Nuclear Localization of Brain-type Glycogen Phosphorylase in Some Gastrointestinal Carcinoma

Our previous reports have demonstrated frequent and strong expression of glycogen phosphorylase (EC 2.4.1.1) activity mainly in the cytoplasm of gastric carcinoma. Although previous studies have suggested the phosphorylase glyco-syltransferase system to be in the nucleus from enzyme histochemical analyses, intranuclear localization of the phosphorylase has not been fully established. The aims of the present study are to investigate the nuclear localization of glycogen phosphorylase and to identify the isoform of phosphorylase in the nucleus of gastrointestinal carcinoma. The activity of glycogen phosphorylase in carcinoma cells corresponding to the nucleus was demonstrated using enzyme cytochemical analysis. The phosphorylase activity coincided with localization revealed by immunocytochemistry using affinity-purified specific anti-human brain-type glycogen phosphorylase antibody. The isoform expressed in the nuclei of carcinoma cells was identified as bei ng only the brain type according to a polymerase chain reaction-based assay using RNA obtained from gastric carcinoma cells and primers specific to muscle, liver and brain types of glycogen phosphorylase. The intranuclear localization of the brain-type isoform was confirmed by immunoelectron microscopical analyses. Further investigation to examine the nuclear localization in human carcinoma tissue (145 and 25 specimens with gastric and colonic carcinoma respectively) was carried out by immunohistochemistry using specific anti-brain-type antibody. Nuclear immunostaining was observed in seven cases out of 145 gastric carcinoma. The present study is the first to clarify the nuclear localization of glycogen phosphorylase with enzymatic activity in gastrointestinal carcinoma. The isoform of the enzyme expressed in the carcinoma was identified as the brain type. These results warrant further studies on the mechanisms for transporting the large molecule of brain-type glycogen phosphorylase to nuclei and its function in the nucleus of carcinoma cells.     

Phe-X-Pro-Arg-Leu-NH(2) peptide producing cells in the central nervous system of the silkworm,Bombyx mori

Members of the neuropeptide family having Phe-X-Pro-Arg-Leu-NH(2) (FXPRLamide; X=Ser, Thr, Val, or Gly) at the C-terminus serve as regulators of oviduct and visceral muscle contraction, sex pheromone production, and diapause induction. Antibody raised against Bombyx mori diapause hormone recognized a variety of FXPRLamide peptides. Using this antibody, the antigen was immunocytochemically localized in the central nervous system (CNS) of the silkworm, Bombyx mori. Immunoreactive somata were observed in all ganglia of the CNS including the brain. Twelve somata localized at the midline of the suboesophageal ganglion (SG) were most intensely stained, and their neurite projections reached the retrocerebral complex. Thus, these cells in the SG exhibited typical features of neuroendocrine neurons. Marked reduction in immunoreactivity was observed in a pair of neurosecretory cells in the labial neuromere in SG of diapause type pupae, which indicates an active release of FXPRLamide peptides from these cells. No clear connection to neurohemal sites were observed in immunoreactive cells in the brain, thoracic or abdominal ganglia, suggesting that the immunoreactive peptides in these organs are likely to serve as neurotransmitters or neuromodulators.     

Ribosomal proteins in the cyanobacterium Anabaena variabilis strain M3: presence of L25 protein

Ribosomal proteins from a cyanobacterium Anabaena variabilis were analyzed by two-dimensional gel electrophoresis. We detected 21 protein spots of the small subunit and 29 protein spots of the large subunit. One of the spots was identified as L25 protein, which suggests that the reading frame sll1824 of Synechocystis is the L25 protein.     

Measurement of early pregnancy factor activity for monitoring the viability of the equine embryo

The viability of embryos before flushing from donor mares (n = 5) and after transfer to recipient mares (n = 7) was monitored in mare serum by detecting early pregnancy factor (EPF) using the rosette inhibition test (RIT). The EPF activity was measured in donor mares before and after natural mating at natural estrus; after ovulation on Days 2, 5 and 8; and after embryo flushing (Day 8) on Days 8, 9, 10 and 13 after ovulation. The collected embryos were transferred immediately after flushing. The EPF activity in recipient mares were measured on the day of transfer and after embryo transfer on Days 1, 2, 3 and 5. Pregnancy was confirmed on Day 12 to 14 after embryo transfer. The mean EPF activity of donor mares was increased to the pregnant level (> an RI titer score of 10) on Day 2 after ovulation. Two days after flushing the embryos, the EPF activity of donor mares had decreased to the nonpregnant level. Among the 7 recipient mares, 3 mares were diagnosed pregnant on Day 12 after embryo transfer with ultrasound. The EPF activity of the pregnant recipient mares was increased above the minimum level observed in pregnant mares on Days 2 to 3 after transfer. However, among the nonpregnant recipient mares after embryo transfer, the EPF activity of 3 mares remained at the pregnant level only 2 to 3 d and then declined to the nonpregnant level. In one recipient mare, EPF activity did not reach the pregnant level throughout the sample collection. The results of this study indicated that equine EPF can be detected in serum of pregnant mares as early as Day 2 after ovulation. From our observation, we conclude that the measurement of EPF activity is useful for monitoring the in vivo viability of equine embryos and early detection of embryonic death.     

Presenilin 1 Mutations Linked to Familial Alzheimer's Disease Increase the Intracellular Levels of Amyloid β-Protein 1–42 and Its N-Terminally Truncated Variant(s) Which Are Generated at Distinct Sites

Abstract: Mutations in the presenilin genes PS1 and PS2 cause the most common form of early-onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid β-protein (Aβ) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuro-blastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1 (M146L or Δexon 10). The induction of mutated PS1 increased the intracellular levels of two distinct Aβ species ending at residue 42 that were likely to be Aβ1–42 and its N-terminally truncated variant(s) Aβx-42. The induction of mutated PS1 resulted in a higher level of intracellular Aβ1–42 than of intracellular Aβx-42, whereas extracellular levels of Aβ1–42 and Aβx-42 were increased proportionally. In addition, the intracellular generation of these Aβ42 species in wt and mutated PS1 -induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular Aβx-42 versus inhibition of intracellular Aβ1–42 generation. These data strongly suggest that Aβx-42 is generated in a proximal Golgi, whereas Aβ1–42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific γ-secretase cleavage that occurs in the normal β-amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to β-secretase cleavage or (b) in the distinct sites where Aβx-42 and Aβ1–42 are generated.     

Class II MHC-expressing cells in the rat adrenal gland defined by monoclonal antibodies

 The detailed distribution and heterogeneity of various immunocompetent cells were characterized in the normal adrenal gland of the rat, with special emphasis on major histocompatibility complex (MHC) class II-expressing cells and macrophages. All adrenals contained at least two different populations of cells reactive with the dendritic cell or the macrophage antibodies. These cells were clearly distinguished from adrenal parenchymal cells by their morphology and location. The majority of dendritic cells were immunoreactive for the MHC class II (Ia) antigen (MRC OX6) and/or the dendritic cell antibodies (MRC OX62), and negative for the macrophage antibodies (ED1, ED2, and/or MRC OX42), whereas the main population of macrophages was immunonegative for the former antibodies and positive for the latter. The OX62-positive cells and the OX42-labeled cells occurred exclusively throughout the medulla. The cellular density of dendritic cells in the adrenal cortex was significantly higher than that of macrophages. Double-immunoperoxidase staining for ED1 and OX6 revealed that positively stained cells could be classified into the following categories: ED1⁺OX6⁺, ED1⁺OX6⁻, and ED1⁻OX6⁺. More then 40% of OX6⁺ cells were immunoreactive for ED1 in the zona glomerulosa, while approximately 15%, 20%, and 30% of OX6⁺ cells were positive for ED1 in the zona fasciculata, zona reticularis and medulla, respectively. ED1⁺ED2⁻ cells were more frequently detected in the zona glomerulosa than in other adrenal zones. Only a few ED1⁻ED2⁺ cells were located in the zona glomerulosa, whereas a large number of them were found in the zona fasciculata. In the zona reticularis and medulla, ED1⁺ED2⁺, ED1⁺ED2⁻, and ED1⁻ED2⁺ cells were detected in the ratio 2:1:3. Our rsults suggest that dendritic cells and macrophages mature during their migration within the adrenal gland. These immunocompetent cells may contribute to a paracrine regulation of adrenal function under physiological conditions. Accepted: 3 November 1997