Association of neuronal nitric oxide synthase (nNOS) with alpha1-syntrophin at the sarcolemma.

alpha1-syntrophin is a PDZ-containing dystrophin-associated protein, expressed predominantly in striated muscle and brain. alpha1-syntrophin null mice generated by gene targeting technique showed no overt muscular dystrophic phenotype. Though other dystrophin-associated proteins were localized at the sarcolemma, neuronal nitric oxide synthase (nNOS) was selectively lost from the membrane fraction but remained in the cytoplasm. Thus, the alpha1-syntrophin null mice are useful in the elucidation of the functional importance of nNOS targeting at the sarcolemma. In addition, the mice would facilitate identification of other signaling molecules, which are targeted to dystrophin complex via interaction with alpha1-syntrophin.     

Expression of Leu-19 (CD56, N-CAM) and nitric oxide synthase (NOS) I in denervated and reinnervated human skeletal muscle.

Neural cell adhesion molecule (N-CAM, Leu-19, CD 56) expression appears during muscle fiber regeneration and after denervation. Sarcolemma-associated nitric oxide synthase (NOS) I, however, disappears from denervated myofibers. The dynamics of expression of both proteins were studied in 5 cases of acute/subacute denervation, 28 cases of chronic denervation with and without collateral reinnervation, 5 cases of the intermediate type spinal muscular atrophy (SMA 2), and in 2 normal biopsies. NOS I and its NADPH diaphorase (NADPHd) activity disappeared from the sarcolemma region shortly after denervation, and before the appearance of denervation atrophy. N-CAM was found diffusely distributed in the sarcoplasm at the most severe phase of denervation atrophy in the majority of highly atrophic fibers. During reinnervation, NOS I expression remained absent and in part of the cases the target/targetoid phenomenon appeared. In parallel with the increase in volume of the reinnervated muscle fibers, the intensity of N-CAM immunoreactivity decreased progressively. After full restitution of muscle fiber caliber, the target/targetoid phenomenon and N-CAM immunostaining disappeared completely, and, finally, NOS I reappeared in the sarcolemma region. The sarcolemmal expression of dystrophin and dystrophin-associated proteins was unchanged during denervation. NOS I was completely absent in children with SMA 2, since the protein does not appear before 5 years of age in skeletal muscle, while N-CAM was very intensely expressed in the sarcoplasm of highly atrophic denervated muscle fibers. In conclusion, this study suggests that innervation is an important factor for selective gene expression and positioning of NOS I and N-CAM in skeletal muscle and gives practical information for the assessment of the phase and developmental stage of the denervation and reinnervation process.     

High resolution scanning electron microscopy of stereocilia in the cochlea of normal,postmortem, and drug-treated guinea pigs

The morphology of hair bundles has been studied by high resolution scanning electron microscopy using a variety of fixatives, including glutaraldehyde, glutaraldehyde-picrate, glutaraldehyde-tannic acid, glutaraldehyde followed by post-fixation in osmium tetroxide, and the osmium thiocarbohydrazide technique. Critical evaluation of several metal coatings, gold, gold-palladium, and platinum has been carried out. Both the surface texture of stereocilia and their cross-links are sensitive to fixation and metal coating. We are of the opinion that glutaraldehyde gives the best general quality of fixation and preservation for all types of cross-links. We have described three major sets of cross-links: first, lateral links connecting stereocilia within the same row; second, lateral links connecting stereocilia of adjacent rows; and third, upward-pointing links, one per stereocilium, connecting the tip of each shorter stereocilium to the lateral surface of the adjacent taller stereocilium. Current physiological and anatomical evidence suggests that the lateral links couple the individual stereocilia within the hair bundle so that they function as a single mechanical unit. The upward-pointing tip links are ideally placed to respond to mechanical deformation of the hair bundle, being stretched when the stereocilia are deflected in the excitatory direction towards the tallest row and relaxed when deflected in the opposite, inhibitory direction. Postmortem morphological changes are detected within 15 minutes of cardiac arrest and become progressively more pronounced in time. These results enabled us to distinguish specific druginduced changes which could not be attributed simply to cell death. Effects of cisplatin and kanamycin upon hair bundles are described. The work reported here is based on studies using the guinea pig cochlea. Some of the postmortem changes described have also been confirmed in human cochleas. It is stressed that many of the postmortem and drug-induced effects can only reliably be studied by high resolution scanning electron microscopy coupled with appropriate prearation procedures.     

Colour display of video information in scanning electron microscopy: Principles and applications to physics,geology, soil science,biology, and medicine

From a colourimetric point of view, colour has two independent aspects: brightness and chromaticity. In black and white images, all elements are of the same chromaticity and can be distinguished only by brightness contrast. In the colour image, elements of the same brightness can be discriminated by chromaticity (colour) contrast. Generally, colour image elements can be discriminated both by brightness and by chromaticity. As the human eye can distinguish a number of hues two orders of magnitude larger than the number of grey levels, it is safe to say that the colour image is much more informative than the black and white image. There are some peculiarities of the colour image and methods of its formation in SEM. Two principles of image formation are used. The first consists of the formation of a real colour image in the cathodoluminescence mode. In this case the colour of an image element is determined by the spectrum of the luminescence emission excited in the corresponding point of an object by the electron beam. The second principle is that of colour coding (quasicolour, pseudocolour), when a video signal in colour (either digital or analog) corresponds to a video signal (amplitude, frequency, phase, etc.) produced by any mode in the scanning electron microscope. We present a review of the methods of colour display of video information in scanning electron microscopy and their applications to physics, geology, soil science, biology, and medicine.     

A quantitative evaluation of the glomerular capillary endothelium in rat and human kidneys: Utilization of vascular perfusion,freeze-cracking of tissue,and scanning electron microscopy

Modern morphological investigation requires the use of a variety of technological approaches and the employment of rigorous morphometric analysis for an adequate evaluation of the structural and ultrastructural features of a tissue or organ. The introduction of the technique of freeze-cracking of tissue to expose new surfaces has made it possible to quantitate the normal surface characteristics of the glomerular capillaries of the mammalian kidney. This report describes the techniques used for the preparation and quantitative assessment of normal glomerular endothelial morphology. The techniques of in vivo and in vitro vascular perfusion of kidneys as a method of fixation and the freeze-cracking of tissue are outlined in detail. In addition, a morphometric analysis of the endothelial surface characteristics are described and values are reported for the control rat and human kidneys from transplant donors.     

Acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine.

Tissue processing for transmission electron microscopy (TEM) is commonly accomplished using ethanol (EtOH) as a dehydrating solvent and propylene oxide (PO) as a transition fluid. Both solvents have some undesirable properties: EtOH solubilizes lipids; PO is highly flammable, volatile, toxic, and potentially carcinogenic. Their replacement by a compound devoid of these characteristics is therefore desirable. Acetonitrile (AN) appears to be such a solvent. It is freely miscible with water, alcohols, acetone, and epoxy resins; it does not interfere with epoxy polymerization; and the resulting cured resins have excellent cutting quality and beam stability. AN is also an excellent dehydrating agent whose use does not necessitate modification of current techniques. Most importantly, the low solubility of phospholipids (PL) in AN limits the loss of membrane lipids and, hence, leads to a better preservation of tissue features.