Rod mutations associated with MYH9-related disorders disrupt nonmuscle myosin-IIA assembly

MYH9-related disorders are autosomal dominant syndromes, variably affecting platelet formation, hearing, and kidney function, and result from mutations in the human nonmuscle myosin-IIA heavy chain gene. To understand the mechanisms by which mutations in the rod region disrupt nonmuscle myosin-IIA function, we examined the in vitro behavior of 4 common mutant forms of the rod (R1165C, D1424N, E1841K, and R1933Stop) compared with wild type. We used negative-stain electron microscopy to analyze paracrystal morphology, a model system for the assembly of individual myosin-II molecules into bipolar filaments. Wild-type tail fragments formed ordered paracrystal arrays, whereas mutants formed aberrant aggregates. In mixing experiments, the mutants act dominantly to interfere with the proper assembly of wild type. Using circular dichroism, we find that 2 mutants affect the alpha-helical coiled-coil structure of individual molecules, and 2 mutants disrupt the lateral associations among individual molecules necessary to form higher-order assemblies, helping explain the dominant effects of these mutants. These results demonstrate that the most common mutations in MYH9, lesions in the rod, cause defects in nonmuscle myosin-IIA assembly. Further, the application of these methods to biochemically characterize rod mutations could be extended to other myosins responsible for disease.     

Formation and involution of Mallory bodies ("alcoholic hyalin") in murine and human liver revealed by immunofluorescence microscopy with antibodies to prekeratin.

Antibodies raised against prekeratin intensely and specifically stain, in immunofluorescence microscopy, Mallory bodies ("alcoholic hyalin") present in livers of human alcoholics and griseofulyin-treated mice. The high sensitivity of this method allows the identification of small distinct cytoplasmic structures that are observed during early stages of Mallory body formation, especially frequent in the perinuclear cytoplasm, as well as during stages of Mallory body disintegration and disappearance, such as after withdrawal of the drug. In the latter situation, the prekeratin-containing small particles exhibit a characteristic pattern of arrangement in the hepatocyte periphery. Electron microscopy illustrates that such small bodies are heap-like aggregates of typical Mallory body filaments. Immunofluorescence studies with antibodies to isolated prekeratin polypeptides from bovine hoof or muzzle epidermis show that Mallory body filaments, in particular those in human liver, are immunologically more closely related to prekeratin of tonofilaments from living epidermal cells (stratum spinosum). The data indicate that Mallory bodies contain a pathologic form of prekeratin-like material. They also suggest that disorders of cytoskeletal structures of the intermediate-sized filament class are associated with specific diseases and can be visualized and characterized by immunofluorescence microscopy by using antibodies to constitutive proteins of such filaments.     

The role of the retinylidene Schiff base counterion in rhodopsin in determining wavelength absorbance and Schiff base pKa.

Glu-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Purified mutant rhodopsin pigments were prepared in which Glu-113 was replaced individually by Gln (E113Q), Asp (E113D), Asn (E113N), or Ala (E113A). E113Q, E113N, and E113A existed as pH-dependent equilibrium mixtures of unprotonated and protonated Schiff base (PSB) forms. The Schiff base pKa values determined by spectrophotometric titration were 6.00 (E113Q), 6.71 (E113N), and 5.70 (E113A). Thus, mutation of Glu-113 markedly reduced the Schiff base pKa. The addition of NaCl promoted the formation of a PSB in E113Q and E113A. An exogenously supplied solute anion replaced Glu-113 to compensate for the positive charge of the PSB in these mutants. The lambda max values of the PSB forms of the mutants in NaCl were 496 nm (E113Q), 506 nm (E113A), 510 nm (E113D), and 520 nm (E113N). To evaluate the effect of different types of solute anions on lambda max values, mutants were prepared in sodium salts of halides, perchlorate, and a series of carboxylic acids of various sizes and acidity. The lambda max values of E113Q and E113A depended on the solute anion present and ranged from 488 nm to 522 nm for E113Q and from 486 nm to 528 nm for E113A. The solute anion affected the lambda max values of E113N and E113D to lesser degrees. The reactivities of the mutants to hydroxylamine were also studied. Whereas rhodopsin was stable to hydroxylamine in the dark, E113N reacted slowly and E113Q reacted rapidly under these conditions, indicating structural differences in the Schiff base environments. The lambda max values and solute anion dependencies of the Glu-113 mutants indicate that interactions between Schiff base and its counterion play a significant role in determining the lambda max of rhodopsin.     

Synaptophysin: a marker protein for neuroendocrine cells and neoplasms.

Synaptophysin is an integral membrane glycoprotein (Mr 38,000) that occurs in presynaptic vesicles of neurons and in similar vesicles of the adrenal medulla. By using a monoclonal antibody to this protein (SY38), we have found, by immunohistochemistry and immunoblotting, that an identical or similar protein is also expressed in neuroendocrine tumors of neural type, such as pheochromocytomas and paragangliomas. In addition, this protein occurs in certain neuroendocrine epithelial cells, such as pancreatic islet cells; in a variety of neuroendocrine epithelial tumors, including isletcell adenomas and carcinomas and several carcinoids and neuroendocrine carcinomas of the gastrointestinal and the bronchial tracts; and in medullary carcinomas of the thyroid. Our results show that synaptophysin, and the vesicles that contain it, can occur in normal and neoplastic neuroendocrine cells of neural type, as demonstrated by colocalization with neurofilaments, as well as in those of epithelial type, as shown by colocalization with cytokeratin filaments and desmoplakins. We conclude that synaptophysin is expressed independently of other neuronal differentiation markers and propose that it be used as a differentiation marker in tumor diagnosis.     

Detection of abnormal aortic elastic properties in asymptomatic patients with Marfan syndrome by combined transoesophageal echocardiography and acoustic quantification.

OBJECTIVE: To evaluate the potential value of transoesophageal echocardiography combined with automated border detection and acoustic quantification for the assessment of elastic properties of the thoracic aorta in patients with Marfan syndrome. SUBJECTS: 16 patients with Marfan syndrome and 12 age matched normal controls. METHODS: Transoesophageal echocardiography was performed in all subjects. Minimum and maximum diameters of the descending thoracic aorta were obtained from M mode images and acoustic quantification was used for the on-line evaluation of cross sectional aortic area and peak positive area changes over time. Compliance, distensibility, and stiffness index were calculated using M mode data and non-invasively measured blood pressure and were compared with the indices derived from acoustic quantification. RESULTS: Aortic dimensions normalised for body surface area were not statistically different between patients and normal controls, but there were significant differences for all elasticity indices except compliance. Marfan patients had a lower distensibility [4.2 (SD 1.8) v 5.8 (2.1) cm2/dyn, P < 0.05] and a higher stiffness index [9.7 (3.0) v 7.1 (1.8), P < 0.05]. The dynamic indices derived from the acoustic quantification were significantly smaller in Marfan patients [peak positive area change: 5.1 (1.0) v 7.7 (1.7) cm2/s; P < 0.001; and normalised peak positive area change: 2.5 (1.2) v 4.0 (0.8) cm2/s respectively, P < 0.001] and were suitable to discriminate between normal and abnormal elastic properties. CONCLUSIONS: In Marfan syndrome elastic properties of the descending aorta are significantly different from normal controls, even in the absence of vessel dilatation. In addition to established static indices, indices derived from acoustic quantification reflect dynamic changes of the cross sectional area for the evaluation of regional vessel mechanics. The on-line assessment of peak positive area change allows differentiation from normal individuals and may be more accurate than standard M mode measurements.     

Cell-based resurfacing of large cartilage defects: long-term evaluation of grafts from autologous transgene-activated periosteal cells in a porcine model of osteoarthritis

OBJECTIVE: To investigate the potential of transgene-activated periosteal cells for permanently resurfacing large partial-thickness cartilage defects. METHODS: In miniature pigs, autologous periosteal cells stimulated ex vivo by bone morphogenetic protein 2 gene transfer, using liposomes or a combination of adeno-associated virus (AAV) and adenovirus (Ad) vectors, were applied on a bioresorbable scaffold to chondral lesions comprising the entire medial half of the patella. The resulting repair tissue was assessed, 6 and 26 weeks after transplantation, by histochemical and immunohistochemical methods. The biomechanical properties of the repair tissue were characterized by nanoindentation measurements. Implants of unstimulated cells and untreated lesions served as controls. RESULTS: All grafts showed satisfactory integration into the preexisting cartilage. Six weeks after transplantation, AAV/Ad-stimulated periosteal cells had adopted a chondrocyte-like phenotype in all layers; the newly formed matrix was rich in proteoglycans and type II collagen, and its contact stiffness was close to that of healthy hyaline cartilage. Unstimulated periosteal cells and cells activated by liposomal gene transfer formed only fibrocartilaginous repair tissue with minor contact stiffness. However, within 6 months following transplantation, the AAV/Ad-stimulated cells in the superficial zone tended to dedifferentiate, as indicated by a switch from type II to type I collagen synthesis and reduced contact stiffness. In deeper zones, these cells retained their chondrocytic phenotype, coinciding with positive staining for type II collagen in the matrix. CONCLUSION: Large partial-thickness cartilage defects can be resurfaced efficiently with hyaline-like cartilage formed by transgene-activated periosteal cells. The long-term stability of the cartilage seems to depend on physicobiochemical factors that are active only in deeper zones of the cartilaginous tissue.     

Non-contrast second harmonic imaging improves interobserver agreement and accuracy of dobutamine stress echocardiography in patients with impaired image quality

OBJECTIVE: To examine the influence of second harmonic imaging during dobutamine echocardiography on regional endocardial visibility, interobserver agreement in the interpretation of wall motion abnormalities, and diagnostic accuracy in patients with reduced image quality. DESIGN: Blinded comparison. SETTING: Tertiary care centre. PATIENTS: 103 consecutive patients with suspected coronary artery disease and impaired transthoracic image quality (>/= 2 segments with poor endocardial delineation). METHODS: Fundamental and second harmonic imaging were performed at each stage of a dobutamine stress echocardiography. Coronary angiography was undertaken within three weeks of dobutamine echocardiography in 75 patients. MAIN OUTCOME MEASURES: Evaluation of regional endocardial visibility (scoring from 0 = poor to 2 = good) and of segmental wall motion abnormalities for both modalities separately. A second blinded examiner analysed 70 studies to determine interobserver agreement. RESULTS: Mean (SD) visibility score for all segments was 1.2 (0.4) using fundamental imaging and 1.7 (0.2) using second harmonic imaging at rest (p < 0.001), and 1.1 (0.4) v 1.6 (0.3), respectively, at peak dobutamine dose (p < 0.001). The average number of segments with poor endocardial visibility was lower for second harmonic than for fundamental imaging (0.6 (1.1) v 3.8 (2.6) at rest, p < 0.001; 0.9 (1.3) v 4.3 (2.9) at peak dose, p < 0.001). Improvement was most pronounced in all lateral and anterior segments. The kappa value for identical study interpretation increased from 0. 40 to 0.69 (p < 0.05). Sensitivity for the diagnosis of coronary artery disease was 64% using fundamental imaging versus 92% using harmonic imaging (p < 0.001), while specificity remained unchanged at 75% for both imaging modalities. CONCLUSIONS: Second harmonic imaging enhances endocardial visibility during dobutamine echocardiography. Consequently, interobserver agreement on stress echocardiography interpretation and diagnostic accuracy are significantly improved compared to fundamental imaging. Thus, in difficult to image patients, dobutamine echocardiography should be performed using second harmonic imaging.     

Persistent vegetative state: evaluation of brain metabolism and brain perfusion with PET and SPECT

Patients in persistent vegetative state (PVS) after severe head trauma were investigated with 99mTc-ECD SPECT and 18F-FDG PET to further characterize the degree of brain damage and to obtain insight into changes of brain perfusion and glucose metabolism. 18F-FDG PET and 99mTc-ECD SPECT were performed in 16 patients in PVS. Quantitative PET data were compared with that obtained from seven normal controls. After spatial normalization into Talairach space, global grey matter values and regional data using predefined ROI sets were derived. For comparison of PET and SPECT, regional data were normalized to their individual mean grey matter values. Patients in PVS showed significantly lower values of cerebral glucose metabolism than did the controls. The mean reduction of grey matter values in cortical and subcortical structures was 58%, except in the vermis cerebelli, where only a reduction of 16% was found compared to the controls. Comparing the glucose metabolism and perfusion within the patient group, the pattern of both modalities was similar in the neocortex and internal ganglia. In the cerebellar hemispheres a relatively higher perfusion than glucose metabolism was found. The overall reduction of 58% of glucose metabolism in grey matter structures is in accordance with other PET studies investigating PVS patients with different disease histories. The relative preserved activity of vermis cerebelli seems to be an uncommon finding not described by other authors up to now.