Granular Immunoreactivity Research Articles

Succinate Dehydrogenase B (SDHB) Immunohistochemistry for the Evaluation of Muscle Biopsies.

Succinate dehydrogenase (SDH) is a key mitochondrial enzyme complex composed of 4 subunits. SDH histochemistry is routinely utilized in the assessment of muscle biopsies to reveal underlying pathology such as subsarcolemmal mitochondrial aggregates. In this study, we evaluated the utility of succinate dehydrogenase B (SDHB) immunohistochemistry (IHC) in 27 muscle biopsies, including 13 mitochondrial myopathies (MMs), 9 inflammatory myopathies, and 5 controls. SDHB IHC was performed on formalin-fixed, paraffin-embedded tissue sections with a mouse monoclonal antibody (Abcam 21A11AE7) in parallel with histochemical SDH stains on a fresh-frozen tissue. In all muscle biopsies, SDHB IHC exhibited granular immunoreactivity and highlighted the dark type 1 and lighter type 2 staining pattern observed by histochemistry. In all cases of MM, SDHB IHC showed subsarcolemmal granular aggregates involving the entire periphery of the fibers that were more distinct than those seen by SDH histochemistry. In 3 extraocular muscle biopsies, SDHB immunoreactive speckles of various sizes were distributed throughout the entire sarcoplasm that were more prominent than those seen on SDH histochemistry. Subsarcolemmal and cytoplasmic granular aggregates seen on SDHB IHC correlated with mitochondrial pathology on electron microscopy. In cases of inflammatory myopathy, there was diffuse sarcoplasmic SDHB immunoreactivity in degenerating fibers, but no evidence of subsarcolemmal aggregates. This study demonstrates that SDHB IHC is highly sensitive and specific in the identification of MM. The automation, reproducibility, and cost efficiency of SDHB IHC offer advantages over the labor-intensive histochemical method requiring frozen sections. As this technique is performed on formalin-fixed, paraffin-embedded tissues, it can be easily applied for retrospective studies.

Increased immunoreactivity of cathepsins in the rat esophagus under chronic acid reflux esophagitis.

We have designed a stable rat chronic acid reflux esophagitis (RE) model. In gastrointestinal lesions, several lysosomal cathepsins are known to participate in epithelial permeability in cell-cell connections, such as tight junctions in ulcerative colitis. However, very few studies have focused on the distribution of cathepsins in the esophageal multilayer squamous epithelium. Therefore to clarify the role of cathepsins in RE, we investigated their immunohistological localization in the esophageal epithelium under normal conditions and after RE. Of the cathepsins examined (cathepsins B, C, D, F, H, L, S, and X), granular immunoreactivity for cathepsins B, C, D and L was observed in the control esophageal epithelia; although, their distribution differed depending on the enzyme examined. In the RE model, immunoreactivity of these cathepsins was increased in esophageal epithelial cells and activated macrophages. The immunoreactivity for cathepsins F, H, S and X was barely detectable in the control esophageal epithelium. However, in the RE model, we noticed a slight increase in the expression of cathepsins H and X in the epithelial cells. Furthermore, activated macrophages of the RE model possessed intense immunoreactivity for these cathepsins, which may have been related to esophageal inflammatory mechanisms.

Immunohistochemical localization of autophagosomal membrane-associated protein LC3 in granular cell tumor and schwannoma

Granular cell tumor (GCT) is a neoplasm derived from Schwann cell or (in cases arising in the neurohypophysis) pituicyte and is characterized by abundant cytoplasm filled with numerous eosinophilic granules, which have been considered autophagolysosomes on the basis of their ultrastructure. To confirm that the formation of these granules is related to an autophagy phenomenon, 12 cases of GCT (including two cases of GCT of the neurohypophysis) were studied immunohistochemically using an antibody against LC3 (microtubule-associated protein 1 light chain 3, a specific marker of autophagy). All cases of GCT showed granular immunoreactivity for LC3 in the cytoplasm of tumor cells, indicating that the formation of intracytoplasmic granules in GCT is closely related to an autophagy phenomenon. For elucidation of the relationship between GCT and schwannoma, 20 cases of schwannoma were similarly studied using the anti-LC3 antibody. In eight of 20 cases, a small number of tumor cells showed granular immunoreactivity for LC3, suggesting an increased autophagic activity in some schwannomas and further reinforcing the close relationship between GCT and schwannoma.

Different Prion Conformers Target the Olfactory Pathway in Sporadic Creutzfeldt–Jakob Disease

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are mammalian neurodegenerative diseases that occur as sporadic, inherited, or iatrogenic forms. Human TSEs exhibit a wide spectrum of phenotypic variability, which is influenced by (1) the conformation of the pathologic prion protein, or PrP(Sc); (2) the polymorphic codon 129 of the prion protein gene (PRNP), involving synonymous or nonsynonymous expression of Met or Val; and (3) the site of formation or entry of the self-replicating PrP(Sc). Brain deposition of PrP(Sc) occurs in a phenotype-specific regional pattern, either as extracellular amyloid plaques and plaque-like aggregates, or as fine granular immunoreactivity at intracellular sites and presynaptic and postsynaptic locations, including dendrites. We previously demonstrated PrP(Sc) deposition in ciliated dendrites of olfactory sensory neurons in sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease. PrP(Sc) immunoreactivity was not limited to the olfactory neuroepithelium. But additionally involved the central olfactory pathway. More recently, we have found that the pathology of the olfactory pathway occurs early in the disease course, either in the myoclonic or classic sCJD or in the ataxic variant. Intriguingly, in the ataxic or cerebellar variant, mainly observed in patients with the Met/Val polymorphism (2) carrying PrP(Sc) type 2, olfactory involvement is accompanied by pathologic changes in the dorsal motor nucleus of the vagus and other brainstem nuclei. These findings suggest that different molecular events and distinct routes of PrP(Sc) spread contribute to the prominent heterogeneity of sCJD, conceivably providing support to the olfactory pathogenesis theory of neurodegenerative diseases.

Proliferation and Tube Formation of Periodontal Endothelial Cells

Angiogenesis is indispensable to guide a regeneration of good periodontal tissue in the wound healing after periodontal surgery. Hepatocyte growth factor is well known for a strong angiogenic factor and it may play important roles in the periodontal tissue during periodontal wound healing. In exploring the promotion of angiogenesis in the periodontal ligament, proliferative and tubulogenic responses of endothelial cells to hepatocyte growth factor and to soluble factors secreted by fibroblasts were investigated. Pavement-shaped cells isolated from a human periodontal ligament were identified as the endothelial cell by their granular immunoreactivity for factor VIII. The proliferation of the endothelial cells was accelerated by the addition of hepatocyte growth factor or fibroblast-conditioned medium, and far more by adding both than either. The endothelial cells seeded on the agar containing both hepatocyte growth factor and fibroblast products formed a dense network in a shorter time than on the agar containing either. The endothelial cells in the dense network took a tube-like structure with lumen and were covered with laminin. These results suggest that hepatocyte growth factor administered into the regenerating periodontal tissue may promote, synergistically with local factors produced by the activated fibroblast, the proliferation and tubulogenesis of the remaining endothelial cells.

Comparison of STAT3 with HIF-1alpha, Ob and ObR expressions in human endometrioid adenocarcinomas

Signal transducer and activator of transcription (STAT3) maintained invasiveness of endometrial cancer cell line. STAT3 mediated signaling for oncogenic growth stimulated by leptin (Ob) and hypoxia-inducible factor 1 (HIF-1). Therefore, we studied STAT3 in relation with HIF-1alpha, Ob, leptin receptor (ObR) and clinical and pathological variables with immunohistochemistry in 48 human endometrioid adenocarcinomas. Nuclear location was a proof of activity of STAT3 and HIF-1 and it was mainly characteristic for granular anti-STAT3 staining and rarely for diffuse HIF-1alpha expression. HIF-1alpha, Ob and ObR presented cytoplasmic granular immunoreactivities. Positive staining for STAT3, HIF-1, Ob and ObR occurred in 75%, 79%, 60% and 31% of cancers, respectively. Anti-STAT3 staining did not significantly vary with grading, staging and patients’ age. STAT3 correlated with Ob ( p = 0.048, r = 0.290) and with HIF-1alpha ( p = 0.004, r = 0.407) in all cancers but it failed to associate with ObR at all. In opposition to the absence of significant relationship between STAT3 and Ob, STAT3 correlated with HIF-1alpha in well differentiated cancers (G1), poorly differentiated tumors (G3), pT1b neoplasms, compound group of pT1c, pT2a, pT2b tumors, and older patients over their sixties. STAT3 mediated signaling pathways that engage leptin and HIF-1alpha could only be partially reflected in correlations between STAT3 and Ob or STAT3 and HIF-1alpha in the examined neoplasms. Nevertheless, STAT3 failed to mark cancer advancement, so progressive significance of STAT3 is questionable in endometrioid adenocarcinomas.

Anterior horn cells with abnormal TDP-43 immunoreactivities show fragmentation of the Golgi apparatus in ALS

Recently, TAR DNA-binding protein of 43-kDa (TDP-43) was identified as a major component of ubiquitinated neuronal cytoplasmic inclusions observed in lower motor neurons in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions. We herein investigated the relationship between TDP-43 immunoreactivities and fragmentation of the Golgi apparatus (GA). Each mirror section of spinal cord tissues in 10 ALS and 3 control cases were immunostained with polyclonal anti-TDP-43 and polyclonal anti-trans-Golgi-network (TGN)-46 antibodies. The neurons were divided into subtypes according to differences in TDP-43 immunoreactivities, and we examined the morphological changes of GA in each type. We divided the neurons into four subtypes according to the observed differences in TDP-43 immunoreactivities, Type A: neurons showing normal nuclear staining, Type B: neurons showing a loss of normal nuclear staining and a few granular cytoplasmic immunoreactivities, Type C: neurons showing a lot of granular immunoreactivities and no inclusions, Type D: neurons with inclusions. All of the neurons in Type A showed normal GA profiles, however, almost all of the neurons with abnormal TDP-43 immunoreactivities (Type B-D) showed GA fragmentation. These results suggest that neurons with abnormal TDP-43 immunoreactivities are associated with dysfunction of the secretory pathway in motor neurons.

Overexpression of CYP2A6 in human colorectal tumors

CYP2A6 metabolizes various nitrosamines, such as those in the diet and in tobacco smoke, which have been implicated as risk factors for colorectal tumors. To determine whether changes in expression levels could contribute to their progression, we carried out immunohistochemistry for CYP2A6 in human colon tumors. Colon specimens (n = 53) were diagnosed as adenoma (n = 16), adenocarcinoma (n = 30) or carcinoma in or with adenoma (n = 7). Colon tumor cells showed cytoplasmic granular immunoreactivity for CYP2A6. Adenocarcinomas and adjacent mucosa showed similar highly elevated degrees of CYP2A6 expression, whereas carcinomas in or with adenoma and adenomas showed lesser increases. To further determine whether CYP2A6 mRNA was expressed at the same level as the CYP2A6 protein, we carried out in situ hybridization of CYP2A6 in two cases of adenocarcinoma. In situ hybridization for CYP2A6 revealed mRNA expression in adenocarcinoma cells. The data indicate that CYP2A6 may have important roles in human colorectal tumorigenesis and progression, so that it could be a candidate therapeutic and chemopreventive target for colorectal cancers.

Differentiation of mouse embryonic stem cells into growth hormone and prolactin expressing cells in vitro

Embryonic stem (ES) cells provide an excellent in vitro model system to investigate the cellular and molecular events that drive lineage-specific commitment and differentiation and have considerable potential as agents for novel cell-based therapies. While culture methods for directed in vitro differentiation of ES into neuroectodermal and mesodermal derivatives are relatively well established, little is known about the capacity of ES cells to generate the hormone secreting lineages of the pituitary. Here we show that extended culture of adherent embryoid bodies (EBs) results in the induction of pituitary-specific progenitor marker genes and pituitary hormonal markers. GH-expressing cells comprise approximately 2% of the total viable cell population and exhibit dense granular cytoplasmic immunoreactivity. These data demonstrate for the first time that cell types which have the characteristics of pituitary somatotropes and lactotropes can be generated by in vitro differentiation of mouse ES cells.

Intranuclear immunolocalization of 14-3-3 protein isoforms in brains with spinocerebellar ataxia type 1

Immunolocalization of 14-3-3 protein isoforms, one of the interacters with ataxin 1, was investigated in spinocerebellar ataxia type 1 (SCA 1) brains using isoform-specific antibodies. Samples from the pons and from the cerebellum of four SCA1 cases and three controls were studied. The intensity of the immunoreactivity (IR) and its subcellular topography were analyzed. In control subjects, granular immunoreactivity for an epitope common to all known isoforms of 14-3-3 proteins (14-3-3 COM) found in the cytoplasm of some pontine and dentate nucleus neurons was weak. It was observed in some Purkinje cells, while its intensity varied. Many nuclei of those neurons and Purkinje cells of SCA1 were intensely immunopositive for 14-3-3 COM, while it was less in their cytoplasm. Expanded polyglutamine epitope was colocalized to 14-3-3 COM epitope in some pontine neurons, sometimes accumulated in intranuclear inclusion-like structures. This findings support previous reports that 14-3-3 proteins stabilize mutant ataxin 1 in nucleus and possibly lead to neurodegeneration. However, nuclear localization of 14-3-3 proteins in SCA1 brains was dependent on its isoforms, i.e. pontine neurons intensely positive for beta, Purkinje cells for tau and dentate nucleus neurons for both, while all of those neurons were consistently positive for zeta isoform, although sigma isoform tended to be located in the cytoplasm. Nuclear accumulation and isoform- and region-dependent subcellular localizations of 14-3-3 proteins may be related to SCA1 pathology, which exhibits marked regional variability.

Altered expression of β‐catenin/E‐cadherin in meningiomas

Meningiomas are generally slow-growing benign tumours representing approximately 20% of all primary intracranial tumours. The hallmark of tumorigenesis of meningiomas is the loss of chromosome 22, including loss of heterozygosity of the neurofibromatosis type 2 (NF2) gene. The NF2 encoded protein merlin appears to function as a tumour suppressor gene by controlling cadherin-mediated cell-cell adhesion. The E-cadherin cell adhesion system includes beta-catenin that indirectly connects cadherin to actin filaments. The aim of this study was to analyse the expression and the subcellular location of E-cadherin and beta-catenin in human meningiomas, including meningiomas of different histomorphological subtypes and different World Health Organization (WHO) grades. Immunohistochemical analysis revealed lack of E-cadherin expression at the cell membrane in 34% of meningiomas independent of their WHO grade. Loss of membranous beta-catenin occurred in 79% of meningiomas. An intense perinuclear granular immunoreactivity of beta-catenin without nuclear location was detected in the majority of meningiomas. Both immunofluorescence and Western blot analysis of fractionated meningioma cells located beta-catenin mostly on the Golgi apparatus and ER/Golgi intermediate compartment (ERGIC). Cytogenetic analysis of meningiomas showed no correlation between NF2 loss and the loss of the proper location of beta-catenin. The lack of membranous beta-catenin and/or membranous E-cadherin in meningiomas may indicate an altered interaction between meningioma cells independent of loss of NF2 and independent of the tumour grade.

Sialin, an anion transporter defective in sialic acid storage diseases, shows highly variable expression in adult mouse brain, and is developmentally regulated

Sialin is a lysosomal membrane protein encoded by the SLC17A5 gene, which is mutated in patients with sialic acid storage diseases (SASD). To further understand the role of sialin in normal CNS development and in the progressive neuronal atrophy and dysmyelination seen in SASD, we investigated its normal cellular distribution in adult and developing mice. Overall, sialin showed granular immunoreactivity, consistent with a vesicular protein. Adult mice showed widespread sialin expression, including in the brain, heart, lung, and liver. High-level immunoreactivity was seen in the neuropil of the hippocampus, striatum, and cerebral cortex, as well as in the perikarya of cerebellar Purkinje cells, globus pallidus, and certain thalamic and brainstem nuclei. In mouse embryos, the highest levels of expression were observed in the nervous system. We discuss the possible role of sialin in normal development and in SASD pathogenesis, as a framework for further investigation of its function in these contexts.

Immunolocalization of 14-3-3 isoforms in brains with Pick body disease

Immunolocalization of 14-3-3 protein isoforms in relation to Pick bodies in Pick body disease (PBD) brains was investigated. Weakly granular immunoreactivity of 14-3-3 proteins was found in neurons in control subjects and in Pick body disease brains. In addition to this granular immunoreactivity, many Pick bodies were immunopositive for 14-3-3 proteins as confirmed with double-immunofluorescence with an anti-PHF tau (AT8) and anti-14-3-3 that recognizes all its isoforms (common). When probed with isoform-specific antibodies, Pick bodies were positive for beta, gamma, epsilon, eta, tau, and zeta isoform and exhibited immunostaining pattern similar to that observed with the anti-14-3-3 proteins (common). In addition, immunoreactivity of sigma isoform, so far considered to be exclusively extraneuronal, was unexpectedly found in Pick bodies, normal hippocampal neurons and brain homogenate from age-matched controls. Although localization of 14-3-3 proteins in Pick bodies suggests their involvement in Pick body formation, their role may be variable dependent on the isoforms differently expressed in different area in the brain.

Immunohistochemical expression of Bid in oral squamous cell carcinoma

Bcl-2 and its congeners play a key role in controlling programmed cell death (PCD, apoptosis). We and others have reported the status of anti-PCD proto-oncogenes Bcl-2, Bcl-X L , Mcl-1, and Bag-1 as well as Bax and Bak (pro-PCD) in oral cancer. The objective of this study was to examine the expression of Bid (allosteric regulator) in oral carcinomas. For this purpose, 5 micron-thick sections from archival paraffin blocks were examined using polyclonal antibodies to Bid oncoprotein. A finely granular Bid immunoreactivity was observed primarily in the cytoplasm of 60% (12 out of 20) of the carcinomas. A varying degree of zonal or diffuse heterogenous reaction was observed generally based on the degree of neoplastic cell differentiation. The mechanisms involving the role of Bid in regulation of apoptosis remain unclear. It has been considered that: (1) full-length Bid translocates Bax to the outer mitochondrial membrane and Bax activation results in induction of mitochondrial permeability transition, (2) caspase 8–cleaved Bid (t-Bid) induces conformational change in Bak, and (3) other mechanisms. These processes enable cytochrome-c release from mitochondria. Cytochrome-c forms apoptosome with caspase 9 leading to caspase activation. Initiating caspases 9 and 8 activate downstream caspase 3, leading to apoptosis. It appears that Bid plays an effector role by inducing functional status to other oncogenes. The elucidation of Bid in oral carcinomas as well as caspases 8 and 3 (previously reported by us) indicate a potential for apoptosis. A higher apoptotic index has been shown to confer favorable response to treatment; therefore, the induction of apoptosis (inherent in tumor cells) could be a valuable tool for oral cancer therapy.

14-3-3 proteins and zeta isoform containing neurofibrillary tangles in patients with Alzheimer's disease.

Immunolocalization of 14-3-3 proteins in Alzheimer's disease (AD) brains was investigated using isoform-specific antibodies. Weak granular immunoreactivity of 14-3-3 proteins was found in neuronal cytoplasm in control subjects and AD brains. Both intracellular and extracellular neurofibrillary tangles (NFTs), as well as neuropil thread-like structures, were immunopositive for 14-3-3 proteins. This was corroborated by triple-fluorolabeling method visualizing paired helical filament (PHF) tau and 14-3-3 epitopes in relation to fibrillary state detected by thiazin red. Pretangle neurons (positive for PHF-tau without fibrillary structure detected by thiazin red) only contained fine granular immunoreactivity (IR) of 14-3-3, which was similarly found in unaffected neurons. Granular cytoplasmic IR of 14-3-3 proteins in pretangle neurons was not colocalized to granular tau-like IR, which suggests that participation of 14-3-3 proteins in NFT formation was restricted to its later stages. Its zeta isoform was most prominent in these NFTs, suggesting that this isoform is a major component involved in the formation of NFTs. In contrast, IR of epsilon isoform was found in the neuropil of the hippocampus and that of sigma isoform was localized to granule cells of the dentate gyrus in AD brains, as seen in the age-matched controls. Expression of 14-3-3 proteins were found to be highly variable and dependent on their isoforms, regions and cell types. Molecular, as well as topographical, dissection of 14-3-3 proteins will provide us with an improved understanding of this molecule in normal and pathological conditions.

Electron microscopy evidence that cytoplasmic localization of the p16INK4A “nuclear” cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas

It is well established that p16INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 INK4A usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16INK4A immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16INK4 represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16INK4A staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16INK4A cytoplasmic expression. We observed p16 INK4A immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16INK4A immunoreactivity was detected only in the nuclei. We have demonstrated that p16INK4A cytoplasmic staining is specific and suggest that it represents a mechanism of p16INK4A inactivation similar to that observed in other tumor suppressor genes.

Immunohistochemical localization of c-mos at the light and electron microscope level in non-small cell lung carcinomas

C-mos is a cytoplasmic upstream activator of the mitogen-activating protein kinase pathway with serine-threonine kinase activity. It plays a well established and vital role in oocyte maturation by participating in metaphase II arrest and meiotic asymmetric division, but little is known about its function in somatic cells. Recently, we observed overexpressed c-mos in a portion of non-small cell lung carcinomas (NSCLCS). In particular, c-mos immunoreactivity was detected in tumor cell nuclei in addition to its expected cytoplasmic localization, and c-mos overexpression was associated with chromosomal instability among other findings. To verify our earlier observations and to clarify further the role of c-mos in NSCLCS, we examined its distribution by both light and electron microscopy. We detected c-mos in the cytoplasm and/or nucleus of a portion of tumor cells and fibroblasts. In particular, granular immunoreactivity was observed in the cytoplasm closely associated with the rough endoplasmic reticulum. Nuclear staining was confirmed and was often found near the nuclear membrane, as well as in some large multilobular, possibly aneuploid, nuclei. C-mos positivity was also found in the nuclei of tumor cells undergoing apoptosis. Furthermore, c-mos was detected in areas with diminished vascularization. It should be noted that nuclear staining was found at the ultrastructural level more extensively than at the light microscope study. This suggests a masking effect by the hematoxylin nuclear counterstain.

Differential expression between synaptic vesicle proteins and presynaptic plasma membrane proteins in the anterior horn of amyotrophic lateral sclerosis.

This study concerns the immunohistochemical investigation of synaptic proteins in the anterior horn of amyotrophic lateral sclerosis (ALS). Antibodies against synapsin 1 and synaptophysin (i.e. synaptic vesicle proteins), and those against syntaxin and the synaptosomal-associated, 25 kDa protein, SNAP25 (i.e. presynaptic plasma membrane proteins) were used for immunostaining, respectively. Lumbar spinal cords from five ALS and eight control patients were examined. In the controls, all four synaptic proteins exhibited fine granular immunoreactivities, distributed throughout the spinal gray matter almost uniformly. In contrast, in all five ALS patients, two of the synaptic vesicle proteins examined decreased in the anterior horn neuropil diffusely, while in the same lumbar segments of these cases the immunoreactivities of the two presynaptic plasma membrane proteins showed no apparent decrease, or were only mildly diminished in the same gray matter area. These results indicate that, during the presynaptic terminal degeneration in the anterior horn of ALS, synaptic vesicle involvement may precede that of the presynaptic plasma membrane.

Amphicrine Gastric Carcinoma

64-year-old man presented with epigastralgia and severe weight loss in the previous 4 months. The patient underwent a gastric endoscopy, which showed a large ulceroinfiltrative lesion in the gastric antrum. A total gastrectomy was performed and was submitted to pathologic examination. The gross specimen showed a large ulceroinfiltrative lesion measuring 5.5 3 4.0 3 2.8 cm in the antrum infiltrating all layers of the gastric wall. The histologic examination showed a neoplasm composed of medium-sized to large cells with anaplastic nuclei, prominent eosinophilic to amphophilic macronucleoli, granular chromatin, and eosinophilic finely granular cytoplasm. These cells were disposed in solid nests, trabeculae, and ribbons (Figure 1). Foci of typical poorly differentiated gastric signet ring cell carcinoma were admixed with these areas (Figure 2), as well as constituting distinct neoplastic foci. Immunohistochemistry was performed using the avidin-biotin-peroxidase technique with antibodies against low-molecular-weight cytokeratin, epithelial membrane antigen, carcinoembryonic antigen, synaptophysin, chromogranin, and neuron-specific enolase. The immunostaining showed 2 distinct patterns of positivity in the solid and the signet ring areas. In the solid area, cells showed positivity for low-molecular-weight cytokeratin, epithelial membrane antigen, synaptophysin, chromogranin, and neuron-specific enolase, whereas the signet ring cell foci were immunoreactive for low-molecularweight cytokeratin, epithelial membrane antigen, and carcinoembryonic antigen. However, some signet ring cells showed distinct granular immunoreactivity for both synaptophysin and chromogranin (Figure 3). Six of the 11

Seasonal Changes in β-Endorphin-like Immunoreactivity in the Olfactory System of the Female Catfish, Clarias batrachus (Linn.)

In the olfactory system of the catfish Clarias batrachus, β-endorphin-like immunoreactivity was seen in several olfactory receptor neurons (ORN) and their fiber projections extending caudally over the olfactory nerve to the olfactory bulb (OB). With β-endorphin-like immunoreactivity as a cellular marker, the olfactory system in the female fish was investigated at different stages of its annual reproductive cycle. The reproductive cycle of the fish is divisible into four distinct phases: preparatory (February–April), prespawning (May–June), spawning (July–August), and postspawning (September–January). The gonosomatic index and the immunocytochemical profile of β-endorphin-like immunoreactivity showed distinct changes as the fish progressed from one phase to another. In the preparatory phase, limited immunoreactivity was seen in the periphery of the bulb. However, the immunoreactivity showed a robust increase as the immunolabeled fibers extended progressively deeper into the bulb toward the mitral cell layer during the prespawning and spawning phases. Significant reduction in the immunoreactivity was noticed in the olfactory nerve layer of the fish in the postspawning phase. Several granule cells showed poor to moderate immunoreactivity during the spawning phase, although no immunoreactivity was seen in the inner cell layer during the rest of the year. The β-endorphin-like immunoreactivity in the ORN also showed season-related changes, although these were less distinct. Whereas weak immunoreactivity confined to a few ORN was noticed in the fish collected in the preparatory phase, those in the prespawning phase showed conspicuous augmentation in immunoreactivity. During the spawning phase, the sensory layer of the olfactory epithelium showed reduced, homogenous immunoreactivity. In the postspawning phase, several ORN revealed distinct granular immunoreactivity, suggesting possibilities of de novo synthesis. These annual cyclic changes in the β-endorphin-like immunoreactivity were consistently observed over a 30-month study period that spanned three consecutive spawning phases. The results suggest that the β-endorphin-containing ORN, their fiber projections to the OB, and several granule cells in the inner cell layer may be involved in the processing of reproduction/reproductive behavior-related signals.