A patient with clinical hypercortisolism and an infiltrating ductal carcinoma of the right mammary gland is presented. Provocative testing of adrenal function demonstrated the pattern of ectopic adrenocorticotropic hormone (ACTH) production. Ultrastructural analysis of the tumor revealed 150-200 nm electron-dense granules that when primarily fixed in Os04 appeared as membrane-bound, centrally dense cored granules. ACTH was extracted from the tumor tissue and immunocytochemically localized in the tumor cell cytoplasm. A clinically significant level of estrogen receptor protein was present in the tumor tissue (120 fmoUmg protein). This case confirms the ability of mammary carcinoma to produce the ectopic ACTH syndrome. Table 1) and ACTH levels were measured. Biopsies were done of the subcutaneous tumor nodules, which were studied by tissue extraction for ACTH (Table 2), immunocytochemistry ( Fig. IB), transmission electron microscopy ( Fig. 21, and estrogen binding protein assay. This supported the carcinoma as the source of ACTH. A bilateral adrenalectomy (left 24 g. right 26 g) was performed. Following adrenal ablation, the chest wall tumor masses reduced in size. A recrudescence of tumor growth was noted at eight months, and she expired 22 months after adrenalectomy. Metastases to bone marrow, lung, cranium, liver, and chest wall were present at the time of her death with no other apparent source of tumor other than the initial breast carcinoma.
Trichohyalin, a protein of Mr between 190 and 220 kDa in different species, was first demonstrated in large granules of the inner root sheath and medulla of hair follicles and may provide a matrix for keratin filaments. We have purified trichohyalin in milligram quantities from a citric acid-insoluble fraction derived from pig tongue epithelium. Trichohyalin was extracted under conditions of low ionic strength from the citric acid-insoluble fraction, separated by gel-filtration chromatography in buffer containing 1 M NaBr, and concentrated by ion-exchange chromatography in buffer containing 4 M urea. The purified material, which is soluble in buffers containing 1 M NaBr, was considered to be trichohyalin because of its characteristic molecular weight and amino acid composition and its localization to hair follicle inner root sheath and medulla by indirect immunofluorescence using antibodies against the purified protein. Immunofluorescence showed that trichohyalin is a major protein of filiform papillae of the tongue. Unlike trichohyalin from other animals examined, the porcine protein is a doublet on SDS polyacrylamide gels of 195 and 210 kDa; both bands are recognized by different antibodies, their two-dimensional peptide maps are nearly identical, and they have nearly identical isoelectric points of about 6.6. Trichohyalin has a Stokes radius of 124 A on gel filtration and a Svedberg constant of 6, consistent with an extended structure. The protein probably associates reversibly in solution, and the native protein we have isolated may be dimeric, because crosslinking of the iodinated purified protein with disuccinimidyl suberate demonstrated the presence of a dimer, which could be dissociated in the presence of high concentrations of urea. Rotary shadowing electron microscopy of the native protein showed a filamentous structure averaging 85 nm in length with a single globular-appearing end-domain. The purification of native trichohyalin provides a basis for future functional studies.
SUMMARY Single, intact, frog skeletal muscle fibres and whole frog hearts were quick‐frozen on a polished, liquid‐He‐cooled copper block and examined in the electron microscope after freeze‐substitution and freeze‐fracture. In both kinds of striated muscle, collapse of the peripheral and intracristal membrane spaces in mitochondria was found to increase with increasing distance from the point of first impact (PFI) of the muscle cells on the cold copper block. The changes correlated with a previously described gradient of Z line and A band cryodamage occurring with distance from the PFI. The findings in thin sections from freeze‐substituted preparations were confirmed by freeze‐fracture preparations. It is concluded that, since the mitochondrial membrane changes are concurrent with, and follow the same spatial distribution of, other manifest cryoarte‐facts, the cryoartefactual nature of the mitochondrial changes must be excluded before functional significance is attributed to them. The collapse of mitochondrial membrane spaces as a sensitive indicator of quality of cryopreservation may apply to non‐muscle cells as well.
When various cations, including Ca2+, are in the fixative, both sarcoplasmic reticulum (SR) of whole skeletal muscle and isolated SR vesicles collapse to form pentalaminate “compound membranes” that result from the apparent fusion of the lumenal lamellae of the membranous envelope of the SR. The process may be reversed by subsequently soaking the tissue in 1 ᴍ NaCl. An identical morphological phenomenon is observed in unfixed quickly frozen isolated frog skeletal muscle fibers, the cation in that case coming from endogenous sources. The hypothesis is advanced that the collapse is an in vivo process mediated by the sequestration of Ca2+ after contraction. The resulting obliteration of the SR lumen would have the effect of displacing the SR contents into the junctional SR, as well as electrically isolating the free SR from the junctional SR during relaxation. As a consequence, resistive coupling between the plasmalemma and the junctional SR becomes a plausible mechanism for the translation of the action potential into Ca2+ release, since the bulk of the SR membrane capacitance would now remain separated from the plasmalemma during relaxation.
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