Alternating laminated array of two types of mucin in the human gastric surface mucous layer

Summary Attempts have been made to develop a procedure for preserving and analysing the surface mucous layer of the human stomach in paraffin sections. Histologically normal gastric mucosae were obtained from 20 surgically removed stomachs. Of the different fixatives tested, Carnoy's solution gave rise to the most satisfactory results. In Haematoxylin-Eosin stained sections, the surface mucous layer appeared as a thick eosinophilic layer coating the gastric mucosal surface and measured 55.4±2.5 m in the fundus and 21.8±1.0 m in the pylorus respectively. A dual staining method consisting of galactose oxidase-cold thionine Schiff and paradoxical concanavalin A staining was applied to the surface mucous layer in order to reveal the distribution pattern of mucins secreted by two types of mucous cell in the gastric mucosa: surface mucous cells and gland mucous cells. As a result of this staining, an alternating laminated layer was visualized which consisted of the particular two types of mucin. In five cases, the surface mucous layer was examined in unfixed frozen sections. This layer was only partially preserved but revealed the same laminated structure. These results indicated that gland mucous cell mucins contribute to form the surface mucous layer.     

A metastasis-associated antigen is present on a 60 kDa glycoprotein in transitional cell carcinoma of the human urinary bladder

Summary We have previously shown that the degree of expression of Le^x-related carbohydrate epitopes, namely,Lotus tetragonolobus agglutinin (LTA) receptors, SSEA-1 and FH6, correlates with the metastatic potential of transitional cell carcinoma of the human urinary bladder. In an effort to obtain a better reagent with which to detect a metastasis-associated epitope, monoclonal antibodies were produced against LTA receptors from BOY bladder carcinoma cells. One antigen defined by such a monoclonal antibody, MM4, indeed showed better correlation with the metastatic potential of the tumour than did other carbohydrate markers. In the LTA receptors, MM4 antigen was located only on a 60 kDa glycoprotein. In extracts from primary carcinomas and lymph node metastases, the 60 kDa glycoprotein was the principal carrier of MM4 antigen. LTA receptors from these sources were composed of arrays of glycoproteins, while the 60 kDa one was invariably present. Metastasis-associated carbohydrate epitopes on the 60 kDa glycoprotein may promote metastasis by interaction with carbohydrate-recognizing proteins such as selectins on host cells.     

A study on senescence in tobacco leaf disks I. Inhibition by benzylaminopurine of decrease in protein level

The drop in protein level as an index of senescence in tobaccoleaf disks was examined in the dark in the presence of BA and/orinhibitors of protein synthesis. Cycloheximide at 10^–6–10^–5M and 10^–5 g/ml actinomycin-D accelerated the senescenceand interfered with the anti-senescence action of 10^–6M BA. Chloramphenicol (10^–8– 10^–4 M) and puromycin(10^–8–10^–4M) did not modify the senescenceor the action of BA. Cycloheximide was more effective at earlierstages of the dark culture of the disks. The rate of ¹⁴C-leucineincorporation into the protein fraction was increased by BAand decreased with senescence, and this drop was removed byBA. The incorporation was also suppressed by cycloheximide equallyin the presence and absence of BA. The drop in labeled proteinof the disks during the chasing period was retarded by not onlyBA but cycloheximide also. The conclusion reached, based onthese and relevant findings, was that BA affects the anti-senescenceaction by promoting synthesis and at the same time inhibitingdegradation of protein. (Received December 2, 1974; )     

Induction of auxin-nonrequiring tobacco calluses and its reversal by treatments with auxins

Auxin-nonrequiring calluses were induced with high frequenciesby short treatments with auxins from auxin-requring ones. Auxin-requiringcalluses T22 and XD6S2, subcultured on a medium containing 1mg/liter IAA as plant growth regulator, required quite differenttreatment for induction from auxin-nonrequiring calluses; fromcalluses T22 and XD6S2, auxin-nonrequiring calluses were inducedby treatments with low concentrations of auxins (0.01–0.1mg/liter of IAA or NAA) and high concentrations of syntheticauxins (10–100 mg/liter of NAA or 1–10 mg/literof 2,4-D), respectively. No auxin-nonrequiring calluses wereobtained when they were transferred directly to the basal medium(hormone-free medium). The transformation of auxin-requiring into auxin-nonrequiringcalluses was fully reversible by treatment with 1 mg/liter ofIAA at an early stage of subculturing on the basal medium, butnot after prologed subculturing. ¹Part XXI in the series, ‘Studies on Plant Tissue Cultures’.Part XX, see Proceedings of the IVth International FermentationSymposium: Ferment. Technol. Today 697 (1972). (Received May 29, 1973; )     

Cytodifferentiation of the adenohypophysis of the domestic fowl

Summary In the chick embryo the first membrane-bound secretory granules occur in the cytoplasm of occasional cells in the cephalic lobe of pars distalis at the 7th day of incubation. On the 8th day most of the cells in both the cephalic and caudal lobes contain secretory granules that are variable in size, form and density.On the 9th day at least two types of glandular cells are distinguishable in the cephalic and in the caudal lobes; however, these cells are not comparable with those of the adult gland. Differentiation of acidophils and basophils occurs, apparently simultaneously, in 11-day embryos.The cells of the cephalic and caudal lobes are morphologically distinct from their first appearance. Thus it is concluded that these two lobes develop independently and differently from an early stage of ontogenesis.The secretory granules are formed in the Golgi area of the hypophysial cells after the 8th day of incubation. However, secretory material may be synthesized also by a process not involving the Golgi apparatus.Nerve fibers containing granules first appear in the superficial layer of the median eminence on the 8th embryonic day and by the 12th day three types of granules and two types of clear vesicles are identifiable.The investigation reported herein was supported by grant from Japan-U.S. Cooperative Science Program of Japan Association for Science Promotion to Professor Mikami and by U.S.-Japan Cooperative Science Program Grant No. GF-33334 to Professor Farner.     

Abnormal flower formation of tobacco plants regenerated from callus cultures

The plantlets regenerated from tobacco calluses, subcultured for prolonged periods, were weak and generally could not be cultivated into mature flowering specimens. However, some of them flowered and various kinds of abnormal flowers as well as leaves were observed in all the tobacco plants bearing flowers. They were sterile at had abnormal immature pollen grains. Some of them occasionally germinated in the anther. Abnormalities were also found in the nucleus of immmature pollen grains. Such drastic floral abnormalities were not found in tobacco plants derived from seeds and callus cultures subcultured for a relatively short period under the same conditions of cultivation. Part XVIII in the series “studies on plant tissue cultures”; for Part XVII, see Chem. Pharm. Bull., in press (1972).     

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25°C. Replacement of K⁺ with Na⁺ effectively prevented the lysis by SCN⁻. K⁺ salts of NO₃⁻, Br⁻, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN⁻ than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN⁻ is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na⁺ observed here was considered to be manifested by specific interactions of Na⁺ with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.     

Examination of protein sequence homologies: I. ElevenEscherichia coli L7/L12-type ribosomal “A” protein sequences from eubacteria and chloroplast

Summary Seven complete and four partial sequences ofEscherichia coli L7/L12-type ribosomal A proteins obtained from various bacteria (E. coli, Bacillus subtilis, Micrococcus lysodeikticus, Rhodopseudomonas spheroides, Desulfovibrio vulgaris, Streptomyces griseus, Bacillus stearothermophilus, Clostridium pasteurianum, Arthrobacter glacialis, andVibrio costicola) and spinach chloroplast have been reexamined using a computer program that searches for homologous tertiary structures. Comparison matrices for the sequences show that they match the sequence ofE. coli L7 (EL7) if one assumes the insertion or deletion of certain residues at sites corresponding to residues 1, 38, 49, and 92 of EL7. That two additional insertion points are found only in the spinach chloroplast protein suggests that the chloroplast protein probably diverged from the bacterial forms. Further phylogenetic relationships among these 11 prokaryote-type A proteins are discussed with respect to average correlation coefficients computed, taking into account the existence of the gaps.     

Bacteriorhodopsin Is a Powerful Light-Driven Proton Pump

The activity of bacteriorhodopsin was investigated with Halobacterium halobium cell envelopes, which lack cytoplasmic constituents. It was found that the physiological concentration of magnesium ion greatly enhanced the light-induced pH change; under optimal conditions, the pH change of the external medium was as large as 3.5 pH units, even though the volume fraction of the envelope vesicles was as low as 0.01. This pH change is about three times larger than the largest change reported thus far. This same effect was observed with transition metal ions, but not with other alkaline divalent cations. That is, divalent cations that formed hydroxides below pH 10 were effective in enhancing the light-induced pH change. This result suggests that some divalent cations acted as buffers against a large increase in the internal pH, and that the internal pH was an important factor in determining the activity of bacteriorhodopsin. It was also shown that a high level of the proton-pump activity was maintained in a wide range of external pHs, at least between 4.5 and 9.4.     

Transmembrane Location of Retinal in Purple Membrane: Fluorescence Energy Transfer in Maximally Packed Donor-Acceptor Systems

Transmembrane location of the retinal chromophore in the purple membrane of Halobacterium halobium was investigated in three different systems in which excitation energy transfer between the chromophore and external dye molecules condensed on the membrane surfaces was observed. In system ii, the energy donor was the retinal chromophore converted in situ to a fluorescent derivative. The fluorescent membranes were embedded in solid cobalt-EDTA, which served as energy acceptors. System iii was similar to system ii, except that the acceptors were tris(2,2′-bipyridyl)ruthenium(II) complex in solid form. The positively charged ruthenium complex had a radius of 0.7 nm, whereas the cobalt complex in system ii was smaller (radius ~0.4 nm) and negatively charged. System iv was stacked sheets of native purple membrane with interspersed ruthenium complex; energy transfer from the luminescent ruthenuim complex to the native retinal chromophore was observed. The energy transfer rates in these three systems, and in two additional systems already described (Kouyama, T., K. Kinosita, Jr., and A. Ikegami, 1983, J. Mol. Biol., 165:91-107), were all consistent with a location of the retinal chromophore at a depth of 1.0 ± 0.3 nm from a surface of the purple membrane. All the analyses in the present work involved an assumption that contacts between the external dye molecules and membrane surfaces were maximal; the depth values obtained cannot be underestimates. The chromophore therefore must be outside the middle one-third of the thickness, ~4.5 nm, of the purple membrane.