Journal of Histochemistry & Cytochemistry

Rao, V V; Anthony, D C; Piwnica-Worms, D

doi: 10.1177/43.12.8537634pmid: 8537634

Multidrug resistance (MDR) is associated with overexpression of a 170 KD plasmamembrane P-glycoprotein (P-gp), a putative energy-dependent efflux transporter thatreduces intracellular accumulation of chemotherapeutic agents. For detection of P-gpexpression in normal and malignant tissues, an MDR1-specific monoclonal antibody(MAb) JSB-1 has been used extensively. In this report we show that MAb JSB-1crossreacts with a protein of M(r) approximately 130,000 present in rat livermitochondrial inner membrane/matrix fractions. Peptide mapping and microsequencingidentify this protein as pyruvate carboxylase (PC), an abundant mitochondrial enzyme.MAb JSB-1 also crossreacts with purified PC from bovine liver. Under immunoblottingconditions, this crossreactivity is partially abolished by pre-incubation of MAbJSB-1 with a 1000-fold molar excess of MAb C494 epitope-specific peptide (PNTLEGN),indicating that the epitope of MAb JSB-1 may either overlap with or be in closeproximity to that of MAb C494. Immunohistochemical cross-reactivity was alsodemonstrated in cryosections of human skeletal muscle, a tissue known not to expressP-gp. MAb JSB-1 strongly immunostained Type 1 fibers, the subtype known to containabundant mitochondria. Use of MAb JSB-1 for detection of MDR1 P-gp expression shouldbe approached with caution.

Connor, J R; Dodds, R A; James, I E; Gowen, M

doi: 10.1177/43.12.8537635pmid: 8537635

Animal model and in vitro cultures suggest that osteoclasts and cells of the mononuclear phagocyte system share a common precursor. However, the human osteoclast precursor has not been positively identified. We attempted to identify the precursor in situ by using a number of osteoclast- and macrophage-selective markers, together with the expression of osteopontin mRNA, previously shown to be abundant in human osteoclasts. Sections of osteophytic bone and a panel of inflammatory connective tissues were processed for in situ hybridization; serial sections were analyzed for tartrate-resistant acid phosphatase (TRAP) and nonspecific esterase (NSE) activity, selective cytochemical markers for the osteoclast and cells of the macrophage/monocyte lineage, respectively. The murine anti-human osteoclast monoclonal antibodies 23C6 (vitronectin receptor) and C35 (osteoclast-selective) were used to further identify the osteoclast phenotype. We compared osteoclasts, giant cells, and their respective putative mononuclear precursors. At resorption sites within osteophytic bone, osteopontin mRNA was expressed in osteoclasts and a distinct population of TRAP+, NSE- mononuclear cells. Adjacent clusters of mononuclear cells were TRAP- and NSE+ or were active for both enzymes; these cells demonstrated variable expression of osteopontin mRNA. In the inflammatory connective tissues, abundant macrophage-like cells (NSE+/TRAP-) did not express osteopontin mRNA. However, TRAP+ mononuclear cells observed among clusters of NSE+ cells did express osteopontin mRNA. At these sites, clusters of putative macrophage polykaryons removing fragments of bone debris were observed. These giant cells and associated mononuclear cells were NSE- and distinctly TRAP+, and expressed osteopontin mRNA, C35, and 23C6 (human osteoclast) reactivity. Therefore, cells involved in the remodeling (resorption) of bone or the removal of bone debris, together with their immediate precursors, switch from being NSE+/TRAP- to NSE-/TRAP+ cells that express osteopontin mRNA. We propose that the clusters of NSE+/TRAP- mononuclear cells represent the immature osteoclast precursor. In support of this, TRAP+/NSE+ cells were occasionally observed in both tissues, representing an intermediate stage in differentiation. These results further suggest that cells of the mononuclear phagocyte lineage within bone and inflammatory connective tissue have the potential to differentiate into osteoclasts.

Man, Y G; Ball, W D; Culp, D J; Hand, A R; Moreira, J E

doi: 10.1177/43.12.8537636pmid: 8537636

In the perinatal submandibular gland (SMG) of the rat, Type I cells secrete protein C(89 KD) and Type III cells secrete B1-immunoreactive proteins (20-30 KD); both celltypes secrete protein D (175 KD). After the disappearance of both perinatal celltypes from the maturing acini, only cells of the intercalated ducts (ID) show strongreactivity for the perinatal antigens. In adult ID, light and electron microscopicimmunocytochemical analysis showed that most cells had either C or B1 reactivity, afew had either C and D or B1 and D reactivities, and some cells were unreactive forall of the perinatal proteins. Occasional clusters of "adult" acini, however, werestrongly positive for B1 and for D, and these clusters were negative for a typicaladult acinar marker, the glutamine/glutamic acid-rich proteins (GRP). Also seen insome preparations were a few anomalous acini with the histological appearance ofsublingual (SLG) acini. These were negative for the perinatal and adult submandibulargland marker proteins but reactive with an antibody against SLG mucin. We suggestthat the B1-positive acini in the adult SMG consist of newly differentiatedreplacement cells that have arisen from the ID, and that the anomalous mucous aciniare, phenotypically, SLG acini that have differentiated within the SMGparenchyma.

Morimoto, Y; Saga, K

doi: 10.1177/43.12.8537637pmid: 8537637

Morphological observations of sweat glands showed degenerated debris of secretorycells in the secretory lumen in both apocrine and eccrine sweat glands. Thissuggested that dead secretory cells of human eccrine and apocrine sweat glands werereleased into the lumen and replaced by other cells. However, we did not know whichtype of cells replaced lost secretory cells. Therefore, we studied the proliferatingcells in human eccrine and apocrine sweat glands by labeling S-phase cells in vitrowith 5-bromo-2'-deoxyuridine (BrdUrd) and by immunostaining proliferation-associatedproliferating cell nuclear antigen (PCNA) with anti-PCNA monoclonal antibody. BrdUrdand anti-PCNA antibody labeled a few secretory cells in eccrine and apocrine sweatglands, but neither method labeled myoepithelial cells. Luminal and peripheral cellsof the eccrine and apocrine coiled duct were labeled with both BrdUrd and PCNA.However, we could not find any highly proliferative germinative cells in coiledducts. Our results suggest that lost secretory cells could be replaced byproliferation of secretory cells themselves rather than by proliferation ofmyoepithelial cells or duct cells.

Jin, C; Nagata, T

doi: 10.1177/43.12.8537638pmid: 8537638

The localization and activity of DNA synthesis of mouse cecal epithelia from prenatal Day 19 embryos to 12-month adults were investigated by light microscopic autoradiography after [3H]-thymidine injection in vivo. The cecal epithelial labeled cells with [3H]-thymidine were located at the lower half of the crypts, and the labeling index (LI) changed with the development of the cecum due to aging. A peak in the LI of absorptive cells was found at embryonic Day 19, but it decreased strikingly at the first postnatal day and then kept an almost constant value until 12 months. The LI of the goblet cells showed the highest value at fetal Day 19, decreased gradually with aging from the first postnatal day, and completely disappeared from 1 month onwards. The basal granulated cells were located only in the base of crypts, and the significant difference of LI was not found from embryonic Day 19 through 12-month adult. The results obtained in this study suggest that there are some differences of DNA synthesis among different kinds of cecal epithelial cells from embryonic stages to postnatal stages as a result of aging.

Mehdizadeh, S; O'Farrell, A; Bitensky, L; Weisz, J; Alaghband-Zadeh, J; Chayen, J

doi: 10.1177/43.12.8537639pmid: 8537639

In the previous communication we described a histochemical method for measuringsoluble guanylate cyclase (sGC) activity in sections of rat liver. In theory, thismethod could be used to assess nitric oxide synthase (NOS) activity by the increasedsGC activity induced by the additional presence of the substrates for NOS activity.We found that this was correct provided that the concentration of the colloidstabilizer in the reaction medium was decreased to just below the concentrationrequired to fully stabilize the guanylate cyclase activity in the sections. This wasrelated to the fact that the site of NOS activity was different from that of the sGCactivity in the hepatocytes, so that the NO generated had to diffuse from the Kupffercells to the hepatocytes as could occur only in partially unstabilized sections.Optimal concentrations of arginine and of NADPH have been determined fordemonstrating NOS activity; the increased reaction was shown to be largely inhibitedby methyl-arginine.

Mehdizadeh, S; O'Farrell, A; Bitensky, L; Weisz, J; Alaghband-Zadeh, J; Chayen, J

doi: 10.1177/43.12.8537640pmid: 8537640

Guanylate cyclase liberates pyrophosphate from guanosine triphosphate (GTP). Instudies published previously, this phosphate is trapped by lead ions even though itis known that free lead ions inactivate a considerable proportion of this enzymaticactivity. To overcome the damaging effects of fixation, this study used freshcryostat sections stabilized with a sufficient concentration of a collagen-derivedpolypeptide to ensure no measurable loss of guanylate cyclase activity. To avoid thedamaging influence of free lead ions, we used a hidden metal capture reagent, i.e., acomplex of lead ammonium citrate/acetate that does not react with GTP but whichrapidly forms a precipitate with the pyrophosphate liberated by the enzyme. The leadprecipitate is then converted into the colored sulfide which is measured inindividual cells by microdensitometry. This system was used to measure guanylatecyclase activity in individual cells in unfixed sections of rat liver.

Nissinen, M J; Karlstedt, K; Castrén, E; Panula, P

doi: 10.1177/43.12.8537641pmid: 8537641

In this study we investigated the developmental expression of histidine decarboxylase(HDC) mRNA and the distribution of histamine-immunoreactive (histamine-ir) cells inthe rat embryonic tissues. We applied Northern blot analysis, in situ hybridizationwith synthetic oligonucleotide probes complementary to the rat HDC cDNA, and indirecthistamine immunocytochemistry. Northern blot analysis revealed the appearance of amajor (2.6 KB) HDC mRNA species in liver on embryonic Day 14. Its hybridization levelpeaked on Day E18, when two minor (1.6 and 3.5 KB) mRNA species were also present.During the periparturition period, a rapid decrease in HDC RNA was apparent, as the2.6 KB mRNA species was expressed at a low level on postnatal Day P1. The embryonicliver expressed HDC on days E14-E20. On days E18 and E20, the periosteum and theepiphyseal growth plates of the endochondrally ossificating bones, and some striatedmuscle cells, showed hybridization signal for HDC. Histamine immunoreactivity wasdetected in many epithelial and neuronal cell types during embryogenesis. An intensehistamine immunoreaction appeared first in essentially all cells of the liverparenchyma on day E12. This parenchymal histamine immunoreactivity disappeared bybirth, after which this immunofluorescence in liver was restricted to a few scatteredmast cells until adulthood. Some neurons in the peripheral sensory, sympathetic andcranial nerve ganglia were histamine-immunoreactive from day E16 to birth. Inaddition, many immunoreactive nerve fibers were detected in the gastrointestinalmuscularis externa, mesentery, salivary glands, kidney, lung, and muscle tissue. Weconclude that during rat embryogenesis histamine is produced and stored transientlyby cells in liver, developing bone, and a few striated muscle cells, in addition topreviously reported neurons in rat brain. Many peripheral neurons, epithelial cells,and mast cells display histamine immunoreactivity during rat embryogenesis but aredevoid of detectable HDC mRNA with the current method. It remains possible thathistamine is formed by another enzyme or is taken up from the extracellular space.The results support the concept that a significant proportion of histamine is formedand stored by embryonic cells other than mast cells.

Moreno, S; Mugnaini, E; Cerù, M P

doi: 10.1177/43.12.8537642pmid: 8537642

Catalase is a marker for peroxisomes, which are ubiquitous cytoplasmic organelles.Although the distribution and features of peroxisomes are well known in liver andkidney, these organelles have been rarely studied in neural tissues. Catalase is animportant scavenging enzyme against reactive oxygen species, as it removes H2O2produced during metabolic processes. Reactive oxygen species are involved in a numberof brain lesions and in brain aging. We investigated the distribution of catalase inrat central nervous system by means of a newly developed immunocytochemical procedurefor signal enhancement, using an affinity-purified polyclonal antiserum. The datashow that catalase immunoreactivity is present in all neural cells, both neuronal andglial, albeit at different concentrations. Among glial cells, ependymal cells andtanycytes of the third ventricle and the median eminence show the most intenseimmunoreaction; positivity is also found in oligodendrocytes and astrocytes. Ingeneral, neurons in the brainstem are relatively more immunoreactive than those inthe forebrain although, within these respective brain regions, there are areas withlow and high staining intensity. Moreover, within the same area, certain types ofneuron appear more immunoreactive than others. The cell bodies in the septal nuclei,pallidum, reticular thalamic nucleus, mesencephalic nucleus of the trigeminal nerve,Deiter's nucleus, locus ceruleus, cranial and spinal motor nuclei, and the Golgicells of the cerebellar cortex are among the most densely stained neurons. Catalaseimmunoreactivity of the cell bodies, which presumably is proportional to catalasecontent, appears to be only partially correlated with cell size or type ofneurotransmitter used in the nerve endings; it is likely that other unknownparameters regulate the abundance of the enzyme. In many cases, highly immunoreactivecells correspond to neurons known to be resistant to ischemia-reperfusion injury,whereas weakly stained cells correspond to neurons that are more susceptible toischemic damage. The amount of catalase may be critical for a protective effectagainst oxidative stress under pathological conditions, such as ischemia-reperfusioninjury.

Riederer, B M; Draberova, E; Viklicky, V; Draber, P

doi: 10.1177/43.12.8537643pmid: 8537643

The microtubule-associated protein MAP2 is essential for development of earlyneuronal morphology and maintenance of adult neuronal morphology. Several splicevariants exist, MAP2a-d, with a lack of MAP2a in cat brain. MAP2 is widely used as aneuronal marker. In this study we compared five monoclonal antibodies (MAbs) againstMAP2. They show differences in the immunocytochemical distribution of MAP2 isoformsduring development of the visual cortex and cerebellum of the cat. Local and temporaldifferences were seen with MAb AP18, an antibody directed against aphosphorylation-dependent epitope near the N-terminal end. In large pyramidaldendrites in visual cortex, the AP18 epitope remained in parts immunoreactive aftertreatment with alkaline phosphatase. Three MAbs, AP14, MT-01, and MT-02, recognizedthe central region of the MAP2b molecule, which is not present in MAP2c and 2d, andreacted with phosphorylation-independent epitopes. During the first postnatal weekthe immunostaining in cerebellum differed between antibodies in that some cellularelements in external and internal granular layers and Purkinje cells were stained tovarious degrees, whereas at later stages staining patterns were similar. At earlystages, antibody MT-02 stained cell bodies and dendrites in cerebral cortex andcerebellum. With progressing maturation, immunoreactivity became restricted to distalparts of apical dendrites of pyramidal cells and was absent from perikarya and finerproximal dendrites in cortex. MT-02 did not stain MAP2 in cerebellum of adultanimals. This study demonstrates that the immunocytochemical detection of MAP2depends on modifications such as phosphorylation and conformational changes of themolecule, and that MAP2 staining patterns differ between MAbs. Phosphorylation andspecific conformations in the molecule may be essential for modulating function andmolecular stability of MAP2, and monoclonal antibodies against such sites may providetools for studying the functional role of modifications.