Journal of Histochemistry & Cytochemistry

Gilardoni, Mónica B.; Ceschin, Danilo G.; Sahores, María M.; Oviedo, Mabel; Gehrau, Ricardo C.; Chiabrando, Gustavo A.

doi: 10.1177/002215540305101201pmid: 14623925

The aim of this work was to evaluate by immunohistochemistry (IHC) the expression of both LRP-1 and urokinase-type plasminogen activator receptor (uPAR) at different developmental stages of rat prostate disease by using a prostate cancer model previously developed in our laboratory. We found that LRP-1 was weakly expressed in normal prostates and in rats with hyperplastic glands. The expression of this receptor increased and correlated with the degree of premalignant lesions (PIN I, II, and III). The IHC for uPAR in normal prostates and in premalignant lesions showed a score of immunostaining that correlated with the expression of LRP-1. On the other hand, in prostates with adenocarcinomas and undifferentiated carcinomas, LRP-1 was undetectable or weakly detected, whereas uPAR showed a significantly higher level of expression. Based on the IHC results in rat prostates with premalignant and malignant lesions and considering that LRP-1, by mediating the internalization of uPAR, is involved in the regulation of extracellular matrix remodeling and cell migration, we conclude that a decreased expression of LRP-1 could be involved with the increasing activation of plasminogen activators shown in cancers.

Casano, Caterina; Gianguzza, Fabrizio; Roccheri, Maria C.; Giorgi, Rossana Di; Maenza, Luigia; Ragusa, Maria A.

doi: 10.1177/002215540305101202pmid: 14623926

In a previous paper we demonstrated that, in Paracentrotus lividus embryos, deciliation represents a specific kind of stress that induces an increase in the levels of an acidic protein of about 40 kD (p40). Here we report that deciliation also induces an increase in Hsp40 chaperone levels and enhancement of its ectodermal localization. We suggest that Hsp40 might play a chaperoning role in cilia regeneration.

Ichimura, Koichiro; Kurihara, Hidetake; Sakai, Tatsuo

doi: 10.1177/002215540305101203pmid: 14623927

The foot processes of podocytes possess abundant microfilaments and modulate glomerular filtration. We investigated the actin filament organization of foot processes in adult rat podocytes and the formation of the actin cytoskeletal system of immature podocytes during glomerulogenesis. Electron microscopy revealed two populations of actin cytoskeletons in foot processes of adult podocytes. One is the actin bundle running above the level of slit diaphragms and the other is the cortical actin network located beneath the plasmalemma. Immunogold labeling for actin-binding proteins demonstrated that oí-actinin and synaptopodin were localized in the actin bundle, whereas cortactin was in the cortical actin network. Immunofluorescence labeling for actin-binding proteins in immature podocyte showed that α-actinin was localized at the level of the junctional complex, whereas cortactin was distributed beneath the entire plasmalemma. Synaptopodin was first observed along the basal plasmalemma from the advanced S-shaped body to the capillary loop stage. We conclude that foot processes have specialized actin filamentous organization and that its establishment is associated with the expression and redistribution of actin-binding proteins during development.

Li, ZL; Abe, H.; Ueki, K.; Kumagai, K.; Araki, R.; Otsuki, Y.

doi: 10.1177/002215540305101204pmid: 14623928

We describe the application of the biomolecular interaction (BIA) technique to detection of the interaction between protein (e.g., c-Jun) and DNA (e.g., two AP-1 motifs from bcl-2 promoter), compared with immunohistochemistry (IHC) of c-Jun. The specific binding assay for the interaction of c-Jun and activating protein-1 (AP-1) motifs was performed using a Biacore 2000 system. Intense immunoreactivity of c-Jun in glandular cells of the human uterine endometrium was observed in the proliferative phase, while c-Jun in stromal cells was expressed throughout the menstrual cycle. In contrast to the IHC of c-Jun, the specific binding of c-Jun to two separate AP-1 motifs in the bcl-2 promoter region was detected only in nuclear extracts of glandular cells, but not in stromal cells, during the proliferative phase. These results indicate that, while transmitting various signals, c-Jun enhances the transcription level of bcl-2, which in turn keeps glandular cells alive and proliferating in normal human endometrium during the proliferative phase. Moreover, the method involving real-time biomolecular interactions such as DNA–protein binding is novel for the study of transcription factors when combined with IHC.

Kirk, Sonnie P.; Oldham, Jenny M.; Jeanplong, Ferenc; Bass, John J.

doi: 10.1177/002215540305101205pmid: 14623929

This study tested whether administration of insulin-like growth factor-II (IGF-II) enhances muscle regeneration. Rat biceps femoris muscle was damaged with notexin and then IGF-II was administered for up to 7 days. Results show that the proportion of nuclei containing or surrounded by immunoreactivity to MyoD, myogenin, and developmental myosin heavy chain (dMHC) is less in the IGF-II treatment group relative to the control group on days 1 (p = 0.057), 2 (p = 0.034), and 3 (p = 0.047), respectively. This indicates a delay in muscle precursor cell (MPC) proliferation and differentiation with IGF-II administration. This effect was not associated with decreased binding capacity of the type 1 IGF receptor, as determined by receptor autoradiography in day 1 muscle sections (NS), but was associated with inhibition of phagocytic processes. The cross-sectional area of regenerating muscle fibers was significantly greater in the IGF-II treatment group than in the control group by day 7 (p = 0.0092). The enhancing effect of IGF-II on late muscle regeneration, when the main process taking place is fiber enlargement, coincides with the period in which IGF-II is normally expressed by regenerating muscle, indicating that greater endogenous production of IGF-II would be associated with improved regeneration.

Immenschuh, Stephan; Baumgart-Vogt, Eveline; Tan, Melly; Iwahara, Shin-Ichiro; Ramadori, Giuliano; Dariush Fahimi, H.

doi: 10.1177/002215540305101206pmid: 14623930

Heme-binding protein 23 (HBP23), also termed peroxiredoxin (Prx) I, and heme oxygenase-1 (HO-1) are distinct antioxidant stress proteins that are co-ordinately induced by oxidative stress. HBP23/Prx I has thioredoxin-dependent peroxidase activity with high binding affinity for the pro-oxidant heme, while HO-1 is the inducible isoform of the rate-limiting enzyme of heme degradation. We investigated the cellular and subcellular localization of both proteins in rat liver. Whereas by immunohistochemistry (IHC) a uniformly high level of HBP23/Prx I expression was observed in liver parenchymal and different sinusoidal cells, HO-1 expression was restricted to Kupffer cells. By immunoelectron microscopy using the protein A–gold technique, HBP23/Prx I immunoreactivity was detected in cytoplasm, nuclear matrix, mitochondria, and peroxisomes of parenchymal and non-parenchymal liver cell populations. In contrast, the secretory pathway, i.e., the endoplasmic reticulum and Golgi complex, was free of label. As determined by immunocytochemical (ICC) studies in liver cell cultures and by Western and Northern blotting analysis, HBP23/Prx I was highly expressed in cultures of isolated hepatocytes and Kupffer cells. In contrast, HO-1 was constitutively expressed only in Kupffer cell cultures but was also inducible in hepatocytes. These data suggest that HBP23/Prx I and HO-1 may have complementary antioxidant functions in different cell populations in rat liver.

Mis, Katarina; Mars, Tomaz; Jevsek, Marko; Brank, Martina; Zajc–Kreft, Katarina; Grubic, Zoran

doi: 10.1177/002215540305101207pmid: 14623931

In spite of intensive investigations, the roles of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) in the central nervous system (CNS) remain unclear. A role recently proposed for BuChE as an explanation for survival of AChE knockout mice is compensation for AChE activity if it becomes insufficient. Neuronal contribution of both enzymes to the cholinesterase pool in the neuromuscular junction has also been suggested. These proposals imply that BuChE expression follows that of AChE and that, in addition to AChE, BuChE is also expressed in α-motor neurons. However, these assumptions have not yet been properly tested. Histochemical approaches to these problems have been hampered by a number of problems that prevent unambiguous interpretation of results. In situ hybridization (ISH) of mRNAs encoding AChE and BuChE, which is the state-of-the-art approach, has not yet been done. Here we describe rapid nonradioactive ISH for the localization of mRNAs encoding AChE and BuChE. Various probes and experimental conditions had been tested to obtain reliable localization. In combination with RT-PCR, ISH revealed that, in rat spinal cord, cells expressing AChE mRNA also express BuChE mRNA but in smaller quantities. α-Motor neurons had the highest levels of both mRNAs. Virtual absence of transcripts encoding AChE and BuChE in glia might reflect a discrepancy between mRNA and enzyme levels previously reported for cholinesterases.

Lips, Katrin S.; Pfeil, Uwe; Reiners, Katja; Rimasch, Christoph; Kuchelmeister, Klaus; Braun-Dullaeus, Ruediger C.; Haberberger, Rainer V.; Schmidt, Rupert; Kummer, Wolfgang

doi: 10.1177/002215540305101208pmid: 14623932

Liyou, Nancy E.; Buller, Kathryn M.; Tresillian, Michael J.; Elvin, Christopher M.; Scott, Heather L.; Dodd, Peter R.; Tannenberg, Anthony E.G.; McManus, Michael E.

doi: 10.1177/002215540305101209pmid: 14623933

Cytosolic sulfotransferases are believed to play a role in the neuromodulation of certain neurotransmitters and drugs. To date, four cytosolic sulfotransferases have been shown to be expressed in human brain. Recently, a novel human brain sulfotransferase has been identified and characterized, although its role and localization in the brain are unknown. Here we present the first immunohistochemical (IHC) localization of SULT4A1 in human brain using an affinity-purified polyclonal antibody raised against recombinant human SULT4A1. These results are supported and supplemented by the IHC localization of SULT4A1 in rat brain. In both human and rat brains, strong reactivity was found in several brain regions, including cerebral cortex, cerebellum, pituitary, and brainstem. Specific signal was entirely absent on sections for which preimmune serum from the corresponding animal, processed in the same way as the postimmune serum, was used in the primary screen. The findings from this study may assist in determining the physiological role of this SULT isoform.

Wei, Chongjuan; Amos, Christopher I.; Rashid, Asif; Sabripour, Mahyar; Nations, Linda; McGarrity, Thomas J.; Frazier, Marsha L.

doi: 10.1177/002215540305101210pmid: 14623934

Germline mutations of the LKB1 gene lead to Peutz–Jeghers syndrome (PJS), which is associated with a predisposition to gastrointestinal polyposis and cancer. In this study we tested for germline mutations of LKB1 in 11 patients with PJS from nine families and analyzed the expression patterns of the LKB1 and cyclo-oxgenase-2 (COX-2) proteins in 28 Peutz–Jeghers polyps (PJPs) and five carcinomas from these patients by immunohistochemical (IHC) analysis. In eight of those families we identified seven different mutations, which consisted of two splice site mutations, two nonsense mutations, one small inframe deletion, one frame-shift mutation, and one silent mutation. Immunostaining revealed nuclear and cytoplasmic expression of LKB1 protein in 23 PJPs and five carcinomas, nuclear expression alone in one PJP, and loss of LKB1 protein expression in four PJPs, indicating a heterogeneous LKB1 expression pattern in PJPs. Overexpression of COX-2 was detected in 23 (82%) of 28 PJPs and in all carcinomas. Despite heterogeneity in staining of LKB1 among individuals and even among samples from the same individual, we found statistically significant correlations in staining of LKB1 relative to COX-2. These results suggest that COX-2 plays a role in tumorigenesis in PJS and may therefore be considered as a potential target for PJS chemoprevention.