Journal of Histochemistry & Cytochemistry

Ozden, S; Aubert, C; Gonzalez-Dunia, D; Brahic, M

doi: 10.1177/39.10.1940303pmid: 1940303

SJL/J mice inoculated intracranially with the DA strain of Theiler's virus exhibit a persistent demyelinating disease of the central nervous system. To investigate the effect of persistent infection of oligodendrocytes on the expression of myelin genes, we analyzed the level of PLP mRNA in infected as well as uninfected oligodendrocytes. This study was performed at the single-cell level using the simultaneous detection of viral antigens by immunocytochemistry and PLP mRNAs by in situ hybridization with 35S-labeled oligonucleotide probes. Our data indicate that viral infection of oligodendrocytes reduces the level of PLP mRNA by about 80%.

Yodozawa, S; Tsunoda, Y; Funai, T; Tashiro, Y

doi: 10.1177/39.10.1940304pmid: 1940304

We investigated spatiotemporal changes in cytoplasmic free Ca2+ concentration ([Ca2+]i) in norepinephrine (NE)-stimulated and fura-2-loaded individual H-35 rat hepatoma cells, using digital imaging microscopy and high time-resolution microspectrofluorometry. Application of NE (5 x 10(-6) M) resulted in an initial transient increase in [Ca2+]i, followed by a small sustained [Ca2+]i plateau above the pre-stimulation level. The initial peak and the small sustained plateau originated from intracellular stores and the extracellular space, respectively. The initial transient evoked by NE was totally blocked by phentolamine, an alpha-adrenergic antagonist, but was not blocked by either pre-incubation with nominally Ca(2+)-free medium or by pre-treatment of cells with La3+. On the other hand, the sustained plateau was eliminated by Ca(2+)-free medium or La3+. Therefore, H-35 cells have a Ca(2+)-signaling pathway which is activated via alpha-adrenergic receptors. Mn2+ entered the cytosol after NE stimulation, as shown by quenching of fura-2. This indicates that H-35 hepatoma cells possess Mn(2+)-permeable Ca2+ channels at the plasma membrane. In addition, the Ca2+ efflux pattern from H-35 cells to the extracellular space during NE stimulation was visualized by digital imaging microscopy when free fura-2 was equilibrated between the cells and the extracellular space. The efflux of Ca2+ from H-35 begins between the initial [Ca2+]i transient and the sustained [Ca2+]i plateau.

Snow, A D; Bramson, R; Mar, H; Wight, T N; Kisilevsky, R

doi: 10.1177/39.10.1940305pmid: 1940305

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.

Voorhout, W F; Veenendaal, T; Haagsman, H P; Verkleij, A J; van Golde, L M; Geuze, H J

doi: 10.1177/39.10.1940306pmid: 1940306

Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice.

Kalina, M; Socher, R

doi: 10.1177/39.10.1658127pmid: 1658127

We investigated the uptake of Lucifer yellow and surfactant complexed with gold (S-G) by isolated alveolar Type II cells. The fluid phase marker Lucifer yellow did not reach lamellar bodies (LB) even after prolonged incubation time, whereas S-G was internalized and found in LB. Treatment of Type II cells with lysosomotropic weak bases (NH4Cl and chloroquine) resulted in dilation of endosomes, lysosomes, and LB. The effect of these agents on LB resulted in disappearance of their lamellar organization, as detected by polarized light and electron microscopy. After incubation in lysosomotropic agent-free medium, endocytosis of Lucifer yellow and S-G in treated cells was mainly directed towards large vacuoles resembling either multivesicular bodies (MVB) or lysosomes. The possible relationship between LB, MVB, and lysosomes in freshly isolated as well as cultured alveolar Type II cells is discussed.

Iida, H; Shibata, Y

doi: 10.1177/39.10.1719068pmid: 1719068

We examined the effects of disassembly of microtubules (MT) on the structure and the functions of the Golgi apparatus (GA) in cultured atrial myocytes. MT disassembly with nocodazole led to fragmentation of the GA into small units. The fragmented Golgi units retained their cis-trans polarity and post-cisternal elements, including the trans-Golgi network (TGN). Neither endocytosis of lectin-labeled membrane nor its delivery to the fragmented Golgi units was interrupted by fragmentation of the GA after MT disassembly with nocodazole treatment. A fraction of the secretory granules associated with the fragmented Golgi units was also labeled with the internalized tracer. These results suggest that in nocodazole-treated cultured atrial myocytes, the fragmented Golgi units appear to be structurally and functionally intact despite the altered geometric arrangement of the GA in the cells.

Usuda, N; Kuwabara, T; Ichikawa, R; Hashimoto, T; Nagata, T

doi: 10.1177/39.10.1940307pmid: 1940307

We examined the distribution of peroxisome-specific membrane polypeptides (PMPs) among peroxisomes of the liver, renal cortex, and jejunal mucosa, using antibodies for 70 KD, 26 KD and 22 KD PMPs. Immunoblot analysis showed signals for 70 KD polypeptide in all three kinds of tissue, but for the other two only in the liver and renal cortex, with neither being detected in jejunal mucosa. The total amounts of PMPs increased in all three organs with DEHP (di-(2-ethylhexyl)phthalate) administration. By immunoelectron microscopic analysis using protein A-gold, the three PMPs were localized along the peroxisomal membrane. Quantitation of the gold particles associated with the peroxisomal membrane showed an increase in the density of 70 KD and 26 KD PMPs but a decrease in 22 KD PMP with the administration of DEHP. The presence of tissue-specific localizations of PMPs suggest the 70 KD PMP is a common constituent of peroxisomes of these three tissues, whereas 26 KD and 22 KD PMPs are absent in microperoxisomes of jejunal mucosal epithelium.

Arbeille, B B; Fauvel-Lafeve, F M; Lemesle, M B; Tenza, D; Legrand, Y J

doi: 10.1177/39.10.1940308pmid: 1940308

We used antisera directed against human platelet thrombospondin (TSP) and microfibril-associated GP 128 to localize the presence of these glycoproteins in fixed sections of human placenta or porcine arteries and skin by immunogold labeling, using electron microscopy. These two antibodies reacted with both human and porcine tissues and always recognized the same structures. In all three tissues the antibodies were associated with the basement membranes and, more precisely, with the microfibrillar structures present at the junction between the basement membrane and the adjacent connective tissue. This localization indicates that GP 128 and TSP are associated with the microfibrils, and suggests their possible role in the attachment of basement membrane to the connective tissue meshwork. Their presence in microfibrils associated with the subendothelial basement membrane in arteries may be important in regard to the thrombogenicity of the subendothelium since, after an endothelial lesion, they may be directly accessible to blood platelets.

Kiyama, H; Emson, P C

doi: 10.1177/39.10.1682362pmid: 1682362

We have developed a method of non-radioactive in situ hybridization histochemistry using alkaline phosphatase-labeled oligonucleotide probes to detect gene expression in the intestine. Because the intestine contains a large amount of endogenous alkaline phosphatase activity, mild acid pretreatment of the tissue sections was required to inactivate the alkaline phosphatase. Acid pre-treatment dramatically reduced the endogenous activity without affecting the efficiency of hybridization or the probe's ability to reveal a positive mRNA signal. Furthermore, the addition of polyvinyl alcohol to the substrate solution helped to keep the background staining low without adversely affecting the intensity of the signal. The current protocol allows rapid and sensitive detection of sites of gene expression in intestinal tissue.

Völker, W; Schön, P; Vischer, P

doi: 10.1177/39.10.1940309pmid: 1940309

We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.