Development of an all-in-one technology for glycan profiling targeting formalin-embedded tissue sections

An ultra-sensitive method for glycan analysis targeting small tissue sections (1.5 mm in diameter) is described as an application of a recently-established lectin microarray technology. The developed system achieved a high level of detection of a tissue section consisting of approximately 500 cells for differential profiling, where both N- and O-glycans attached to a pool of glycoproteins are subjected to multiplex analysis with 43 lectins. By using an optimized protocol for differential glycan analysis, sections of adenocarcinoma (n = 28) and normal epithelia (n = 12) of the colon were analyzed in an all-in-one manner. As a result, Wisteria floribunda agglutinin (WFA) was found to clearly differentiate cancerous from normal epithelia with P < 0.0001. The obtained results correlated well with the subsequent histochemical study using biotinylated WFA. Thus, the developed technology proved to be valid for expanding the lectin microarray applications to tissue-based glycomics, and hence, should accelerate a discovery phase of glycan-related biomarkers.     

Ubiquitin signals in the developing acrosome during spermatogenesis of rat testis: an immunoelectron microscopic study.

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4-5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6-8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9-11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.     

Activation of Phospholipase A2 by the Nociceptin Receptor Expressed in Chinese Hamster Ovary Cells

Abstract: To gain insight into the molecular mechanism for nociceptin function, functional coupling of the nociceptin receptor expressed in Chinese hamster ovary (CHO) cells with phospholipase A₂ (PLA₂) was examined. In the presence of A23187, a calcium ionophore, activation of the nociceptin receptor induced time- and dose-dependent release of arachidonate, which was abolished by pretreatment of the cells with pertussis toxin (PTX). Immunoblot analysis using anti-Ca²⁺-dependent cytosolic PLA₂ (cPLA₂) monoclonal antibody demonstrates that activation of the nociceptin receptor induces a time- and dose-dependent electrophoretic mobility shift of cPLA₂, suggesting that phosphorylation of cPLA₂ is induced by the nociceptin receptor. Pretreatment of the cells with PD98059, a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 inhibitor, or staurosporine, a potent inhibitor of serine/threonine protein kinases and tyrosine protein kinases, partially inhibited the nociceptin-induced cPLA₂ phosphorylation and arachidonate release. These results indicate that the nociceptin receptor expressed in CHO cells couples with cPLA₂ through the action of PTX-sensitive G proteins and suggest that cPLA₂ is activated by phosphorylation induced by the nociceptin receptor via mechanisms partially dependent on p44 and p42 mitogen-activated protein kinases.     

Growth and phosphate uptake of a high phosphate accumulating bacterium, Arthrobacter globiformis PAB-6 in continuous culture

A high phosphate accumulating bacterium, Arthrobacter globiformis PAB-6, was grown in a chemostat under glucose-limitation. Two different growth patterns at steady state with various dilution rates were obtained. In one case, cells having a coccus shape tended to washout at a low dilution rate, 0.2 (h(-1)). In another, cells with a rod shape grew faster and gave a good steady-state growth at a dilution rate of 0.4. Such a close relationship between growth rate and cell morphology was found both in continuous and batch cultures. The amount of phosphate uptake per cell mass was almost constant irrespective of the dilution rate, but the rate of the uptake was maximum at about the dilution rate of 0.4. A clone of PAB-6 was isolated from the continuous culture with high dilution rate and had maximum specific growth rate of 0.7 in a simple glucosesalt medium.     

A newly isolated heterotrophic bacterium,Xanthomonas sp. DY44, to oxidize hydrogen sulfide to polysulfide

Summary A newly isolated heterotrophic bacterium,Xanthomonas sp. DY44, was found to be capable of oxidizing hydrogen sulfide (H₂S). Cells made non-viable by heat treatment (120°C, 20 min) did not show H₂S oxidation. However, both cells sterilized by -rays irradiation and cell-free extract oxidized H₂S, suggesting the existence of the heat-labile intracellular enzymatic system for H₂S oxidation. AsXanthomonas sp. DY44 exhibited no autotrophic growth with H₂S in basal mineral medium, the H₂S oxidation was judged not to be a consequence of chemolithotrophic activity. Using X-rays photoelectron spectroscopy (XPS), the metabolic product of H₂S oxidation was assessed to be polysulfide.     

Folate binding in intestinal brush border membranes: evidence for the presence of two binding activities

Binding of [(3)H]folic acid by isolated rat jejunal brush border membranes (BBMs) was analyzed by chromatography on small Biogel P-30 columns. Folic acid binding to BBMs exhibited a prominent pH effect with a sharp maximum at pH 5.5 to 6.0. After acid treatment to strip the BBMs of bound folate, the membranes demonstrated a wider pH optimum (5.5 to 7.5) of folate binding and a higher binding capacity. Scatchard analysis of binding experiments performed at pH 6.0 revealed the existence of two components: one with a high affinity (kd = 12 to 25 nM) and low capacity (V(max) for non-acidified BBMs = 0.259 to 0.264 pmol/mg protein, V(max) for acidified BBMs = 0.41 to 0.71 pmol/mg protein) and the other with a low affinity (kd = 1.1 to 5.1 microM and high capacity (V(max) for non-acidified BBMs = 0.93 to 1.93 pmol/mg protein, V(max) for acidified BBMs = 4.05 to 7.69 pmol/mg protein). Phosphatidylinositol-specific phospholipase C preferentially detached the high affinity component from jejunal BBMs. Phosphatidylinositol-specific phospholipase C-released folate binding protein was precipitated by antibodies to the high-affinity folate-binding protein from rat kidney. These data suggest the existence of two different folate-binding proteins in isolated rat jejunal BBMs. The high-affinity folate-binding protein shares epitopes with the folate-binding protein in the kidney.     

Degradation of hydrogen sulfide by Xanthomonas sp. strain DY44 isolated from peat.

Xanthomonas sp. strain DY44, capable of degrading H2S, was isolated from dimethyl disulfide-acclimated peat. This bacterium removed H2S either as a single gas or in the presence of the sulfur-containing compounds methanethiol, dimethyl sulfide, and dimethyl disulfide. The maximum specific H2S removal rate, obtained in the late stationary phase, was 3.92 mmol g of dry cells-1 h-1 (6.7 x 10(-16) mol cell-1 h-1) at pH 7 and 30 degrees C through a batch experiment in a basal mineral medium. Since Xanthomonas sp. strain DY44 exhibited no autotrophic growth with H2S, the H2S removal was judged not to be a consequence of chemolithotrophic activity. By using X-ray photoelectron spectroscopy, the metabolic product of H2S oxidation was determined to be polysulfide, which has properties very similar to those of elemental sulfur. Autoclaved cells (120 degrees C, 20 min) did not show H2S degradation, but cells killed by gamma-irradiation and cell extracts both oxidized H2S, suggesting the existence of a heat-labile intracellular enzymatic system for H2S oxidation. When Xanthomonas sp. strain DY44 was inoculated into fibrous peat, this strain degraded H2S without lag time, suggesting that it will be a good candidate for maintaining high H2S removability in the treatment of exhaust gases.     

Degradation of hydrogen sulfide by Xanthomonas sp. strain DY44 isolated from peat.

Xanthomonas sp. strain DY44, capable of degrading H2S, was isolated from dimethyl disulfide-acclimated peat. This bacterium removed H2S either as a single gas or in the presence of the sulfur-containing compounds methanethiol, dimethyl sulfide, and dimethyl disulfide. The maximum specific H2S removal rate, obtained in the late stationary phase, was 3.92 mmol g of dry cells-1 h-1 (6.7 x 10(-16) mol cell-1 h-1) at pH 7 and 30 degrees C through a batch experiment in a basal mineral medium. Since Xanthomonas sp. strain DY44 exhibited no autotrophic growth with H2S, the H2S removal was judged not to be a consequence of chemolithotrophic activity. By using X-ray photoelectron spectroscopy, the metabolic product of H2S oxidation was determined to be polysulfide, which has properties very similar to those of elemental sulfur. Autoclaved cells (120 degrees C, 20 min) did not show H2S degradation, but cells killed by gamma-irradiation and cell extracts both oxidized H2S, suggesting the existence of a heat-labile intracellular enzymatic system for H2S oxidation. When Xanthomonas sp. strain DY44 was inoculated into fibrous peat, this strain degraded H2S without lag time, suggesting that it will be a good candidate for maintaining high H2S removability in the treatment of exhaust gases.