Phaseolus Vulgaris Leukoagglutinin Research Articles

Is the central mesencephalic reticular formation a purely horizontal gaze center?

Historically, the central mesencephalic reticular formation has been regarded as a purely horizontal gaze center based on the fact that electrical stimulation of this region produces horizontal saccades, it provides monosynaptic input to medial rectus motoneurons, and cells recorded in this region often display a peak in firing when horizontal saccades are made. We tested the proposition that the central mesencephalic reticular formation is purely a horizontal gaze center by examining whether this region also supplies terminals to superior rectus and levator palpebrae superioris motoneurons, both of which fire when making vertical eye movements. The experiments were carried out using dual tracer techniques at the light and electron microscopic level in macaque monkeys. Injections of biotinylated dextran amine or Phaseolus vulgaris leukoagglutinin into the central mesencephalic reticular formation produced anterogradely labeled terminals that were in synaptic contact with superior rectus and levator palpebrae superioris motoneurons that had been retrogradely labeled. These results indicate that this region is not purely connected with horizontal gaze motoneurons. In addition, we found that the number of contacts on vertical gaze motoneurons increased with more rostral injections involving the mesencephalic reticular formation adjacent to the interstitial nucleus of Cajal. This suggests that there is a caudal to rostral gradient for horizontal to vertical saccades, respectively, represented within the midbrain reticular formation. Finally, we utilized post-embedding immunohistochemistry to show that a portion of the labeled terminals were GABAergic, indicating they likely originate from downgaze premotor neurons.

A Phaseolus vulgaris Leukoagglutinin Biosensor as a Selective Device for the Detection of Cancer‐associated N‐glycans with Increased β1→6 Branching

AbstractA Phaseolus vulgaris leukoagglutinin (PHA−L) impedimetric biosensor was developed for the selective detection of cancer‐associated N‐glycans presenting increased β1→6 GlcNAc branching. The increase in the biosensor‘s impedance after sample incubation was indicative of lectin recognition and complex formation between PHA−L and N‐glycans with increased β1→6 GlcNAc branching, present in serum glycoproteins. Bovine thyroglobulin was used as a model glycoprotein and a linear correlation was found between glycoprotein concentration and analytical signal from 0.05 to 2.0 mg ml−1. The biosensor was validated by analyzing serum samples from patients with diverse types of carcinomas.

Effect of vitamin D supplementation on N-glycan branching and cellular immunophenotypes in MS.

ObjectiveTo investigate the effect of cholecalciferol (vitamin D3) supplementation on peripheral immune cell frequency and N‐glycan branching in patients with relapsing‐remitting multiple sclerosis (RRMS).MethodsExploratory analysis of high‐dose (20 400 IU) and low‐dose (400 IU) vitamin D3 supplementation taken every other day of an 18‐month randomized controlled clinical trial including 38 RRMS patients on stable immunomodulatory therapy (NCT01440062). We investigated cholecalciferol treatment effects on N‐glycan branching using L‐PHA stain (phaseolus vulgaris leukoagglutinin) at 6 months and frequencies of T‐, B‐, and NK‐cell subpopulations at 12 months with flow cytometry.ResultsHigh‐dose supplementation did not change CD3+ T cell subsets, CD19+ B cells subsets, and NK cells frequencies, except for CD8+ T regulatory cells, which were reduced in the low‐dose arm compared to the high‐dose arm at 12 months. High‐dose supplementation decreased N‐glycan branching on T and NK cells, measured as L‐PHA mean fluorescence intensity (MFI). A reduction of N‐glycan branching in B cells was not significant. In contrast, low‐dose supplementation did not affect N‐glycan branching. Changes in N‐glycan branching did not correlate with cell frequencies.InterpretationImmunomodulatory effect of vitamin D may involve regulation of N‐glycan branching in vivo. Vitamin D3 supplementation did at large not affect the frequencies of peripheral immune cells.

Highlights of This Issue

[18F]FLT (3′-Fluoro-3′ deoxythymidine)–positron emission tomographic imaging ([18F]FLT–PET) has been used to measure in vivo tumor cell proliferation; however, its use in clinical tumor staging is limited due to the lower overall uptake of FLT. Zhang and colleagues evaluated the factors other than tumor cell proliferation that could affect the outcome of imaging to understand the translational aspects of the [18F]FLT–PET imaging modality. The preclinical use of [18F]FLT–PET imaging for tracking therapeutic response was also assessed. The results of this study strongly agreed with the clinical outcome. This work highlights the preclinical understanding of [18F]FLT–PET imaging and provides insights into the technologic applications in the oncology arena.Glioma stem cells (GSC) are a critical therapeutic target of glioblastoma multiforme (GBM). Miyazaki and colleagues addressed the hypothesis that a G-quadruplex ligand, telomestatin (TMS), eradicates GSCs. TMS treatment induced apoptosis of GSCs in vitro and in vivo through both telomeric and nontelomeric DNA damage. A proto-oncogene, c-Myb, was identified as a novel target of TMS by the cDNA microarray and pharmacodynamic analysis. c-Myb knockdown significantly reduced the growth of GSCs both in vitro and in vivo, and c-Myb expression in surgical specimens of GBM patients was statistically significantly elevated. These data suggest a novel GSC-directed therapeutic strategy for GBM through telomere disruption and c-Myb inhibition.The prostate cancer field is critically lacking a metastasis xenograft model that respects the entire physiologic metastatic cascade and provides evidence for its clinical relevance. Lange and colleagues established subcutaneous xenograft models using 4 prostate cancer cell lines and quantified tumor development and spontaneous metastasis formation. Metastatic xenografts revealed an increased activity of Mgat5b, a β(1,6)- branching glycosyltransferase previously found in neurons and testes, as detected by Phaseolus vulgaris leukoagglutinin (PHA-L) binding. According to a clinical part of the study (n = 2,085), PHA-L intensity was correlated with serum prostate-specific antigen (PSA) levels, and a cytoplasmic localization of PHA-L binding adversely affected PSA recurrence-free survival.Few studies have explored the connection between tumor imaging results and gene expression. To identify signaling pathways associated with treatment-related changes in tumor 2[18F]fluoro-2-deoxy-D-glucose–positron emission tomographic (FDG–PET) imaging, Schwarz and colleagues performed gene expression profiling using 62 pretreatment cervical cancer biopsies from patients treated with definitive chemoradiation. All patients underwent pre- and post-treatment FDG–PET. Alterations in expression of genes from the phosphoinositide 3-kinase (PI3K)/Akt pathway were associated with abnormal FDG uptake on the posttreatment PET. These results were validated using immunohistochemistry for p-Akt and comparing results with patient survival outcome data after treatment (n = 174). These results suggest that PI3K/Akt inhibition may improve response to chemoradiation.

Human Prostate Cancer in a Clinically Relevant Xenograft Mouse Model: Identification of β(1,6)-Branched Oligosaccharides as a Marker of Tumor Progression

To establish xenograft mouse models of metastatic and nonmetastatic human prostate cancer and to apply these models to the search for aberrant glycosylation patterns associated with tumor progression in vivo and in patients. Prostate cancer cells (LNCaP, PC-3, LuCaP 23.1, and DU-145) were xenografted subcutaneously into immunodeficient pfp(-/-)/rag2(-/-) mice. Tumor growth and metastasis formation were quantified and as altered glycosylation patterns have been associated with metastasis formation in several other malignancies, prostate cancer cells were profiled by a quantitative real-time PCR (qRT-PCR) glycosylation array and compared with normal human prostate cells. The activity of upregulated glycosyltransferases was analyzed by their sugar residues end products using lectin histochemistry on primary tumors and metastases in the animal experiments and on 2,085 clinical samples. PC-3 cells produced the largest number of spontaneous lung metastases, followed by LNCaP and LuCaP 23.1, whereas DU-145 was nonmetastatic. qRT-PCR revealed an upregulation of β1,6-N-acetylglucosaminyltransferase-5b (Mgat5b) in all prostate cancer cell lines. Mgat5b products [β(1,6)-branched oligosaccharides] were predominantly detectable in metastatic xenografts as shown by increased binding of Phaseolus vulgaris leukoagglutinin (PHA-L). The percentage of prostate cancer patients who were PHA-L positive was 86.5. PHA-L intensity correlated with serum prostate-specific antigen and a cytoplasmic staining negatively affected disease-free survival. We show a novel xenograft mouse model for human prostate cancer respecting the complete metastatic cascade. Specific glycosylation patterns reveal Mgat5b products as relevant markers of both metastatic competence in mice and disease-free survival in patients. This is the first description of Mgat5b in prostate cancer indicating a significant biologic importance of β(1,6)-branched oligosaccharides for prostate cancer progression.

Control of TNF-Induced Dendritic Cell Maturation by Hybrid-Type N-Glycans

The activity of α-1,2-mannosidase I is required for the conversion of high-mannose to hybrid-type (ConA reactive) and complex-type N-glycans (Phaseolus vulgaris-leukoagglutinin [PHA-L] reactive) during posttranslational protein N-glycosylation. We recently demonstrated that α-1,2-mannosidase I mRNA decreases in graft-infiltrating CD11c(+) dendritic cells (DCs) prior to allograft rejection. Although highly expressed in immature DCs, little is known about its role in DC functions. In this study, analysis of surface complex-type N-glycan expression by lectin staining revealed the existence of PHA-L(low) and PHA-L(high) subpopulations in murine splenic conventional DCs, as well as in bone marrow-derived DC (BMDCs), whereas plasmacytoid DCs are nearly exclusively PHA-L(high). Interestingly, all PHA-L(high) DCs displayed a strongly reduced responsiveness to TNF-α-induced p38-MAPK activation compared with PHA-L(low) DCs, indicating differences in PHA-L-binding capacities between DCs with different inflammatory properties. However, p38 phosphorylation levels were increased in BMDCs overexpressing α-1,2-mannosidase I mRNA. Moreover, hybrid-type, but not complex-type, N-glycans are required for TNF-α-induced p38-MAPK activation and subsequent phenotypic maturation of BMDCs (MHC-II, CD86, CCR7 upregulation). α-1,2-mannosidase I inhibitor-treated DCs displayed diminished transendothelial migration in response to CCL19, homing to regional lymph nodes, and priming of IFN-γ-producing T cells in vivo. In contrast, the activity of α-1,2-mannosidase I is dispensable for LPS-induced signaling, as well as the DCs' general capability for phenotypic and functional maturation. Systemic application of an α-1,2-mannosidase I inhibitor was able to significantly prolong allograft survival in a murine high-responder corneal transplantation model, further highlighting the importance of N-glycan processing by α-1,2-mannosidase I for alloantigen presentation and T cell priming.

Cocaine and amphetamine-regulated transcript-containing neurons in the nucleus accumbens project to the ventral pallidum in the rat and may inhibit cocaine-induced locomotion

We have previously demonstrated that cocaine- and amphetamine-regulated transcript (CART) peptide colocalizes with GABA, dynorphin, D1 receptors, and substance P in some neurons in the nucleus accumbens (NAcc). One of the main nuclei that receive accumbal efferents is the ventral pallidum (VP), and both dynorphin and substance P have been shown to be present in the cell bodies and terminals of this projection. Thus, we investigated whether CART peptide is also present in the VP in terminals that originate in the accumbens. The anterograde tracer Phaseolus vulgaris leukoagglutinin (PHA-L) colocalized with CART in neuronal processes in the VP when injected into the NAcc. Also, CART colocalized with the retrograde tracer r-BDA in accumbens cell bodies after the tracer was injected into the VP. Using electron microscopic immunocytochemistry, we examined CART terminals in the VP and found that CART-immunoreactive terminals formed symmetric synapses consistent with inhibitory GABAergic synapses. These synapses closely resemble GABAergic synapses in the substantia nigra pars reticulata (SNr), another nucleus that receives some CART–containing accumbal efferents. Lastly, we found that intra-pallidal injection of CART 55–102 inhibited cocaine-induced locomotion, indicating that CART peptide in the VP can have functional effects.

N-glycoproteins bearing ??1???6 branched oligosaccharides from the A375 human melanoma cell line analysed by tandem mass spectrometry

Tumour-associated alterations of cell surface glycosylation play a crucial role in the adhesion and metastasis of cancer cells. It is well known that the metastatic potential is associated with increased GlcNAc beta1-6 branching in N-glycans of tumour cells specifically recognized by a lectin from Phaseolus vulgaris leukoagglutinin (PHA-L). We identified proteins bearing GlcNAc beta1-6 branched N-glycans in the A375 human melanoma cell line by affinity chromatography separation on a PHA-L agarose column, followed by immunoidentification and tandem mass spectrometry (MS/MS) analysis. Amongst the proteins identified were integrin subunits alpha2, alpha3, alpha5 and beta1, as well as N-cadherin and lysosome-associated membrane proteins (LAMP-1 and LAMP-2). In addition, L1, Mac-2 binding protein (Mac-2-BP), activated leukocyte cell adhesion molecule/CD166 (ALCAM) and melanotransferrin were shown to react with PHA-L. Some of these proteins are connected mainly with nervous tissues or the immune system and play a crucial role in cell adhesion processes. The presence of GlcNAc beta1-6 branched oligosaccharides in these proteins may influence their adhesion properties, reducing adhesion of the cells to the extracellular matrix (ECM) and thus facilitating tumour cell invasion.

Glycosylation of urinary prostate-specific antigen in benign hyperplasia and cancer: assessment by lectin-binding patterns

Objectives: In the present study, we examined the glycosylation of urinary prostate-specific antigen (PSA) from benign prostatic hyperplasia (BPH) and prostate cancer (PCa) subjects, specifically looking at alterations in its oligosaccharide chain as a potential biomarker of these pathophysiological conditions. Design and methods: First morning urine voids were collected from subjects with PCa and BPH before initiation of any treatment. Urinary PSA was characterized by ion-exchange chromatography, followed by lectin affinity chromatography on the columns using immobilized plant lectins. Results: Four isoforms of urinary PSA from both BPH and PCa samples were separated by ion-exchange chromatography. The elution profiles from lectin-affinity columns reflected molecular heterogeneity of PSA isoforms and the main differences observed were in the reactivity to Ulex europaeus agglutinin, Aleuria aurantia agglutinin, Phaseolus vulgaris erythroagglutinin and Phaseolus vulgaris leukoagglutinin. Conclusions: The observed differences in the lectin reactivities between BPH PSA and PCa PSA may be of clinical importance in the evaluation of prostate health.

Projections from the periamygdaloid cortex to the amygdaloid complex, the hippocampal formation, and the parahippocampal region: a PHA-L study in the rat.

The periamygdaloid cortex, an amygdaloid region that processes olfactory information, projects to the hippocampal formation and parahippocampal region. To elucidate the topographic details of these projections, pathways were anterogradely traced using Phaseolus vulgaris leukoagglutinin (PHA-L) in 14 rats. First, we investigated the intradivisional, interdivisional, and intra-amygdaloid connections of various subfields [periamygdaloid subfield (PAC), medial subfield (PACm), sulcal subfield (PACs)] of the periamygdaloid cortex. Thereafter, we focused on projections to the hippocampal formation (dentate gyrus, hippocampus proper, subiculum) and to the parahippocampal region (presubiculum, parasubiculum, entorhinal, and perirhinal and postrhinal cortices). The PACm had the heaviest intradivisional projections and it also originated light interdivisional projections to other periamygdaloid subfields. Projections from the other subfields converged in the PACs. All subfields provided substantial intra-amygdaloid projections to the medial and posterior cortical nuclei. In addition, the PAC subfield projected to the ventrolateral and medial divisions of the lateral nucleus. The heaviest periamygdalohippocampal projections originated in the PACm and PACs, which projected moderately to the temporal end of the stratum lacunosum moleculare of the CA1 subfield and to the molecular layer of the ventral subiculum. The PACm also projected moderately to the temporal CA3 subfield. The heaviest projections to the entorhinal cortex originated in the PACs and terminated in the amygdalo-entorhinal, ventral intermediate, and medial subfields. Area 35 of the perirhinal cortex was lightly innervated by the PAC subfield. Thus, these connections might allow for olfactory information entering the amygdala to become associated with signals from other sensory modalities that enter the amygdala via other nuclei. Further, the periamygdalohippocampal pathways might form one route by which the amygdala modulates memory formation and retrieval in the medial temporal lobe memory system. These pathways can also facilitate the spread of seizure activity from the amygdala to the hippocampal and parahippocampal regions in temporal lobe epilepsy.

Carbohydrate moieties of N-cadherin from human melanoma cell lines.

Expression of N-cadherin an adhesion molecule of the cadherin family, in tumor cells is associated with their increased invasive potential. Many studies suggested the role of N-linked oligosaccharides as important factors that contribute to metastasis by influencing tumor cell invasion and adhesion. N-cadherin is a heavily glycosylated protein. We have analysed the carbohydrate profile of this protein synthesized in human melanoma cell lines: WM35 from the primary tumor site and WM239, WM9, and A375 from different metastatic sites. N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterisation of its carbohydrate moieties was carried out by SDS/PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and analysis of their glycans using highly specific digoxigenin or biotin labelled lectins. The positive reaction of N-cadherin from the WM35 cell line with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and Sambucus nigra agglutinin (SNA) indicated the presence of high-mannose type glycans and biantennary complex type oligosaccharides with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines gave a positive reaction with Phaseolus vulgaris leukoagglutinin (L-PHA) and lotus Tetragonolobus purpureas agglutinin (LTA). This indicated the presence of tri- or tetra-antennary complex type glycans with alpha-fucose. In addition, N-cadherin from WM9 (lymphomodus metastatic site) and A375 (solid tumor metastatic site) contained complex type chains with alpha2-3 sialic acid (positive reaction with Maackia amurensis agglutinin--MAA). The results demonstrated that N-glycans of N-cadherin are altered in metastatic melanomas in a way characteristic for invasive tumor cells.

Dentate Granule Cells in Reeler Mutants and VLDLR and ApoER2 Knockout Mice

We have studied the organization and cellular differentiation of dentate granule cells and their axons, the mossy fibers, in reeler mutant mice lacking reelin and in mutants lacking the reelin receptors very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). We show that granule cells in reeler mice do not form a densely packed granular layer, but are loosely distributed throughout the hilar region. Immunolabeling for calbindin and calretinin revealed that the sharp border between dentate granule cells and hilar mossy cells is completely lost in reeler mice. ApoER2/VLDLR double-knockout mice copy the reeler phenotype. Mice deficient only in VLDLR showed minor alterations of dentate organization; migration defects were more prominent in ApoER2 knockout mice. Tracing of the mossy fibers with Phaseolus vulgaris leukoagglutinin and calbindin immunolabeling revealed an irregular broad projection in reeler mice and ApoER2/VLDLR double knockouts, likely caused by the irregular wide distribution of granule cell somata. Mutants lacking only one of the lipoprotein receptors showed only minor changes in the mossy fiber projection. In all mutants, mossy fibers respected the CA3–CA1 border. Retrograde labeling with DiI showed that malpositioned granule cells also projected as normal to the CA3 region. These results indicate that (1) reelin signaling via ApoER2 and VLDLR is required for the normal positioning of dentate granule cells and (2) the reelin signaling pathway is not involved in pathfinding and target recognition of granule cell axons.

Lectin Histochemistry of Resected Adenocarcinoma of the Lung: Helix pomatia Agglutinin Binding Is an Independent Prognostic Factor

The worldwide incidence of adenocarcinoma of the lung is rising. Unfortunately, no significant prognostic marker beyond the classical TNM staging exists to stratify these patients for appropriate therapy. Because lectins, carbohydrate-binding proteins, have been shown to be useful prognostic markers in several other adenocarcinomas, a panel of lectins [Helix pomatia agglutinin (HPA), Phaseolus vulgaris leukoagglutinin, Ulex europaeus agglutinin, Maackia amurenis agglutinin, Sambucus nigra agglutinin] with different carbohydrate-binding specificities were tested for their prognostic relevance. Paraffin wax sections of 93 patients with adenocarcinomas of the lung who had undergone surgery between 1990 and 1995 were investigated by lectin histochemistry. Lectin-binding data and other known prognostic factors were correlated with survival. In univariate analysis, binding of HPA, Phaseolus vulgaris leukoagglutinin, and Ulex europaeus agglutinin to adenocarcinoma cells were prognostic indicators for overall and relapse-free survival, whereas Maackia amurenis agglutinin and Sambucus nigra agglutinin binding had no prognostic value. However, in a multivariate analysis next to stage and gender only HPA was a significant independent prognostic factor on survival. In conclusion, HPA binding was the primary marker-based predictor of prognosis in our patient population and allows to stratify patients with adenocarcinomas of the lung into a low- and a high-risk group.

Lectin cytochemistry of rat cerebral and cerebellar neuronal cells.

Lectin cytochemistry of normal rat central nervous system (CNS) was performed on formalin-fixed, paraffin-embedded sections by using 14 lectins and light and electron microscopy. Hippocampal neurons reacted positively with Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I), Griffonia (Bandeiraea) simplicifolia lectin II (GSL-II) and Vicia villosa agglutinin (VVA); neurons in the cornu ammonis (CA) 2 region reacted positively with GSL-I and GSL-II, and neurons in the CA2 and CA3 regions reacted with VVA. Cortical neurons reacted positively with GSL-I and VVA. Cerebellar Purkinje cells and granule cells were reactive with Phaseolus vulgaris leukoagglutinin (PHA-L). Cerebellar nucleus neurons were also reactive with PHA-L and VVA. Reactivity with GSL-I, GSL-II, VVA and PHA-L was observed only on the neuronal cell surface. The present findings demonstrate the differences between cerebral and cerebellar neurons in reacting with glycoconjugates.

Lectin cytochemistry of rat cerebral and cerebellar neuronal cells

Lectin cytochemistry of normal rat central nervous system (CNS) was performed on formalin‐fixed, paraffin‐embedded sections by using 14 lectins and light and electron microscopy. Hippocampal neurons reacted positively with Griffonia (Bandeiraea) simplicifolia lectin I (GSL‐I), Griffonia (Bandeiraea) simplicifolia lectin II (GSL‐II) and Vicia villosa agglutinin (VVA); neurons in the cornu ammonis (CA) 2 region reacted positively with GSL‐I and GSL‐II, and neurons in the CA2 and CA3 regions reacted with VVA. Cortical neurons reacted positively with GSL‐I and VVA. Cerebellar Purkinje cells and granule cells were reactive with Phaseolus vulgaris leukoagglutinin (PHA‐L). Cerebellar nucleus neurons were also reactive with PHA‐L and VVA. Reactivity with GSL‐I, GSL‐II, VVA and PHA‐L was observed only on the neuronal cell surface. The present findings demonstrate the differences between cerebral and cerebellar neurons in reacting with glycoconjugates.

Subcortical projections of area 25 (subgenual cortex) of the macaque monkey

In several species, including primates, stimulation studies indicate that the infralimbic cortex, the most caudal part of the ventromedial prefrontal cortex, functions as a visceral motor region. In addition, recent positron emission tomography studies implicate the subgenual region in depression and mania. To determine the subcortical projections of this region in primates, injections of Phaseolus vulgaris leukoagglutinin, biotinylated dextran amine, or rhodamine-labeled dextran amine were placed in area 25 in three monkeys. In contrast to the efferents from area 25 previously described in the rat, there were no projections to autonomic effector regions, such as the nucleus of the solitary tract, magnocellular neurosecretory cell groups in the hypothalamus, ventrolateral medulla, or intermediolateral column of the spinal cord. However, projections were shown to a number of structures with probable roles in autonomic function and direct connections to some of the abovementioned autonomic effector regions, including bed nucleus of the stria terminalis, perifornical and anterior hypothalamus, periaqueductal gray, and lateral parabrachial nucleus. In addition, there were projections to several forebrain structures that receive projections from other components of the medial prefrontal network including the medial part of the caudate nucleus, lateral septum, midline and mediodorsal thalamic nuclei, the lateral parvocellular part of the basal accessory amygdaloid nucleus, and the magnocellular part of the basal amygdaloid. None of the injections resulted in labeling in the medulla. These connections support the idea of a role for cortical area 25 in emotional and autonomic responses, albeit less direct than that described in rodents.

Differentiation of islet cells in long-term culture.

Our previous studies in the hamster pancreatic cancer model have shown that exocrine pancreatic cancer arises from ductal/ductular cells, as well as from within the islets, most probably from islet precursor (stem) cells. To identify and characterize these cells, we established a long-term culture from isolated hamster islets and investigated their growth, differentiation, and expression of biomarkers. Islets maintained their original form and structure within the first 14 days in culture. However, beginning at day 7, ductular structures began to form within the islets. At day 21 in culture, acinar cells, intermediary cells, oncocytes, and cells comparable to pancreatic hepatocytes also appeared between ductular and endocrine cells. The number of duct-like cells gradually increased, whereas the number of hormone-producing cells decreased. After 35 days in culture, the exocrine cells disappeared, and undifferentiated cells formed a monolayer. These cells expressed cytokeratins, alpha1-antitrypsin, transforming growth factor-alpha, epidermal growth factor receptor, carbonic anhydrase II, vimentin, laminin, and showed binding to tomato lectin and Phaseolus vulgaris leukoagglutinin. They did not express the regulatory transcriptional factors, insulin-promoting factor 1, NKx6.1, Pax6, and NeuroD. The results thus indicate that islet cells have potential to form exocrine cells. At present, it is not clear whether these cells originate from preexisting stem cells or from transdifferentiated islet cells.

A novel evaluation system of metastatic potential of oral squamous cell carcinoma according to the histopathological and histochemical grading.

We established a new evaluation system for metastatic potential of oral squamous cell carcinoma (SCC), utilizing a combined examination of histopathological grades of the carcinomas based on cell differentiation and invasive mode according to Yamamoto's criteria, and the cellular expressions of CD44, E-cadherin (E-cad), heparan sulfate glycosaminoglycan (HS-GAG) and Phaseolus vulgaris leukoagglutinin (L-PHA)-binding oligosaccharides on the carcinomas. Histochemical patterns of expression of these markers were classified into positive (+2), weakly positive (+), and negative (-). The histopathological grades and the histochemical patterns of the SCC were estimated on a 0-2 point scale, i.e. point 2 for poorly differentiated, mode 4D, CD44++, E-cad-, HS-GAG++, or L-PHA++; point 1 for moderately differentiated, mode 4C, CD44+, E-cad+, HS-GAG+, or L-PHA+; and point 0 for well differentiated, mode 1, mode 2, mode 3, CD44-, E-cad++, HS-GAG-, or L-PHA-. As a result, incidence of metastasis in the cases with a total score of more than 6 (62.8%) was significantly higher than that with a total score of less than 5 (9.3%). This evaluation system will yield useful information concerning the prognosis of patients with oral SCC.

Suprachiasmatic efferents avoid phenestrated capillaries but innervate neuroendocrine cells, including those producing dopamine.

The key role of the suprachiasmatic nucleus in the diurnal regulation of anterior pituitary hormone secretions, including PRL, is well established. However, the pathway via suprachiasmatic signals reach the pituitary is ill defined. To determine whether suprachiasmatic efferents innervate neuroendocrine cells, the anterograde tracer, Phaseolus vulgaris leukoagglutinin, was injected iontophoretically into the suprachiasmatic nucleus in parallel with ip administration of fluorogold (20 mg/BW in saline). After visualization of anterogradely labeled processes with a dark blue chromogen. Vibratome sections were immunostained for fluorogold. As fluorogold labeling resulted in dense immunopositive granules without diffuse cytoplasmic labeling, selected sections were further immunostained for cytoplasmic tyrosine hydroxylase (dopamine). Anterogradely labeled suprachiasmatic efferents were observed in the medial preoptic area, periventricular regions, and the lateral aspects of the arcuate and ventromedial nuclei of the hypothalamus, whereas the median eminence and organum vasculosum laminae terminalis lacked labeled suprachiasmatic projections. All of the aforementioned regions contained a high number of cells immunoreactive for fluorogold. However, immunolabeling for fluorogold revealed no retrogradely labeled (ergo neuroendocrine) cells in the suprachiasmatic nucleus. Retrogradely labeled cells in all of these hypothalamic sites, with the exception of the median eminence and organum vasculosum laminae terminalis, were targets of suprachiasmatic nucleus axon terminals. In the preoptic area, anterior hypothalamus, periventricular area, and arcuate nucleus, subpopulations of dopamine cells were retrogradely labeled. In all of these areas, both retrogradely labeled and nonlabeled dopamine cells were frequently found to be in contact with dark blue, anterogradely labeled suprachiasmatic efferents. Electron microscopic examination confirmed the putative connections to be synaptic. This experiment provided evidence that the circadian pacemaker suprachiasmatic nucleus sends efferents onto neuroendocrine cells, but has no contacts with fenestrated capillaries. It was found that a population of median eminence-projective cells targeted by suprachiasmatic axons in the hypothalamus contains dopamine. These observations indicate no direct effect of the circadian pacemaker on the anterior hypophysis, but offer an indirect pathway via circadian signals, mediated by hypothalamic neural systems, that may regulate pituitary hormone secretion, in particular PRL.

Expression of Phaseolus vulgaris leukoagglutinin-binding oligosaccharides in oral squamous cell carcinoma: possible association with the metastatic potential.

The expression of -GlcNAc beta 1-6Man-(beta 1-6) branched oligosaccharides in carcinoma cells has been considered to influence their metastatic potentials. In the present paper, the lectin histochemistry of oral squamous cell carcinomas obtained in biopsy from 34 patients with Phaseolus vulgaris leukoagglutinin (L-PHA), which potentially binds to N-glycosidic carbohydrates with beta 1-6 linked lactosamin antennae, was studied in order to analyze the relationship between their staining patterns and metastases. The L-PHA-binding oligosaccharides of the carcinomas were expressed on the cell surface in the following patterns: (i) all cells were positive for the staining ('positive'); (ii) some cells were positive but the rest of the carcinoma cells were negative ('weakly positive'); and (iii) all were negative ('negative'). Statistical analysis revealed that the incidence of the metastasis to regional lymph nodes in the 'positive' cases was significantly higher than that in the 'negative' cases. Moreover, the number of the CD14 positive cells including macrophages in the stroma adjacent to the carcinomas in the 'positive' cases was less than that in the 'negative' or 'weakly positive' cases. The expression of L-PHA-binding oligosaccharides in oral squamous cell carcinoma may be responsible for their metastatic potential to regional lymph nodes, possibly including their ability to escape macrophage recognition.