Kaori Ihida

Subcellular localization of N-acetylglucosaminide beta 1-4 galactosyltransferase revealed by immunoelectron microscopy.

We prepared a monoclonal antibody (MAb) against N-acetylglucosaminide beta 1----4 galactosyltransferase purified from F9 embryonal carcinoma cells. The MAb recognized the protein portion of the enzyme, since it inhibited galactosyltransferase activity, reacted with the enzyme both from F9 cells and from bovine milk, and did not exhibit anti-carbohydrate activity. Using this MAb, we studied the subcellular localization of the enzyme by immunoelectron microscopy. Intense staining was observed in trans-Golgi stacks within testicular interstitial cells and mucous neck cells, confirming the specificity of the immunological reaction. Cell surface galactosyltransferase was detected in the following regions: cultured cells such as F9 embryonal carcinoma cells, testicular interstitial cells, seminiferous tubule epithelial cells, Sertoli cells, the head of the epididymal sperm, epididymal epithelial cells, and apical surfaces of epithelial cells in the fundic gland and of intestinal goblet cells. The use of Triton X-100 intensified the cell surface immunoreactivity, and in certain cases the mode of distribution of the cell surface enzyme was different from that described in previous reports. In addition, nuclear envelopes of cultured cells were distinctly stained. The possible significance of the latter finding is discussed in relation to recent advances in nuclear localization of glycoproteins.

Cationic colloidal gold — a probe for light- and electron-microscopic characterization of acidic glycoconjugates using poly-l-lysine gold complex

SummaryCationic colloidal gold (CCG) was used to characterize acidic glycoconjugates in semithin and ultrathin sections of rat large intestine and salivary glands embedded in hydrophilic Lowicryl K4M resin. It was prepared from poly-l-lysine and 10 nm colloidal gold solution. The staining of CCG in semithin sections was amplified after photochemical silver reaction using silver acetate as a silver ion donor and examined under bright-field and epi-illumination microscopy. CCG adjusted to various pH levels was tested on various rat tissues whose histochemical characteristics with regard to acidic glycoconjugates are well known. At pH 2.5 CCG labelled tissues containing sialylated and sulphated acidic glycoconjugates such as the apical cell surface, mucous cells in the distal and proximal colon, and acinar cells of the sublingual gland. In contrast, CCG at pH 1.0 labelled tissues containing sulphated acidic glycoconjugates such as mucous cells in the upper crypt of the proximal colon and mucous cells in the whole crypt of the distal colon. This specificity of CCG was verified by the alteration of CCG staining following several types of cytochemical pretreatment. These results were further confirmed by electron microscopy. CCG staining is thus a useful postembedding procedure for the characterization of acidic glycoconjugates at both the light- and electron-microscopic levels.

Current Status of PACAP as a Regulator of Insulin Secretion in Pancreatic Isletsa

PACAP-27 and PACAP-38 as low as 10(-13) M stimulate insulin release from rat islets in a glucose-dependent manner. PACAP also glucose dependently increases cAMP and [Ca2+]i in rat islet beta cells. The [Ca2+]i and insulin secretory responses to PACAP exhibit a similar concentration-response relationship, exhibiting a peak at 10(-13) M. When the [Ca2+]i response is abolished by nitrendipine, a blocker of L-type Ca2+ channels, the insulin response is also inhibited. Insulinotropic peptides glucagon, GLP-1, and VIP also increase [Ca2+]i in beta cells, but only in the nanomolar concentration range. PACAP is 4 logs more potent that VIP, a peptide that exhibits 68% amino acid homology and shares the type II PACAP receptor with PACAP. Immunoreactivity for the type I PACAP receptor is demonstrated in rat islets. Furthermore, PACAP immunoreactivity is demonstrated in nerve fibers and islets in rat pancreas. Based on these findings, we can draw the following conclusions: (1) PACAP is localized in pancreatic nerve fibers and islets; (2) PACAP in the subpicomolar range stimulates insulin release from islets; (3) the stimulation of insulin release is mediated by the cAMP-dependent increase in [Ca2+]i in beta cells; (4) all the PACAP effects are glucose-dependent; (5) PACAP is the most potent insulinotropic hormone known, and (6) the type I PACAP receptor appears to mediate the action of PACAP in the subpicomolar range. Finally, we hypothesize that PACAP is a pancreatic peptide of both neural and islet origin and functions as an intrinsic potentiator of glucose-induced insulin secretion in pancreatic islets (FIG 6).

Subcompartment Sugar Residues of the Gastric Surface Mucous Cells Studied with Labeled Lectins.

SummaryWe examined the intracellular localization of sugar residues of the rat gastric surface mucous cells in relation to the functional polarity of the cell organellae using preembedding method with several lectins.In the surface mucous cells, the nuclear envelope and rough endoplasmic reticulum (rER) and cis cisternae of the Golgi stacks were intensely stained with Maclura pomifera (MPA), which is specific to α-Gal and GalNAc residues. In the Golgi apparatus, one or two cis side cisternae were stained with MPA and Dolichos biflorus (DBA) which is specific to terminal α-N-acetylgalactosamine residues, while the intermediate lamellae were intensely labeled with Arachis hypogaea (PNA) which is specific to Galβ 1,3 GalNAc. Cisternae of the trans Golgi region were also stained with MPA, Ricinus communis I (RCA I) which is specific to β-Gal and Limax flavus (LFA) which is specific to α-NeuAc. Immature mucous granules which are contiguous with the trans Golgi lamellae were weakly stained with RCA I, while LFA stained both immature and mature granules.The differences between each lectins reactivity in the rough endoplasmic reticulum, in each compartment of the Golgi lamellae and in the secretory granules suggest that there are compositional and structural differences between the glycoconjugates in the respective cell organellae, reflecting the various processes of glycosylation in the gastric surface mucous cells.