional analysis. We demonstrate that in the absence of Ca(2+), troponin bound to both tropomyosin and actin can act as a latch to constrain tropomyosin in a position on actin that inhibits actomyosin ATPase. In addition, we find that on Ca(2+) activation the actin-troponin connection is broken, allowing tropomyosin to assume a second position, initiating actomyosin ATPase and thus permitting contraction to proceed.

Effects of a cardiomyopathy-causing troponin t mutation on thin filament function and structure.

Familial hypertrophic cardiomyopathy (FHC) is caused by missense or premature truncation mutations in proteins of the cardiac contractile apparatus. Mutant proteins are incorporated into the thin filament or thick filament and eventually produce cardiomyopathy. However, it has been unclear how the several, genetically identified defects in protein structure translate into impaired protein and muscle function. We have studied the basis of FHC caused by premature truncation of the most frequently implicated thin filament target, troponin T. Electron microscope observations showed that the thin filament undergoes normal structural changes in response to Ca(2+) binding. On the other hand, solution studies showed that the mutation alters and destabilizes troponin binding to the thin filament to different extents in different regulatory states, thereby affecting the transitions among states that regulate myosin binding and muscle contraction. Development of hypertrophic cardiomyopathy can thus be traced to a defect in the primary mechanism controlling cardiac contraction, switching between different conformations of the thin filament.

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments.

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.

Three-dimensional reconstruction of thin filaments containing mutant tropomyosin.

Interactions of the components of reconstituted thin filaments were investigated using a tropomyosin internal deletion mutant, D234, in which actin-binding pseudo-repeats 2, 3, and 4 are missing. D234 retains regions of tropomyosin that bind troponin and form end-to-end tropomyosin bonds, but has a length to span only four instead of seven actin monomers. It inhibits acto-myosin subfragment 1 ATPase (acto-S-1 ATPase) and filament sliding in vitro in both the presence and absence of Ca(2+) (, J. Biol. Chem. 272:14051-14056) and lowers the affinity of S-1.ADP for actin while increasing its cooperative binding. Electron microscopy and three-dimensional reconstruction of reconstituted thin filaments containing actin, troponin, and wild-type or D234 tropomyosin were carried out to determine if Ca(2+)-induced movement of D234 occurred in the filaments. In the presence and absence of Ca(2+), the D234 position was indistinguishable from that of the wild-type tropomyosin, demonstrating that the mutation did not affect normal tropomyosin movement induced by Ca(2+) and troponin. These results suggested that, in the presence of Ca(2+) and troponin, D234 tropomyosin was trapped on filaments in the Ca(2+)-induced position and was unable to undergo a transition to a completely activated position. By adding small amounts of rigor-bonded N-ethyl-maleimide-treated S-1 to mutant thin filaments, thus mimicking the myosin-induced "open" state, inhibition could be overcome and full activation restored. This myosin requirement for full activation provides support for the existence of three functionally distinct thin filament states (off, Ca(2+)-induced, myosin-induced; cf.;, J. Mol. Biol. 266:8-14). We propose a further refinement of the three-state model in which the binding of myosin to actin causes allosteric changes in actin that promote the binding of tropomyosin in an otherwise energetically unfavorable "open" state.

Magnetic resonance imaging features of allografts.

OBJECTIVE: To investigate the magnetic resonance imaging (MRI) features of allografts at various time intervals after surgery in patients with osteoarticular allografts. DESIGN AND PATIENTS: Sixteen patients who were treated with osteoarticular allografts and who were followed over time with MRI studies as part of their long-term follow-up were retrospectively selected for this study. T1-weighted images were obtained both before and after gadolinium administration along with T2-weighted images. All images were reviewed by an experienced musculoskeletal radiologist, with two other experienced radiologists used for consultation. Imaging studies were organized into three groups for ease of discussion: early postoperative period (2 days to 2 months), intermediate postoperative period (3 months to 2 years), and late postoperative period (greater than 2 years). RESULTS: In the early postoperative period, no gadolinium enhancement of the allograft was visible in any of the MR images. A linear, thin layer of periosteal and endosteal tissue enhancement along the margin of the allograft was visible in images obtained at 3-4 months. This enhancement appeared gradually to increase in images from later periods, and appears to have stabilized in the images obtained approximately 2-3 years after allograft placement. The endosteal enhancement diminished after several years, with examinations conducted between 6 and 8 years following surgery showing minimal endosteal enhancement. However, focal enhancement was noted adjacent to areas of pressure erosion or degenerative cysts. All the cases showed inhomogeneity in the marrow signal (scattered low signal foci on T1 with corresponding bright signal on T2), and a diffuse, inhomogeneous marrow enhancement later on. CONCLUSION: We have characterized the basic MRI features of osteoarticular allografts in 16 patients who underwent imaging studies at various time points as part of routine follow-up. We believe that the endosteal and periosteal enhancement observed on MRI during the first few months to 2 years following surgery represents vascular ingrowth and early skeletal repair. The zone of periosteal enhancement could also include the new bone laid on the surface of the allograft through which the soft tissues bind to the cortex. The exact reason for the inhomogeneity in the marrow signal, and the diffuse, inhomogeneous marrow enhancement is not clear. This may represent saponified and/or necrotic marrow fat interspersed with the fibrovascular tissue. The features noted here should provide radiologists with useful information regarding imaging characteristics they can expect to see in other allograft replacement patients.

Using quantitative CT to assess adipose distribution in adult men with acquired hypogonadism.

OBJECTIVE: Quantitative CT is a powerful tool that may be used to assess distribution of adipose and lean mass and bone mineral density in specific anatomic compartments. Testosterone deficiency (hypogonadism) is increasingly recognized in adult men and is associated with osteoporosis, diminished strength, and an increase in cardiovascular risk. We used quantitative CT to determine whether hypogonadism is associated with fat redistribution and altered bone density. SUBJECTS AND METHODS: Quantitative CT was performed at the level of the L4 vertebra in 26 men with adult onset testosterone deficiency and 17 eugonadal men of similar body mass index and age. Adipose area in the subcutaneous, visceral, and skeletal muscle areas was determined and trabecular bone density was measured. Values between the groups were compared using t tests. RESULTS: The ages of the hypogonadal and eugonadal men were 52 +/- 14 years and 51 +/- 8 years (p value not significant), respectively. Subcutaneous fat area was higher in the testosterone-deficient men than in the control subjects (270 +/- 101 cm2 versus 202 +/- 111 cm2; p = .046). Muscle fat area was higher in the hypogonadal men (6 +/- 3 cm2 versus 2 +/- 1 cm2; p = .001). Measurements of visceral fat were similar for both groups. Trabecular bone density was lower in the hypogonadal than in the eugonadal men (112 +/- 38 mg K2HPO4/dl versus 148 +/- 34 mg K2HPO4/dl, respectively; p = .003). CONCLUSION: Our findings indicate that testosterone deficiency is associated with a decrease in bone density and a redistribution of fat. Quantitative CT is a sensitive method that may be useful in determining alterations in regional adipose deposition in hypogonadal men and in evaluating the benefit of interventional therapy such as testosterone replacement.

Vertebral morphometry derived from digital images.

OBJECTIVE: We describe a method for capturing measurement data directly from digitized images using specialized software and high-resolution workstations. We have evaluated the reliability, accuracy, and reproducibility of this method in an international clinical trial involving vertebral morphometry. MATERIALS AND METHODS: Accuracy was determined using clinical radiographs measured with vernier calipers and a film phantom. Intra- and interobserver variabilities were assessed, and longitudinal reproducibility was evaluated. As part of the trial, spinal radiographs were collected from more than 200 international health care facilities and digitized at four screening centers. Digitized images were stored and sent to our central facility for morphometry and archiving. Timeliness and variability of the process were tracked. RESULTS: Relative accuracy was nearly 100%. Correlation with clinical measurements was high (r = .96; p < .05). The mean coefficient of variation for interobserver variability was 2%. Intraobserver variation was 3-5%. The coefficient of variation for longitudinal reproducibility ranged from 4% to 6%. After 9 months of operation, our trial included 9494 patients. Of approximately 36,000 radiographs, 98% passed quality review. Only 1% of vertebral levels were not measurable. Hardware and software problems were minimal. CONCLUSION: The use of digitized images for morphometry is accurate, reproducible, and convenient. When applied to a large-scale clinical trial, it offers unique advantages that may justify the cost and complexity that exceed those of conventional radiographs.

Quantification of articular cartilage in the knee with three-dimensional MR imaging.

RATIONALE AND OBJECTIVES: To determine the volume of articular cartilage in cadavers, patients, and healthy volunteers by using a volumetric, fat-suppressed spoiled gradient-recalled signal acquisition in the steady state (SPGR) magnetic resonance (MR) sequence. METHODS: Sagittal MR images were obtained with a fat-suppressed SPGR sequence (repetition time, 52 msec; echo time, 10 msec; 60 degrees flip angle; 3.0-3.5-mm partitions, 256 x 192 matrix, two signals acquired). The cartilaginous surfaces of the tibia, femur, and patella were planimetrically defined with a three-dimensional workstation. A three-dimensional model volume was created by threshold segmenting the cartilage from the adjacent tissues. The volume as calculated by using MR imaging was compared with the actual volume of the cartilage specimens. RESULTS: Observed measurements correlated with actual weight and volume displacement measurements with an accuracy of 82%-99% and linear correlation coefficients of 0.99 (P = 2.5e-15) and 0.99 (P = 4.4e-15). Precision of segmentation in healthy volunteers yielded a coefficient of variation of 0.4% for interobserver variability and 0.3% for intraobserver variability. CONCLUSION: This pilot study suggests that accurate volumetric calculations of knee articular cartilage are possible with currently available MR imaging pulse sequences and a commercially available work station.

[Studies on the increase in the incidence of endometrial and ovarian in cancer in Czechoslovakia during 1960-1973]. / Untersuchungen zum Inzidenzanstieg der Endometrium- und Ovarialkarzinome in der CSSR während der Jahre 1960 bis 1973

During the time period of the last 12 years the incidence of the endometrium carcinoma in the whole CSSR rised to 155,5% in the consideration of the incidence in the years 1961 to 1963. In the same time period the incidence of the ovarial carcinoma has grown up to 117,8% only, what seems to be still in the limits of average incidence shift of all female malignancies in the CSSR (117,1%). The rising incidence of endometrium carcinoma affects primarily the elderly women, but testiefies also that this malignancy is among them today much more frequent. Therefore the rising incidence cannot be explained only as a manifestation of elderly of the female population. In agreement with this reality the endometrium carcinoma is today diagnosed significantly more frequently also among the younger women (at least since 40 years of age). The totals of all endometrium carcinomas rised even more than incidence. The gynaecologists meet today 162% of such malignancies in comparison with the numbers in the years 1961-1963. It means that in the whole CSSR yearly 500 endometrium carcinomas more are diagnosed than before 12 years. The incidence of cervix carcinoma dropped during the same period significantly and the endometrium carcinoma became therefore nearly as frequent as cervical malignancy (1:1,2). Possible reasons of these frequency changes are discussed.

[Precancerous conditions in the cervix uteri in young women]. / Präkanzerosen des Gebärmutterhalses bei jungen Frauen

The author presents the increasing securing of various degrees of atypiae of the epithelium in the uterine neck including carcinoma in situ, and even carcinoma invasivum in young females in connection with preventive examinations being extended even on young females without age limit. In the years 1961 to 1973 only in the district Hradec Králové and its environment, a total of 181 females with a pathological finding in the cervix was found. Out of them in 19 females carcinoma in situ with with the onset of invasion was observed, in 13 females progressive carcinoma invasivum was found. Mutual relations are shown in Table I and Table II. In Table III the author wants to show that all preventive examinations in all females coming to the prenatal consulting centre for the first time are worth being carried out thoroughly. Thus 51 females--very young--with a finding in the cervix were discovered. The performance of preventive examinations as late as after the thirtieth year of age is considered to be incorrect. When searching for precanceroses of the uterine neck it is not possible to state any limits. The sooner and the more frequent the prebioptic examination methods are employed, the more probably the changes which may be, however, reversible, but which never exclude any possibility of malignant development may be found.