Anterograde Axonal Transport of Peptidylglycine α-Amidating Monooxygenase in Rat Sciatic Nerves

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.     

Stage-specific localization of cytoskeletal actin mRNA in murine seminiferous tubules and intestinal epithelia as demonstrated by in-situ hybridization

Summary In-situ hybridization experiments have been performed using isoactin ( and )-specific riboprobes in various tissues of the rat and mouse. Distribution of the grains of actin mRNAs for both and types was similar throughout sections of the rat testis. Although both mRNAs were evenly distributed in the seminiferous tubule, extremely heavy labeling was observed in about 10% of the seminiferous tubules that could be identified as stage XII of spermatogenesis. At high magnification, grains of the mRNA were found in the cytoplasm of elongating spermatids and in the Sertoli cell cytoplasm at the adluminal side. Much higher density of the grains of mRNA was observed in the neck region of the spermatids at stage XII. Thus, the dense distribution of cytoskeletal actin mRNAs is stage-specific in the tubule during spermatogenesis in the rat. The high expression of both and actin mRNAs was also observed in the epithelial cells of the intestinal crypts.     

Studies on peptides CLVIII. Model experiments for the synthesis of open-chain unsymmetrical cystine-peptides

Treatment of a mixture of Cys(R)(O) and Cys(R) with an acid was found to generate cystine in fairly good yields, when suitable R, R, and an acid were selected. An unsymmetrical cystine peptide was prepared by treatment of a mixture of Z(OMe)-Cys(R) (0)-Ala-NH₂ (R=Acm or MBzl) and Z(OMe)-Cys(MBzl)-Gly-OBzl with TFA or 1 M TFMSA/TFA.³ Oxytocin was obtained in an excellent yield by TFA treatment of the protected peptide containing Cys(Acm)(0) and Cys(MBzl). Thus, formation of the disulfide bond was found feasible at the position of Cys(R) (0).The following abbreviations are used Boc t-butyloxycarbonyl - Z(OMe) p-methoxybenzyloxycarbonyl - MBzl p-methoxybenzyl - Acm acetamidomethyl - Bzl benzyl - Ad l-adamantyl - tBu t-butyl - TFA trifluoroacetic acid - TFMSA trifluoromethanesulfonic acid - TMSOTf trimethylsilyl trifluoromethane sulfonate     

Progression of the phenotype of transformed cells after growth stimulation of cells by a human papillomavirus type 16 gene function.

Alteration of the growth properties of the established murine fibroblast cell lines NIH 3T3 and 3Y1 was studied in monolayer cultures and in cells suspended in semisolid medium after introduction of a cloned human papillomavirus type 16 (HPV16) DNA. HPV 16 DNA stimulated both cell lines to grow beyond their saturation densities in monolayer cultures without any apparent morphological changes or tendency to pile up. These cells were also stimulated to grow in soft agar. Since essentially all the cells that received the viral gene were stimulated to grow, the growth-stimulatory activity of HPV16 appeared to be due to the direct effect of a viral gene function. The NIH 3T3 cells showed an additional change in growth properties upon prolonged incubation of dense monolayers of cells containing the HPV16 DNA; morphologically recognizable dense foci appeared at a frequency of about 10(-3). These cells, when cloned from the foci, grew more rapidly in soft agar than the parental cells and were morphologically transformed. In other words, there were two sequential steps in cell transformation induced by HPV16. Practically all the viral DNAs were present in the cells as large rearranged multimers and were integrated into host chromosomal DNA. There was no obvious difference in the state of viral DNA in the cells of the original clone or the three subclones derived from it as dense foci. There was no difference in the amount or the number of viral RNA species expressed in the cells at these two stages. The secondary changes in the growth properties of NIH 3T3 cells appear to be due to some cellular alterations.     

Alkaline Bismuth Solution as an En Bloc Stain for Formaldehyde-Glutaraldehyde Potassium Permanganate Fixed Fungal Spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Accuracy of endometrial aspiration in the diagnosis of endometrial cancer

A study was performed to evaluate the cytologic criteria for recognizing endometrial cancer and to determine the accuracy of endometrial aspiration in its detection. In addition to the conventional cytologic criteria for the diagnosis of endometrial cancer, the irregular chromatin distribution around the macronucleoli proved to be a useful criterion. Using all criteria, cytology was reported as positive in 18 of 19 patients (94.7%) with endometrial cancer. In 856 of 12,563 high-risk outpatients at Kinki University Hospital, aspiration using the Masubuchi apparatus was carried out in screening for endometrial cancer. Cancer was detected in 18 patients (2.1%), with 94.7% of the cancers detected by the cytologic screening. This result indicates that endometrial aspiration using the Masubuchi apparatus is a reliable and safe method of screening for endometrial cancer.     

A role of the B-oligomer moiety of islet-activating protein, pertussis toxin, in development of the biological effects on intact cells

Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein (Tamura, M., Nogimori, K., Murai, S., Yajima, M., Ito, K., Katada, T., Ui, M., and Ishii, S. (1982) Biochemistry 21, 5516-5522), the biggest subunit (Mr = 28,000, referred to as the A-protomer) of which catalyzes transfer of the ADP-ribose moiety of NAD to the membrane Mr = 41,000 protein. The pentamer, termed the B-oligomer, consisting of the residual subunits was the moiety of IAP that was responsible for binding to the cell surface, as revealed by competitive inhibition of the development of the IAP actions on intact rat C6 glioma cells and rat adipocytes. The binding of the B-oligomer to its receptor proteins was divalent via the constituent two dimers; it stimulated mitosis of lymphocytes and caused an insulin-like action to enhance glucose oxidation in adipocytes, just as did concanavalin A, presumably as a result of cross-linking or aggregation of the membrane proteins. The A-promoter displayed its biological action on adipocytes only when the B-oligomer had been bound to the cells. Thus, IAP is a typical A-B toxin in which the B-oligomer is first bound to the cell surface proteins to enable the A-protomer to reach to the site of its action within the cell. Diverse biological actions of pertussis toxin may be accounted for by the mitogenic action of the B-oligomer as well as ADP-ribosyltransferase activity of the A-promoter.     

Cytoprotective effect of TRK-100, a prostacyclin analogue, against chemical injuries in cultured human vascular endothelial cells

We studied the cytoprotective effect of TRK-100, a chemically stable analogue of prostacyclin (PGI2), in the cultured human endothelial cells from umbilical vein. TRK-100 (10 and 100 nM) stimulated significantly proliferation of endothelial cells but did not affect PGI2 production in endothelial cells. Exposure of cultured endothelial cells to homocysteine (2.5 mM) or glucose (50 mM) caused concentration-dependent cytotoxicity, as evidenced by a decrease in number of viable cells. When endothelial cells were treated with TRK-100 simultaneously or prior to, but not after, exposure to injury substances, decreases in viable cell were significantly suppressed. The protective effect of TRK-100 against homocysteine-induced cytotoxicity also appeared in endothelial cells treated with acetylsalicylic acid, suggesting that endogenous PGI2 did not involve in the protective effect of TRK-100.