Differential membrane localization and intermolecular associations of alpha-dystrobrevin isoforms in skeletal muscle

alpha-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length alpha-dystrobrevin-1 (84 kD), and COOH-terminal truncated alpha-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that alpha-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. alpha-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, alpha-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro-translated proteins, alpha-dystrobrevin-2 binds dystrophin, whereas alpha-dystrobrevin-1 binds both dystrophin and utrophin. alpha-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, alpha-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of alpha-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the alpha-dystrobrevins.     

Immunohistochemical localization of human tear lysozyme

Possible local sources of human tear lysozyme were investigated using an indirect immunofluorescence technique. Lysozyme was identified in 20% to 50% of acinar and ductular epithelial cells of both main and accessory lacrimal glands. The staining was granular in character and confined to the apices of the cells. Cells that stained positive tended to be grouped. Interstitial tissues of main and accessory lacrimal tissues did not stain. Conjunctiva and all other ocular tissues examined were unstained by antilysozyme antisera. Our findings are compatible with lysozyme either being produced in lacrimal tissue or being concentrated from plasma. The absence of any other lysozyme-specific fluorescence in the interstitial elements of the lacrimal tissues supports the notion of local synthesis by acinar lacrimal tissue.     

The comparison of seven different methods to quantify the amorphous content of spray dried lactose

The purpose of this work was to verify the usefulness, advantages and disadvantages of seven methods that are widely used to detect, and quantify the amorphous contents in pharmaceutical solids. Here, StepScan DSC, a type of modulated temperature calorimetry method, was applied for the first time to quantify amorphicity. The comparison of the analytical methods was undertaken with real (non-artificial) test samples, i.e. spray-dried lactose samples with various degrees of crystallinity. In these samples, it was essential that the amorphous and the crystalline portions are not present as separate particles, which is the case when physical (artificial) mixtures of totally amorphous and totally crystalline materials are used to produce the calibration curves. X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), StepScan DSC (SS-DSC), isothermal microcalorimetry (IMC), solution calorimetry (SC), Raman spectrometry and gravimetric moisture sorption (GMS) were utilized to quantify the amorphous contents. The results obtained with IMC, SC, Raman, SS-DSC and GMS were in good agreement with each other. Generally, XRPD is the only method that reveals the disordered/amorphous nature of the samples in the crystallographic sense and, in the present study, it also provided consistent results. The conventional DSC method resulted in great variations for the mean amorphicity values since the method is not suitable for lactose where the dehydration of the hydrate and recrystallization of the amorphate overlap during the measurement of the partially amorphous samples. The recrystallized products of totally and partially amorphous lactose samples were found to be different, the former also including crystalline anhydrate. This complicated the interpretation of the IMC and GMS results, where the recrystallization process was monitored and analyzed. Thus, caution is warranted when the amorphicity of real samples is quantified by means of the calibration curves obtained with the physical mixtures of amorphous and crystalline lactose. With SS-DSC, where the quantification was based on the measurement of the heat capacity change associated with glass transition, this problem was not encountered. Utilizing only one method may lead to erroneous conclusions and hence, on the basis the present results, it is advisable to employ a combination of spectroscopic (Raman) and calorimetric (IMC) or gravimetric (GMS) methods.     

Arterial vascular remodeling: the endothelial cell''s central role

As a measurement of the level of anxiety in psychiatric outpatients with anxiety, we determined the saliva level of free 3-methoxy-4-hydroxyphenyleglycol (MHPG) using gas chromatography- mass spectrometry and scored the levels of anxiety with the Hamilton Anxiety Scale (HAS) in patients, before and after drug treatment with alprazolam for 1 week. The saliva level of free-MHPG at first visit to hospital was significantly higher than that of control individuals and disease control individuals and was reduced by alprazolam treatment for 1 week. There was no correlation between MHPG level and the HAS score at the first hospital visit. The MHPG levels after treatment correlated with the HAS scores. The reduction of the anxiety level as scored by the HAS correlated with the reduction of MHPG level. These results indicate that the free saliva MHPG level may be a useful indicator for assessing not only the level of anxiety, but also the response to drug treatment for anxiety in these patients.