Hajime Haimoto

S100a0, (αα) protein in cardiac muscle

S100 protein, an acidic and calcium-binding protein, was believed to be localized in the nervous tissue, but recently it has been reported to be mainly present in the cardiac and the skeletal muscles of various mammals in the alpha alpha form (S100a0) at much higher levels than the nervous tissues. We isolated here S100 protein from human cardiac muscles. The isolated cardiac muscle S100 protein showed a single band on electrophoresis at the same position as that of human skeletal muscle S100a0. The amino acid composition of the purified S100 protein was quite similar to that of human skeletal muscle S100a0 or bovine brain S100a0. The immunohistochemical study by use of antibodies monospecific to the alpha subunit of S100 protein (S100-alpha) revealed that S100-alpha was strongly labeled in human myocardial cells, whereas the beta subunit of S100 protein (S100-beta) was not detected in the cells. These results suggest that a predominant form of S100 protein in human myocardial cells is not S100a (alpha beta) or S100b (beta beta), but S100a0 (alpha alpha). In order to determine the ultrastructural localization of S100a0 in mouse cardiac muscle, the direct peroxidase-labeled antibody method was employed. S100a0 was mainly localized in the polysomes in the interfibrillar spaces, the fine filamentous structure of the Z line and fascia adherens of the intercalated disc and in the lumen of junctional sarcoplasmic reticulum.

S100a0 (αα) Protein: Distribution in Muscle Tissues of Various Animals and Purification from Human Pectoral Muscle

Abstract: When the concentrations of α‐S100 (α subunit of S100 protein) and β‐S100 (β subunit) proteins in various tissues of human and rat were determined by the immunoassay method, immunoreactive β‐S100 was present at high levels in the CNS, adipose tissue, and cartilaginous tissue. In contrast, the α‐S100 was found in the heart and skeletal muscles at concentrations much higher than in the CNS. The concentration of α‐S100 protein was also high in the heart and skeletal muscles of bovine, porcine, canine, and mouse. Since β‐S100 protein levels in those tissues were low, it was suggested that S100 protein in the muscle tissues is present mainly as the αα form (S100a0 protein). To confirm the above findings, immunoreactive α‐S100 protein was purified from human pectoral muscle by employing column chromatographies with butyl‐Sepharose, diethylaminoethyl (DEAE)‐Sepharose, Sephadex G‐75, and finally with an anion‐exchange Mono Q column in a HPLC system. The elution profile of α‐S100 protein from the Mono Q column suggested some heterogeneity of the final preparation. However, each of these fractions traveled with a single band at a position similar to that of bovine S100a0 protein on slab‐gel electrophoresis. The amino acid composition of the final preparation was very similar to the composition of bovine S100a0 protein. The purified α‐S100 protein was eluted from a gel‐filtration column (Superose 12) in the same fraction as bovine S100a0 protein. These results indicate that the α‐S100 protein purified from human pectoral muscle is S100a0 protein, and that S100a0 protein is present universally in the heart and skeletal muscles in a variety of animals at much higher levels than in the brain.

S100a0 (αα) Protein, a Calcium-Binding Protein, Is Localized in the Slow-Twitch Muscle Fiber

Abstract: We previously showed that, in contrast to the distribution of S100b (ββ), S100a0 (αα) is mainly present in human skeletal and heart muscles at the level of 1–2 μg/mg of soluble protein and is universally distributed at high levels in skeletal and heart muscles of various mammals. To elucidate cellular and ultrastructural localizations of the α subunit of S100 protein (S100‐α) in skeletal muscle, we used immunohistochemical and enzyme immunoassay methods. The immunohistochemical study revealed that S100‐α is mainly localized in slow‐twitch muscle fibers, whereas the β subunit of S100 protein (S100‐β) was not detected in both types of muscle fibers, an observation indicating that the predominant form of S100 protein in the slow‐twitch muscle fiber is not S100a or S100b, but S100a0. The quantitative analysis using enzyme immunoassay corroborates the immunohistochemical finding: The S100‐α concentration of mouse soleus muscle (mainly composed of slow‐twitch muscle fibers) is about threefold higher than that of mouse rectus femoris muscle (mainly composed of fast‐twitch muscle fibers). At the ultrastructural level, S100‐α is associated with polysomes, sarcoplasmic reticulum, the plasma membrane, the pellicle around lipid droplets, the outer membrane of mitochondria, and thin and thick filaments, by immunoelectron microscopy.

Evaluation of γ‐enolase as a tumor marker for lung cancer

The α‐enolase and γ‐enolase in tumor tissues and sera of patients with lung cancer were determined with an enzyme immunoassay system. Tissue γ‐enolase in small cell carcinoma of the lung (SCCL, n = 11), large cell carcinoma (n = 11), and non‐SCCL (except for large cell carcinoma) (n = 34) were enhanced approximately 35‐fold, ninefold, and fourfold, respectively; tissue γ/α + γ value of SCCL was significantly higher than that of normal lung tissue (P < 0.01). Serum γ‐enolase level was elevated (>6.0 ng/ml) in 14/18, 3/10, and 15/60 patients with SCCL, large cell carcinoma, and non‐SCCL (except for large cell carcinoma), respectively, and serum γ/α + γ value of SCCL was significantly higher than that of healthy subjects (P < 0.01). Immunohistochemically, the γ‐enolase was positive in 29/31 of the lung cancers. Serum γ‐enolase value is a useful tumor marker for staging and monitoring treatment of patients with lung cancer, and serum γ/α + γ value may be useful for differential diagnosis of SCCL from non‐SCCL or in differentiating lung cancers possessing neuroendocrine features from other lung cancers.

Immunohistochemical study of so-called sclerosing haemangioma of the lung

To elucidate the histogenesis of sclerosis haemangioma of the lung, we examined 7 cases with the immunoperoxidase method using antibodies against several useful marker antigens; secretory component (SC), cytokeratins, epithelial membrane antigen (EMA) for epithelial cells, factor VIII related antigens (factor VIII) for endothelial cells, vimentin and desmin for mesenchymal cells. The results were compared with those of histologically normal lung tissues. Both the characteristic round cells arranged in sheets, which are present predominantly in the solid area and are reported to be neoplastic, and the flattened cells lining blood lakes show positive staining for EMA only, with negative staining for the other marker antigens. These observations suggest that these cells are derived from epithelium rather than mesothelium or from endothelium, and are analogous to type I pneumocytes. This conclusion is supported by their immunohistochemical characteristics, in comparison with the localization patterns of the marker antigens in normal lung tissues. However, the lining epithelial cells of papillary projections in the papillary area and of ducts in the solid area stained for SC and cytokeratins as well as EMA, and their immunohistochemical characteristics are analogous to those of bronchiolar epithelial cells or type II pneumocytes in normal lung tissues.

An immunochemical and immunohistochemical study of S100 protein in renal cell carcinoma

The authors localized S100 protein in renal tubules and renal cell carcinoma by immunohistochemical study and quantitative analysis by enzyme immunoassay. The α subunit of S100 protein (S100‐α) was localized in epithelial cells of proximal tubules, thin limbs of loops of Henle, collecting tubules, and a few of Bowmans capsules. The β subunit (S100‐β) was immunostained in the distal tubules, thick and thin loops of Henle, collecting tubules, and a few proximal tubules. In renal cell carcinoma, S100‐α was immunohistochemically demonstrated in 82% (41/50) of patients including sarcomatoid variants, whereas S100‐β was detected in 46% (23/50). Both the number of positively stained tumor cells and the staining intensity were greater in S100‐α than in S100‐β. Concentrations of S100‐α in the cortex were 80.3 ± 22.5 ng/mg protein (n = 7), whereas those of renal cell carcinoma were 387 ± 533 ng/mg protein (n = 19), i.e., about five times higher. Concentrations of S100‐β in both normal kidney (1.96 ± 0.74 ng/mg protein) and renal cell carcinoma (2.05 ± 2.16 ng/mg protein) were much lower than those of S100‐α. The authors also localized S100‐α and S100‐β in tissues of other renal tumors and tumors arising from other organs. S100ao appears to be a useful immunohistochemical marker for renal cell carcinoma.

Evaluation of Gamma-Enolase as a Tumor Marker for Renal Cell Carcinoma

To evaluate whether serum gamma-enolase is a useful marker for renal cell carcinoma alpha and gamma-enolases in tissues of 36 renal cell carcinomas and 13 normal kidneys, and in sera of 103 renal cell carcinoma patients were determined with an enzyme immunoassay system. Tissue gamma and alpha-enolase levels were 34 and 2.3 times higher, respectively, in renal cell carcinoma than in normal renal cortex. The tissue gamma enolase-to-total enolase value of renal cell carcinoma (5.3 per cent) was significantly higher than that of normal cortex (0.29 per cent) and medulla (0.51 per cent). Over-all serum gamma-enolase levels were elevated (more than 6.0 ng. per ml.) in 53 of 103 patients (51 per cent) with renal cell carcinoma. In regard to stage the positive rates were 34 per cent (12 of 35) of patients with stage I, 22 per cent (2 of 9) with stage II, 80 per cent (12 of 15) with stage III, 61 per cent (22 of 36) with stage IV and 61 per cent (5 of 8) with recurrent disease. The mean value of serum gamma-enolase in renal cell carcinoma (8.0 +/- 5.7 ng. per ml.) was significantly higher than that of normal subjects (3.1 +/- 0.9 ng. per ml., p less than 0.001). The mean value of serum gamma-enolase in patients with high stage tumors (III and IV, 9.9 +/- 6.8 ng. per nl.) was significantly higher than that of low stage tumors (I and II, 5.8 +/- 3.0 ng. per ml., p less than 0.001). In 39 patients treated by complete surgical excision serum gamma-enolase was significantly reduced postoperatively (p less than 0.01). Furthermore, 7 of 8 patients whose serum gamma-enolase levels were determined serially had levels within the normal range postoperatively that increased when distant metastases appeared. These results indicate that serum gamma-enolase could be a useful tumor marker to stage disease and monitor treatment in patients with renal cell carcinoma.

Immunoelectronmicroscopic study on the transport of secretory IgA in the lower respiratory tract and alveoli.

To define the immunocytochemical localization of secretory component (SC), IgA and J chain in human bronchioles and alveoli, a direct peroxidase-labeled antibody method was used. SC was found in non-ciliated cells of the bronchioles including respiratory bronchioles and type II alveolar epithelial cells, whereas SC was rarely present in ciliated cells and type I alveolar epithelial cells and was absent from goblet cells. In the positively reacting cells, SC was found in secretory protein synthetic organelles such as perinuclear spaces and endoplasmic reticulum, Golgi complexes, and on the external surfaces of the apical and basolateral plasma membranes. IgA and J chain were localized in the epithelial cells where SC was found. Ultrastructually IgA was present on the apical and basolateral plasma membranes, in pinocytic invaginations of the membranes, and in vesicles distributed through the cytoplasm, especially in the apical cytoplasm of the epithelial cells where SC was found. In addition, IgA and J chain were found to be associated with the endothelial cells of the capillaries, plasma cells and the surrounding interstitium. These observations suggest that SC is synthesized and secreted by epithelial cells, especially non-ciliated cells of the bronchioles including respiratory bronchioles and type II alveolar epithelial cells. They also suggest that secretory IgA (sIgA) is transported into alveolar spaces and the bronchiolar lumen through these cells by SC-mediated transport mechanism. This sIgA may play an important role in defense mechanisms of the lower respiratory tract and alveoli.

Aldolase C in neuroendocrine tumors: an immunohistochemical study

SummaryThe expression of cerebral type aldolase C was investigated immunohistochemically in six varieties of neuroendocrine (n=57) and six types of non-endocrine tumor (n=76) using the avidin-biotin complex method. Aldolase C expression in the neuroendocrine tumors was also compared with those of chromogranin and gamma enolase. Aldolase C was detected in all the islet cell (7/7) and carcinoid tumors (10/10), thyroid medullary carcinomas (7/7), and pheochromocytomas (10/10), as well as in the majority of neuronal tumors (8/10) and bronchial small cell carcinomas (10/13). Chromogranin immunoreactivity was restricted to the tumors with abundant neuroendocrine granules. Gamma enolase positivity was generally similar to that of aldolase C, but there were some differences. Amongst the bronchial small cell carcinomas, three tumors negative for gamma enolase were positive for aldolase C, while another three tumors were positive for gamma enolase only. However all the small cell carcinomas were positive for at least one of these two enzymes. Aldolase C was detected in 28 (37%) of the 76 non-endocrine tumors and tended to be expressed preferentially in the differentiated portions of these tumors. Although aldolase C was expressed in many bronchial squamous cell carcinomas, the immunoreactivity was localized mainly in keratinizing foci and the less differentiated parts of these tumors expressed the enzyme only occasionally. Thus aldolase C, in conjunction with other neuroendocrine-associated markers, may be of value in identifying tumors of neuroendocrine type.