A new fixative which primarily stabilizes carbohydrate moieties was developed for immunoelectron microscopy. It contains periodate, lysine and paraformaldehyde. Theoretically, the carbohydrates are oxidized by periodate and cross-linked by lysine. The fixative can preserve antigenicity as well as paraformaldehyde and ultrastructure as well as glutaraldehyde. Using this fixative and the peroxidase-labeled antibody technique, basement membrane antigen was localized within the cisternae of endoplasmic reticulum of parietal yolk sac cells and in extracellular basement membranes with adequate tissue successfully accomplished by conventional

Periodate-lysine-paraformaldehyde fixative a new fixative for immunoelectron microscopy

Biotin was covalently attached to antibodies, antigens and enzymes, and the effects of this labeling on the antigen and antibody binding capacity and on enzymatic activity were tested. Based on avidin-biotin interaction, the labeled proteins were used in quantitative enzyme-immunoassay and enzyme-immunohistochemical staining procedures. Two procedures were developed. In the first procedure, named the Bridged Avidin-Biotin (BRAB) technique four steps were used sequentially in order to quantify or detect an immobilized antigen: 1) incubation with biotin-labeled antibody; 2) incubation with avidin; 3) incubation with biotin-labeled enzyme; 4) measurement or histochemical staining of the enzyme. The technique is based on the observation that avidin possesses four active sites. In the second procedure, named the Labeled Avidin-Biotin (LAB) technique, biotin-labeled antibody and enzyme-labeled avidin are used sequentially. Enzyme-associated antigen is then quantified or revealed immunohistochemically. The optimal conditions for enzyme-immunoassay and enzyme-immunohistochemical staining using BRAB and LAB procedures were established.

The Use of Avidin-Biotin Interaction in Immunoenzymatic Techniques

Fine structural localization of glutamine synthetase in astrocytes of rat brain.

The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others, localized changes in Ca(o)(2+) within the ECF can originate from several mechanisms, including fluxes of calcium ions into or out of cellular or extracellular stores or across epithelium that absorb or secrete Ca(2+). In any event, the CaR and other receptors/sensors for Ca(o)(2+) and probably for other extracellular ions represent versatile regulators of numerous cellular functions and may serve as important therapeutic targets.

Extracellular Calcium Sensing and Extracellular Calcium Signaling

Oxidative stress is considered a major contributor to etiology of both "normal" senescence and severe pathologies with serious public health implications. Mitochondria generate reactive oxygen species (ROS) that are thought to augment intracellular oxidative stress. Mitochondria possess at least nine known sites that are capable of generating superoxide anion, a progenitor ROS. Mitochondria also possess numerous ROS defense systems that are much less studied. Studies of the last three decades shed light on many important mechanistic details of mitochondrial ROS production, but the bigger picture remains obscure. This review summarizes the current knowledge about major components involved in mitochondrial ROS metabolism and factors that regulate ROS generation and removal. An integrative, systemic approach is applied to analysis of mitochondrial ROS metabolism, which is now dissected into mitochondrial ROS production, mitochondrial ROS removal, and mitochondrial ROS emission. It is suggested that mitochondria augment intracellular oxidative stress due primarily to failure of their ROS removal systems, whereas the role of mitochondrial ROS emission is yet to be determined and a net increase in mitochondrial ROS production in situ remains to be demonstrated.

/pdf/mitochondrial-metabolism-of-reactive-oxygen-species-4bf4e010ud.pdf

Mitochondrial metabolism of reactive oxygen species.

A new method of conjugating horseradish peroxidase with proteins was developed. The carbohydrate moiety of fluorodinitrobenzene-blocked peroxidase was oxidized with so- dium periodate to form aldehyde groups. The peroxidase-aldehyde was then bound to free amino groups of proteins unidirectionally at high efficiencies. Peroxidase-labeled immune- globulin retained its immunologic as well as enzymatic activities. Horseradish peroxidase (HRPO), when cou- pled to immunoglobulin G (IgG), has proven to be a useful marker for immunohistochemistry (13). In contrast to other immunohistochemical markers, such as fluorescein (7) and ferritin (20), HRPO may be used for both light and electron microscopy. It is especially suitable for the intracellular localization of antigens at the ultrastructural level since HRPO (40,000 molec- ular weight) is considerably smaller than fer- ritin (650,000 molecular weight); thus, HRPO- labeled IgG (HRPO-IgG) has superior penetra- tion properties. HRPO is usually coupled to IgG using bifunc- tional reagents. With earlier methods, the con- jugation reactions were carried out in the pres- ence of HRPO, IgG, and p,p'-difluoro-m,m - din tro-diphenyl sulfone (FNPS) (13), (1-cyclohexyl- 3-(2-morpholinoethyl)) carbodiimide metho-p- toluenesulfonate (4), cyanuric chloride (1), bis-

Peroxidase-labeled antibody. A new method of conjugation

Thyroglobulin (TG), the primary synthetic product of the thyroid, is the macromolecular precursor of thyroid hormones TG synthesis, iodination, storage in follicles, and degradation control thyroid hormone formation and secretion into the circulation Thyrotropin (TSH), via its receptor (TSHR), increases thyroid hormone levels by up-regulating expression of the sodium iodide symporter (NIS), thyroid peroxidase (TPO), and TG genes TSH does this by modulating the expression and activity of several thyroid-specific transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, which coordinately regulate NIS, TPO, TG, and the TSHR Major histocompatibility complex class I gene expression, which also is regulated by TTF-1 and Pax-8 in the thyroid, is decreased simultaneously This helps maintain self-tolerance in the face of TSH-increased gene products necessary for thyroid hormone formation In this report we show that follicular TG counter-regulates TSH-increased, thyroid-specific gene transcription by suppressing expression of the TTF-1, TTF-2, and Pax-8 genes This decreases expression of the TG, TPO, NIS, and TSHR genes, but increases class I expression TG acts transcriptionally, targeting, for example, a sequence within 115 kb of the 5′ flanking region of TTF-1 TG does not affect ubiquitous transcription factors regulating TG, TPO, NIS, and/or TSHR gene expression The inhibitory effect of TG on gene expression is not duplicated by thyroid hormones or iodide and may be mediated by a TG-binding protein on the apical membrane We hypothesize that TG-initiated, transcriptional regulation of thyroid-restricted genes is a normal, feedback, compensatory mechanism that limits follicular function and contributes to follicular heterogeneity

https://www.pnas.org/content/pnas/95/14/8251.full.pdf

Autoregulation of thyroid-specific gene transcription by thyroglobulin

Expression of thyroid transcription factor-1 in normal and neoplastic lung tissues.

Growth activity in hyperplastic and neoplastic human thyroid determined by an immunohistochemical staining procedure using monoclonal antibody MIB-1

Thyroid transcription factor-1 (TTF-1) has been known to regulate the transcriptional activity of thyroid-specific genes in thyroid follicular cells. We recently identified TTF-1 mRNA expression in rat thyroid C cells. The current study was undertaken to elucidate how TTF-1 is expressed in human C cells and medullary thyroid carcinomas (MTCs), and how this expression influences the functions and clinical behavior of these cells. By immunohistochemistry, the nuclei of normal and hyperplastic C cells distinctively reacted with antibody against TTF-1, whereas the immunostaining intensity in C cells was rather weak and heterogeneous in comparison with that in follicular cells. Identical TTF-1 immunoreactivity was observed in all 15 MTC specimens examined. The reaction intensity did not depend on tumor patterns or cell features. In nonisotopic in situ hybridization, an antisense riboprobe clearly hybridized the cytoplasms of C cells and MTC cells, which concurrently showed immunohistochemical positivity for calcitonin. Northern blot analysis indicated a marked hybridization with TTF-1 mRNA of approximately 2.3 kb in an MTC specimen. Furthermore, the presence of TTF-1 mRNA was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in the human MTC cell line, TT. Our results suggest that human thyroid C cells and MTC cells express TTF-1 in connection with their functional ability. Therefore, TTF-1 expression can be a functional marker not only for follicular cells and follicular cell tumors but also for C cells and medullary C cell carcinomas.

Akira Kawaoi

Papers

Peroxidase-labeled antibody. A new method of conjugation

Autoregulation of thyroid-specific gene transcription by thyroglobulin

Expression of thyroid transcription factor-1 in normal and neoplastic lung tissues.

Growth activity in hyperplastic and neoplastic human thyroid determined by an immunohistochemical staining procedure using monoclonal antibody MIB-1

Expression of Thyroid Transcription Factor-1(TTF-1)in Human C Cells and Medullary Thyroid Carcinomas