Staining Pattern Of Antibody Research Articles

Porcine spermatozoa contain more than one membrane progesterone receptor

Progesterone has been shown to be a physiologically relevant inducer of the sperm acrosome reaction. A novel protein intrinsic to microsomal membranes, membrane progesterone receptor (mPR, now termed progesterone membrane receptor component 1, PGMRC1) that binds progesterone with high affinity has been cloned from porcine liver previously, and corresponding antibodies mitigate the progesterone induced acrosome reaction. In this study we aimed at the localization of mPR in porcine spermatozoa. Immunostaining suggested the exclusive occurrence of mPR in a hardly accessible place, possibly the inner acrosomal membrane, with digitonin dramatically increasing the number of positively stained cells. Consistent with the structure prediction for mPR, its short N-terminus (NT) but not the large C-terminal part becomes accessible from outside after digitonin treatment as evidenced by the staining pattern of antibodies directed against different regions of the protein. However, digitonin treatment solubilizes a progesterone binding activity of approximately 140 kDa molecular weight, that is different from mPR, which remains in the cell membrane as demonstrated by Western blotting. Ligand binding studies confirm the dissimilarity of mPR and the digitonin-soluble progesterone binding protein. Chemical modification studies also indicate that the digitonin-soluble progesterone binding protein has a binding site that differs from that of mPR. It is concluded that more than one progesterone receptor is present in porcine spermatozoa.

Porcine spermatozoa contain more than one membrane progesterone receptor

Progesterone has been shown to be a physiologically relevant inducer of the sperm acrosome reaction. A novel protein intrinsic to microsomal membranes, membrane progesterone receptor (mPR, now termed progesterone membrane receptor component 1, PGMRC1) that binds progesterone with high affinity has been cloned from porcine liver previously, and corresponding antibodies mitigate the progesterone induced acrosome reaction. In this study we aimed at the localization of mPR in porcine spermatozoa. Immunostaining suggested the exclusive occurrence of mPR in a hardly accessible place, possibly the inner acrosomal membrane, with digitonin dramatically increasing the number of positively stained cells. Consistent with the structure prediction for mPR, its short N-terminus (NT) but not the large C-terminal part becomes accessible from outside after digitonin treatment as evidenced by the staining pattern of antibodies directed against different regions of the protein. However, digitonin treatment solubilizes a progesterone binding activity of approximately 140 kDa molecular weight, that is different from mPR, which remains in the cell membrane as demonstrated by Western blotting. Ligand binding studies confirm the dissimilarity of mPR and the digitonin-soluble progesterone binding protein. Chemical modification studies also indicate that the digitonin-soluble progesterone binding protein has a binding site that differs from that of mPR. It is concluded that more than one progesterone receptor is present in porcine spermatozoa.

The Role of AMPA Receptor Gating in the Development of High-Fidelity Neurotransmission at the Calyx of Held Synapse

During early postnatal development of auditory synapses, the decay time course of AMPA receptor (AMPAR) EPSCs accelerates markedly, but the mechanisms underlying this process remain uncertain. Using the developing calyx of Held synapse in the mouse auditory brainstem, we have examined presynaptic and postsynaptic elements that may regulate decay kinetics of AMPAR EPSCs. We found that the decay time kinetics was voltage dependent in both immature and mature synapses, being slower at positive potentials than negative potentials. By recording evoked miniature events in extracellular Ca2+ or Sr2+, we revealed a significant decrease in decay time constants of EPSCs as maturation progresses. On the basis of internal and external polyamine block of AMPAR EPSCs and immunohistochemistry assays with subunit-specific antibodies, we demonstrated that the glutamate receptor (GluR) 2 subunit is virtually absent at all developmental ages. Antibody staining patterns suggest a gradual shift in subunit composition from GluR1- to GluR3/4-dominant phenotypes. Kinetic analyses of deactivation, desensitization, and recovery from desensitization in outside-out patches in response to ultrafast application of glutamate lend supportive evidence that such a shift in the gating phenotype likely accounts for the accelerated time course throughout development. Finally, by pharmacologically manipulating AMPAR gating and using simulated EPSCs to evoke action potentials, we demonstrated that rapid decay kinetics of AMPAR EPSCs is essential for this synapse to accommodate high-frequency firing without compromising spike amplitude. Hence, developmental alterations in the subunit composition likely dictate changes in the time course of AMPAR EPSCs and play an indispensable role in the refinement of high-fidelity neurotransmission at the calyx of Held synapse.

Detection of shared antigenic determinants between Mycobacterium leprae heat shock protein 65 and human heat shock protein 60

In these studies, it was investigated whether M. leprae and man share antigenic determinants which may be located on Heat Shock Proteins (HSPs), and which may be responsible for tissue destruction. Using immunoperoxidase singlestaining technique on cryostat sections it was observed that three antibodies which are directed against HSP 60 (polyclonal antibodies SPA 804 and SPA 805 and monoclonal antibody SPA 807) probably reacted specifically with macrophages and epitheloid cells in leprosy skin sections. On Western Blotting, it was observed that the antibodies against human HSP 60 and monoclonal antibodies (MoAbs) against M. leprae HSP 65 (F 47-10, F 67-18, F 88-1) all reacted strongly with sonicated M. leprae proteins with a molecular mass of 65 kDa indicating similarity of some antigenic determinants between human HSP 60 and M. leprae HSP 65. Subsequently, a comparative immunohistochemical study of the staining patterns of antibodies against human HSP 60 and antibodies against M. leprae HSP 65 using cryostat skin sections of paucibacillary (PB) leprosy, multibacillary (MB) leprosy and other granulomatous skin disorders revealed that the MoAbs F 47-10 and F 67-18 reacted only weakly with the granulomas in PB leprosy and in other granulomatous skin diseases, but stained MB leprosy granuloma strongly. The MoAb F 88-1 and the polyclonal antibodies SPA 804, SPA 805 and the MoAb SPA 807 stained granulomas of PB patients and of other granulomatous skin disorders with the same intensity as that MB patients. Using a double-staining technique, it was observed that the antigenic determinants recognized by the MoAb against human HSP 60 (SPA 807) and the MoAbs against M. leprae HSP 65 (F 67-18, F 47-10, F 88-1) were mostly located in the macrophages. These findings do not contradict our suggestion, that similarities between antigenic determinants on Heat Shock Proteins of M. leprae and the human host may be at least in part responsible for the induction of an autoimmune reaction causing granuloma formation with subsequent tissue damage in leprosy. The results of this study also indicated that some of these determinants are probably located on HSP 60. A similar explanation possibly applies to the findings in the other granulomatous diseases, e.g. sarcoidosis probably mycobacterial induced and necrobiosis lipoidica related to diabetis, in which antigenic similarities between bacterial HSP 65 and human HSP 60 are considered to play a part.

Basic studies on a labeled anti-mucin antibody detectable by infrared-fluorescence endoscopy

We developed a fluorescent dye, indocyanine green (ICG)-sulfo-OSu, which was excited by infrared rays and conjugated to various antibodies. We attempted to clarify the staining patterns of antisulfomucin and anti-MUC1 antibodies in gastrointestinal cancer. We then evaluated the potential of the dye as a fluorescent label for antibodies specific to cancer, to be used as a diagnostic method for microcancer, with infrared fluorescence endoscopy. Paraffin sections of samples collected from 10 patients with esophageal cancer, 30 patients with gastric cancer, and 20 patients with colorectal cancer were immunohistologically stained using an anti-sulfomucin antibody and an anti-MUC1 antibody, and the staining patterns were examined. If a section had a high staining intensity, it was reacted with the ICG-suflo-OSu-labeled antibody and evaluated with infrared fluorescence imaging. The staining patterns with the antibodies varied depending on the organs and the histological types and depth of the cancers, but the staining was generally good and the staining on the mucosal surface of cancer tissues was retained. Good images of cancer cells could be obtained by infrared fluorescence observation using the ICG-sulfo-OSu-labeled anti-MUC1 antibody. The anti-MUC1 antibody stained gastrointestinal cancer cells well, and nearly specific infrared fluorescence in cancer tissues was observed using the labeled anti-MUC1 antibody. The ICG-sulfo-OSu-labeled anti-MUC1 antibody has possible usefulness for the screening of cancer via infrared fluorescence endoscopy.

Partial Revertant Mosaicism of Keratin 14 in a Patient with Recessive Epidermolysis Bullosa Simplex

A patient with recessive epidermolysis bullosa simplex due to a previously described homozygous KRT14 1842-2A-->C splice-site mutation was re-examined, because we unexpectedly found signs of revertant mosaicism. The germline mutation resulted in different aberrant transcripts containing premature termination codons, all leading to truncated keratin 14 proteins. Basal keratinocytes in skin and in culture completely lacked keratin 14 and intermediate filaments. From this keratin 14-/- patient we started cultures from a new skin biopsy and here, we serendipitously found keratinocytes that spontaneously expressed keratin 14. This biopsy had been taken from an area of skin that was clinically affected, because blisters could simply be evoked by gentle rubbing. Immunofluorescence and electron microscopy of additional biopsies from this skin area revealed a mosaic expression of keratin 14 and reappearance of intermediate filaments in basal keratinocytes. Immunoblotting showed a revertant keratin 14 polypeptide with seemingly normal molecular weight. DNA analysis of exon 2 and its flanking intron borders showed no additional mutations in the genomic KRT14 sequence. Analysis of mRNA isolated from mosaic skin keratinocytes revealed an additional in-frame transcript (1844T-->G, 1845Delta6) that codes for an abnormal keratin 14 polypeptide with a two residue deletion and one amino acid change. The re-expression of a revertant, albeit abnormal, keratin 14 polypeptide, so-called partial revertant mosaicism, accounts for the antibody staining pattern and for the reappearance of intermediate filaments, which however, are semifunctional and not able to revert the clinical phenotype. The combination of a keratin 14-positive and a keratin 14-negative cell population in epidermis as well as in cultured keratinocytes suggests that the cellular reversion might be caused by an endogenous factor. We hypothesize that a second somatic modulating factor in the genome that affects the processing of the mutant KRT14 pre-mRNA may underlie this phenomenon.

Identification of AHNAK as a Novel Autoantigen in Systemic Lupus Erythematosus

To identify candidate autoantigens associated with arthritis, a rat chondrocyte cDNA library was immunoscreened with serum from a patient with rheumatoid arthritis. One isolated cDNA encoded part of AHNAK, a 700-kDa phosphoprotein with DNA binding properties, that appears to be involved in several signal transduction pathways. Immunoreactivity against an in vitro translated human AHNAK fragment was detected in 4.6% (5/109) of patients with rheumatoid arthritis, 29.5% (18/61) of patients with systemic lupus erythematosus (SLE), and 1.2% (2/172) of blood donors. Anti-AHNAK antibodies reacted with a recombinant human AHNAK fragment and with native AHNAK from C32 cell lysates. In vitro translated AHNAK fragment could be cleaved by granzyme B and caspase-3. Anti-AHNAK positive SLE patients had a higher frequency of homogeneous antinuclear antibody staining patterns and a lower frequency of recent mucosal ulcerations. This is the first report that AHNAK can be targeted by the immune system in autoimmune disease.

A Monoclonal Antibody Specific to a Song System Nuclear Antigen in Estrildine Finches

This paper describes a monoclonal antibody that recognizes a molecule whose expression is mostly restricted to some of the forebrain areas that control singing behavior in adult estrildine species studied, including the zebra, Bengalese, and spice finches. When the song system displays extreme sexual dimorphism, as in these species, antibody staining occurs only in the male's song nuclei. However, protein expression is identical in both sexes of estrildine finches, in which females also have a well-developed song system. Canaries appear to lack the protein, but it can be induced in female zebra finches by early estrogen treatment. Antibody staining patterns in the zebra finch show that the protein's expression is developmentally regulated to coincide with the abrupt increase in the volume and cell size of the male's or the estrogen-treated female's song system.

Histochemical characterization of primary capillary endothelial cells from porcine brains using monoclonal antibodies and fluorescein isothiocyanate-labelled lectins: implications for drug delivery

Primary endothelial cells isolated from cerebral microvessels by combined mechanical and enzymatic treatment from porcine brains were characterized with regard to identity, purity and membrane surface characteristics. Cells were grown in culture to adherent monolayers and characterized morphologically and histochemically by their binding for fluorescently-labelled lectins and monoclonal antibodies detected by indirect immunofluorescence. The binding patterns of the cells were compared with the affinity of frozen tissue sections of porcine brain cortex for the markers. Endothelial cells in culture were characterized by the binding of von Willebrand factor, vimentin and fibronectin antibodies. They failed to react with anti-glial fibrillary acid protein, anti-galactocerebroside C and anti-neurofilament 160 antibodies characteristic for astrocytes, oligodendrocytes and neurons, respectively. Cell cultures were stained by the lectins, wheat germ agglutinin, horse gram agglutinin and soybean agglutinin, demonstrating the presence of N-acetylglucosamine and N-acetylgalactosamine residues on membrane surface. Binding sites for concanavalin A and peanut agglutinin characteristic for mannose and galactose could not be detected. Cell age and differentiation had no effect on lectin and antibody staining. Cell cultures gave staining results similar to those of microvessels in frozen tissue sections. The results of morphology, antibody and lectin staining pattern indicate that our in vitro endothelial cell culture model retained many histological characteristics observed for capillary microvessels in vivo and appears to be suitable for studying uptake and targeting properties of drug carrier systems with regard to the blood–brain barrier.

MIB-1 monoclonal antibody to determine proliferative activity of Ki-67 antigen as an adjunct to the histopathologic differential diagnosis of Spitz nevi

Background: The histopathologic differential diagnosis of Spitz nevus (SN) from malignant melanoma (MM) may be difficult. Objective: Our purpose was to determine the staining pattern and usefulness of MIB-1 antibody, which recognizes Ki-67 antigen in formalin-fixed, paraffin-embedded tissue, as an adjunct to the histopathologic differential diagnosis of SN. Methods: Twenty-five compound SNs, 27 MMs, and 26 compound nondysplastic melanocytic nevi (MNs) were immunostained with the MIB-1 antibody. Results: The mean counts of MIB-1-stained tumor cells of the epidermal and dermal components, both alone and together, were significantly lower in SNs and MNs than in MMs (P < .0001). The dermal counts showed the best discriminating power. In addition, the mean dermal/epidermal count ratios for MIB-1 in SNs and MNs (0.25 and 0.23, respectively) were significantly lower than the corresponding ratio (0.94) in MMs (P < .0001). Conclusion: MIB-1-stained tumor cell counts, especially of the dermal component, and dermal/epidermal MIB-1 count ratios may be helpful as an adjunct to the histopathologic differential diagnosis of SN. (J Am Acad Dermatol 2001;44:500-4.)

Characterization of the vasculogenic block in the absence of vascular endothelial growth factor-A

AbstractVascular endothelial growth factor (VEGF) signaling is required for both differentiation and proliferation of vascular endothelium. Analysis of differentiated embryonic stem cells with one or both VEGF-A alleles deleted showed that both the differentiation and the expansion of endothelial cells are blocked during vasculogenesis. Blood island formation was reduced by half in hemizygous mutant VEGF cultures and by 10-fold in homozygous mutant VEGF cultures. Homozygous mutant cultures could be partially rescued by the addition of exogenous VEGF. RNA levels for the endothelial adhesion receptors ICAM-2 and PECAM were reduced in homozygous mutant cultures, but ICAM-2 RNA levels decreased substantially, whereas PECAM RNA levels remained at hemizygous levels. The quantitative data correlated with the antibody staining patterns because cells that were not organized into vessels expressed PECAM but not ICAM-2. These PECAM+ cell clumps accumulated in mutant cultures as vessel density decreased, suggesting that they were endothelial cell precursors blocked from maturation. A subset of PECAM+ cells in clumps expressed stage-specific embryonic antigen-1 (SSEA-1), and all were ICAM-2(−) and CD34(−), whereas vascular endothelial cells incorporated into vessels were PECAM(+), ICAM-2(+), CD34(+), and SSEA-1(−). Analysis of flk-1 expression indicated that a subset of vascular precursor cells coexpressed PECAM and flk-1. These data suggest that VEGF signaling acts in a dose-dependent manner to affect both a specific differentiation step and the subsequent expansion of endothelial cells.

Millipore filter cell block preparation: an alternative to cell block in nongynecologic specimens of limited cellularity.

The use of membrane filters to concentrate cytology specimens was first described by Seal (Cancer 1956; 9:866-868). We report on a technique in which a portion of Papanicolaou-stained Millipore filter (Millipore Corp, Bedford, MA) preparation can be converted into a paraffin block for hematoxylin-eosin (H&E) preparations and immunocytochemical analysis (ICC). Seven cases with moderate cellularity and no cell block preparation were retrieved from our cytopathology files. The specimens included: 4 pleural effusions with metastatic adenocarcinoma, and 3 FNA specimens (1 metastatic melanoma, 1 metastatic adenocarcinoma, and 1 thyroid/papillary carcinoma). The filter was removed from the slide, cut in half, and subjected to paraffin embedding in the usual fashion (postfixed in 10% neutral-buffered formalin). Four-micron-thick sections were cut onto Probe-On (Fisher Scientific, Pittsburgh, Pa) slides. ICC was performed using the avidin-biotin-peroxidase complex technique and capillary gap technology. Antibodies included CAM5.2, AE1/AE3, CEA, CD15, LCA, PanCK, S100, HMB45, and thyroglobulin. All cases showed excellent preservation of cellular morphology on H&E. ICC performed on Millipore filter cell block preparations showed specific antibody staining patterns with preservation of cellular details. All antibodies showed their specific staining patterns with clean background and lack of nonspecific staining. This technique has the following advantages: 1) offers an alternative to cell blocks in moderately cellular specimens; 2) clean background; 3) preservation of cytology specimens for future studies.

Heterogeneity of the human exocrine pancreas: Antidigestive enzyme antibody and lectin binding

We have previously reported the existance of fundamental heterogeneity of the exocrine pancreas in animals; binding of specific anti-digestive enzyme antibodies and lectins to intact pancreatic tissue and isolated acinar cells of rabbit and rat revealed highly heterogenous patterns of distribution of acinar cell digestive enzyme contents (J Histochem Cytochem 1995;44:215 and Gastroenterol 1997;112:A435). Aim: To determine if the adult human pancreas is heterogeneous. Methods: Areas of normal pancreas were obtained without alteration of the surgical procedure from the margins of resected specimens in 5 patients ages 56-76, two with serous cystadenoma, and three with pancreatic cancer. Tissue was fixed and sectioned by standard methods. Sections were permeabilized with saponin, and treated with commerciallyavailable anti-digestive enzyme antibodies employed singly or in pairs directed against amylase, lipase, chymotrypsin and trypsinogen, or with a panel of lectins. Vizualization was by both diaminobenzidine and fluoresence with FITC or Texas red. Routine hematoxylin and eosin staining was employed to ascertain tissue integrity, morphology and absence of pathology. Results: The human pancreas was distinctly heterogeneous at the morphological level as demonstrated by antidigestive enzyme antibody binding. Several patterns of heterogeneity were readily discerned: first, a pattern unique to the human wherein whole lobules were clearly labeled by one of a pair of antibodies -highly lipase-, chymotrypsinand amylasedominant lobules were noted to contrast with other areas where enzymes were co-localized. Second, an autumn leaves-type pattern was noted in which individual acini were found to contain either amylase or lipase predominantly. A third pattern wherein individual acinar cells differed in digestive enzyme content from neighboring cells within the same acinus, and a fourth pattern of anti-digestive enzyme antibody staining in halos of lipase-rich acinar cells surrounding the islets of Langerhans were also noted. Lectins LTL and UEA also strongly showed lobular patterns of heterogeneous binding. Lectins HPA, ACL, WFA, SBA and SWGA showed heterogeneous patterns from cell-tocell in the zymogen granule region. Lectin PHA showed distinct halos of acinar cells surrounding the islets. Numerous species differences in the patterns of heterogeneity were found; the human gland appeared even more broadly heterogeneous than rabbit or rat, and showed an extensive lobular pattern not seen as readily in the other species. Conclusion: The exocrine pancreas in the human adult is fundamentally heterogeneous with respect to the distribution of individual digestive enzyme species within acinar cells, acini, and pancreatic lobules. The heterogeneous interand intra-acinar patterns as well as the peri-insular halos have been found in all species examined; the highly lobular pattern for lipase, amylase and chymotrypsin is, to date, unique to the human.

Dysregulation of cytokine expression in tubulointerstitial nephritis associated with murine malaria

Dysregulation of cytokine expression in tubulointerstitial nephritis associated with murine malaria

An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.

Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six-amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

Differential expression of MAG, MBP and L1 in the developing lateral superior olive

The aim of this study was to investigate whether glial-associated molecules exhibit a pattern of expression that could influence oriented dendrite outgrowth in the gerbil lateral superior olive (LSO). In particular, we have previously noted that axon fascicles are oriented parallel to isofrequency laminae in the medial limb of the LSO, as are LSO dendrites, a phenotype that emerges postnatally. Therefore, we examined the immunocytochemical staining pattern of antibodies directed against three proteins that are found along axons: myelin basic protein (MBP), myelin-associated glycoprotein (MAG), and neuron-glia cell adhesion molecule (L1). MAG staining was first observed at postnatal day (P) 4 on the axon fibers surrounding the LSO. By P7 there was a differential pattern of MAG staining within the LSO, and immunopositive fibers were observed solely in the medial limb (e.g., high frequency projection region). Between P7 and P12, MAG-staining was restricted largely to fascicles in the medial limb, and these were oriented parallel to the isofrequency axes. Few positive fibers of irregular orientation were observed in the lateral limb (e.g., low frequency projection region). Significant MAG-staining was not observed in the lateral limb until P15. The MAG immunoreactivity extended throughout the LSO by P21, although it was no longer restricted to axon fascicles. In contrast, MBP-positive fibers were uniformly distributed within the LSO by P12. Finally, L1 was found on oriented axon fascicles at P0, but became sparsely distributed throughout the LSO neuropil after P7, and was restricted to neuron cell bodies in the adult. Taken together, the results suggest that oriented axon fascicles bearing MAG and L1 may contribute to the developmental refinement of dendrite and axon arbors within the LSO.

The pattern of HMB-45 antibody staining in compound Spitz nevi.

We studied the staining pattern of HMB-45 antibody in 29 compound Spitz nevi (SNs) of the epithelioid cell variety, 17 of which showed extension of nevus cells into the reticular dermis (i.e., "deep"); 20 ordinary compound nevi (CNs), all with a deep dermal component; and 22 primary cutaneous invasive malignant melanomas (MMs) (excluding the desmoplastic and spindle cell types), 12 of which extended into Clark level IV or V. Of the 29 SNs, eight (28%) stained negatively; five (17%), including two deep SNs, stained in the epidermal component only; and 16 (55%), including 10 deep SNs, stained in both the epidermal and dermal components. Of the latter 10 deep SNs, eight stained in the upper dermis only, and in the remaining two lesions, a smaller number of positively stained nevus cells were detectable in the lower dermis as well; these two SNs were not atypical histologically. Of the 20 CNs, four (20%) stained negatively, two (10%) stained in the epidermal component only, and 14 (70%) stained in the epidermal component and the upper dermis only. Of the 22 MMs, one stained negatively, and 21 (95%) stained positively in both the epidermal and dermal components. The pattern was variable in frequency of both staining and distribution, but showed no stratification. We conclude that the majority of our positively stained deep compound SNs showed a stratified pattern of HMB-45 staining, similar to ordinary CNs and different from MMs, and that this pattern might be used as an adjunct in the histopathologic differential diagnosis of compound SN and MM, in the proper clinicopathological context.

Immunolocalization of triadin, DHP receptors, and ryanodine receptors in adult and developing skeletal muscle of rats.

The dihydropyridine receptors (DHPR) and the ryanodine receptors (RyR) are well-characterized proteins of the triad junctions of skeletal muscle fibers. Recently, a newly discovered 95-kDa protein, triadin, has been purified from rabbit skeletal muscle heavy sarcoplasmic reticulum (SR) vesicles. WE have used indirect immunogold EM to localize triadin to the junctional face of the SR in isolated triads. In addition, we have used indirect immunofluorescence to localize triadin in relation to the DHPR and the RyR in adult and developing rat skeletal muscle. In double immunolabelling experiments of longitudinally oriented adult rat skeletal muscle tissue, triadin-specific and RyR-specific antibodies resulted in a characteristic striated staining pattern. The staining arising from these antibodies completely overlapped when examined by computer analysis of digitized laser scanning confocal microscopy images. A similar result was obtained in double staining experiments using antibodies raised against the DHPR and the RyR suggesting that all three proteins are located in the triads in situ. The developmental expression of the three triad proteins was examined using double labeling of skeletal muscle tissue from several fetal and early postnatal ages. The staining patterns of triadin, RyR, and DHPR antibodies were overlapping throughout development, suggesting that from their earliest appearance the three proteins are components of the triads.

A C. elegans mutant screen based on antibody or histochemical staining

A method has been developed for isolating mutations in Caenorhabditis elegans that alter antibody or histochemical staining patterns. The basis for this method is a new procedure for making C. elegans permeable that does not kill the eggs contained within the uterus of gravid adult hermaphrodites. A mutagenized population of gravid hermaphrodites is made permeable and then stained with either an antibody or a histochemical stain. Animals that stain aberrantly are picked to individual petri plates and the eggs within the uterus of the stained mother hatch and establish a new genetic line. Antibody and histochemical stains are especially useful phenotypes because the staining pattern will usually directly reflect the gene expression pattern of the gene that codes for the antigen or enzyme. This method was used to isolate mutants that alter the expression of a mec-7 lacZ fusion gene. Transgenic animals that contained the mec-7 lacZ gene integrated into chromosome I were treated with the mutagen ethylmethanesulfonate, allowed to self-fertilize for two generations and then stained with X-gal or antibodies against β-galactosidase. Gravid animals that stained abnormally were picked to fresh petri plates and offspring were used to establish new mutant lines.

Characteristics of antitumor activity of 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]- 2(1H)-quinolinone (vesnarinone) against a human adenoid squamous carcinoma-forming cell line grown in athymic nude mice.

The tumors produced by transplantation into nude mice of human adenoid squamous carcinoma-forming cell line TYS, presumably derived from a minor salivary gland, were treated with a differentiation-inducing agent, vesnarinone, which was given per o.s. daily at a dose of 200 mg/kg for 35 days. They were then examined morphologically and immunohistochemically. The vesnarinone treatment resulted in a significant suppression of tumor growth. In addition, tumor nests indicating keratinocyte and acinar cell differentiation were often observed in the treated tumors, but not in untreated controls. Tissue sections from vesnarinone-treated and untreated TYS tumors were stained with monoclonal antibody (NAb) directed to carbohydrate antigen LeY or proliferating cell nuclear antigen (PCNA) and with rabbit polyclonal antibody to p53. Antibody staining patterns were compared with morphological characteristics of cells as revealed by hematoxylin and eosin staining, and DNA fragmentation patterns as revealed by 3'-OH nick-end labelling techniques. Tissue sections from vesnarinone-treated TYS tumors showed positive reaction with nick-end labelling and were extensively stained strongly by anti-LeY MAb, whereas the untreated tumors showed negative reaction with nick-end labelling and were infrequently stained by anti-LeY MAb. Within LeY-positive areas of tissue sections from the vesnarinone-treated tumors, keratinocyte and acinar cell differentiation as well as DNA fragmentation were frequently observed, although not all LeY-positive cells showed such signs of apoptosis. LeY-positive cells showed consistent negative staining by anti-PCNA MAb and anti-p53 rabbit serum. From these findings, it can be considered that vesnarinone has differentiation and apoptosis-inducing activity against TYS cells grown in athymic nude mouse.