Muscle membrane markers

Muscle biopsy shows vacuolar myopathy of very severe degree affecting all fibers in Pompe s disease but of varying degree and distribution in childhood and adult AMD. In adult AMD, biopsy specimens from unaffected muscles may appear normal by light microscopy. The vacuoles contain PAS-positive material, a marker for glycogen. Electron microscopy shows abundant glycogen, both within membranous sacs, presumably lysosomes, and free in the cytoplasm. 

Fung and colleagues examined the metabolic conversion of organic nitrates in sub-cellular fractions of bovine coronary artery smooth muscle cells [66, 67]. They found NO-generating capacity to be present in membrane fractions and, with the use of marker enzymes, identified plasma membrane as the primary location. The enzyme involved in bioconversion was not glutathione-S-transferase [68] and differed from those that catalyse activation of organic nitrites [69]. Partial purification [70] established that the molecular sizes of the native enzyme and subunits were approximately 200 kDa and 58 kDa respectively, and that enzymic activity depends on the presence of a free thiol group. 

The enzyme responsible for this topping-up ATP in active muscle is CK. CK is found in high concentration in muscle cells, both free within the sarcoplasm and also associated with membranes of mitochondria and the sarcoplasmic reticulum. Structurally, creatine kinase is a dimeric enzyme of B and/or M subunits, each of about 40 kDa. Three quaternary structure isoenzyme forms arise CK-MM, CK-BB and CK-MB. The predominant form in all muscles is CK-MM, but cardiac muscle also contains a significant amount of CK-MB and this isoenzyme can be used as a specific marker of myocardial damage (see Case Notes at the end of this chapter). 

All the major cell types (epithelial, endothelial, smooth muscle cells, pneumocytes, chondrocytes, fibroblasts) capable of producing connective tissues (e.g. cartilage, basement membrane, parenchymal stroma) are susceptible to oxidative injury in vitro [29- 33], and over the past decade the mechanism(s) of oxidative stress to these cell types has been the focus of intense research. Unfortunately, few of these studies have been specifically extended to examine the biochemical evidence for oxidative injury to connective tissue producing cells in vivo [34], Our most recent work has concentrated on determining the precise biochemical footprints of oxidative injury found within chondrocytes (also colonic epithelial cells) and attempting to correlate the presence or absence of these oxidative-injury markers seen in vitro with inflamed material from animal models and human pathological material. 

Evidence for the link between PKC activation and cell proliferation was initially provided by the demonstration that two intracellular events associated with cell replication - a rise in cytosolic pH and the expression of the proto-oncogenes c-fi/s and c-tnyc - are controlled by PKC PKC stimulates the membrane bound Na /H exchange mechanism in smooth muscle which extrudes intracellular H in exchange for extracellular Na this leads to a rise in intracellular pH, a prerequisite for cellular DNA replication (Mitsuka and Berk, 1991). c-fbs and c-myc are proto-oncogenes whose transcription to mRNA is one of the earliest markers of cell proliferation they encode for proteins, found in the cell nucleus, which initiate the sequence of events leading to DNA synthesis (Rozen-gurt, 1991). 

Immunohistochemical staining is fairly consistent and straightforward in ONE. The tumor cells are positive for synaptophysin and neuron-specific enolase and, occasionally, for chromogranin (Fig. 9.5). Up to 30% of cases may be positive for CAM 5.2. However, ONEs are uniformly negative for epithelial membrane antigen, muscle markers, and CD 99 (MIC-2). Elongated cells often observed at the periphery of the lobules, so-called sus-tentacular cells, are positive for S-100 protein and glial fibrillary acidic protein (Fig. 9.6, Tables 9.4 and 9.5). 

Perineurioma of the GI tract is a relatively recently described entity that rarely presents as a polypoid lesion. These lesions are composed of a bland spindle cell proliferation with delicate cytoplasmic protrusions that emanate from either side of an elongated nucleus. Like neurofibroma, this lesion grows around and entraps adjacent intestinal crypts. Analogous to perineuriomas of the soft tissues, intestinal perineuriomas almost uniformly stain with epithelial membrane antigen (EMA) a subset of these lesions expresses claudin-1. These lesions are negative for smooth muscle markers, S-100 protein, and c-kit. 

Phosphorus NMR is also used for vascularly perfused preparations, such as heart, skeletal muscle, bladder, or uterus. In these cases, the contributions to the spectra by the perfusate may be important. Phe-nylphosphonate or similar derivatives are often used for such experiments. PPA has a direct C-P bond, unlike phosphates that have O-P bonds. As a result, PPA resonates downfield (to the left) of the naturally occurring phosphorus compounds. In mechanical and NMR experiments, the effects of PPA on the isolated, perfused bladder were measured (Fisher and Dillon, 1987a). PPA in concentrations up to 20 mM did not produce a significant reduction in force generation. Its NMR peak position was shown to be pH sensitive with a pK of 7.09, making it ideal for measurements of extracellular pH. It did not produce any alteration in the natural phosphorus spectrum, and it was able to be washed into and out of the perfused tissue without measurable residue, indicating that it did not cross cell membranes. In addition to being a pH indicator, it is therefore also useful as a marker for extracellular space. 

The involvement of pericytes in physiological or tumour angiogenesis is a matter of debate. Papoutsi et al. (2000) studied the expression of pericyte, smooth muscle cell and matrix markers in experimental tumours of the mammary ductal adenoma MDA-MB231 cell line grown on chick or quail chorioallantoic membrane. Pericyte-like cells may be attracted by MDA-MB231 cells during tumour angiogenesis but failed to interact properly with endothelial cells in the tumour environment (Lauer et al. 2000). 

The use of REE in biological systems can be subdivided into three categories. First, a large amount of work has been devoted to the use of rare earths, in particular La, as a probe in systems of biological interest (Reuben 1979), particularly for Ca in biomembranes (Mikkelson 1976). Second, many studies have utilized REE as markers to trace the movement and deposition of elements in tissues. These include the use of La to monitor the movement of Ca and water within isolated membranes, whole cells and whole organisms both animals and plants [for a review see Mikkelson (1976) and Weiss (1974)]. Finally, lanthanides have been used in investigating the role of Ca in muscle and nerve activity. This has been done by using La and other REE as competitive inhibitors of Ca ". ... 

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