Mouse lymphoma L1210 cells acquire a new cystine transport activity upon adaptation in vitro

Summary Mouse lymphoma L1210 cells maintained in vitro at a high cell density for a certain time period adapted themselves to the in vitro environment and were able to grow indefinitely. From these adapted cells, more than 30 clones were isolated. They all had much higher activity to take up cystine than the original L1210 cells, supporting a previous view that the deficiency of the cystine uptake limits the survival and growth of L1210 cells in vitro. The cystine uptake of one cloned cell line was characterized. The enhanced uptake of cystine in these cells was mainly mediated by a Na⁺-independent, saturable system and was potently inhibited by glutamate and some other anionic amino acids, but less by aspartate. Such activity of cystine uptake was not observed in the original L1210 cells. The results suggest that, upon adaptation in vitro, L1210 cells acquire a new cystine transport activity necessary for survival and growth in vitro.     

Immunological Determination of Labeling Index on Human Tumor Tissue Sections Using Monoclonal Anti-Brdurd Antibody

The use of a monoclonal antibody against the thymidine analogue bromodeoxyuridine together with an in vitro labeling technique allowed rapid determination of the labeling index in human tumors. The labeling index estimated by these relatively simple immunofluorexence or immunoenzymatic staining methods was equivalent to that obtained by autoradiography. The interpretation of the preparations is easy since there is a minimum of background staining. This immunohistochemical technique combined with in vitro labeling provides a suitable alternative for determining the labeling index of human tumors.     

Effect of temperature and cell density on cellular protein content in temperature-sensitive mutants of rat 3Y1 diploid fibroblasts

Summary We examined cellular protein content in four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) under various conditions of culture that affect cell proliferation. When proliferation of the ts mutants was inhibited at a nonpermissive temperature (39.8°C) in the G₁ phase, prominent accumulation of cellular protein occurred in three mutants (3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) but not in 3Y1tsD123. The over-accumulation of protein at 39.8°C in the former three mutants was inhibited at high cell densities. At low cell densities there was an upper limit in the protein accumulation at 39.8°C. When the three mutants, proliferation-arrested at high cell densities at 33.8°C, were replated sparsely in fresh medium and shifted to 39.8°C, proliferation was completely inhibited whereas over-accumulation of protein occurred. These results indicating dissociation of protein accumulation and cell proliferation suggest that the two events are regulated by different mechanisms. This work was supported in part by a Grant-in-Aid for Encouragement of Young Scientists (1984) to K. Y. from the Ministry of Education, Science, and Culture, Japan.     

Embryonic development,larvae and juvenile of elkhorn sculpin,Alcichthys alcicornis

The eggs ofAlcichthys alcicornis were spawned in tank at the laboratory and reared for the studies of embryonic, larval and juvenile development. This species takes place entosomatic fertilization, and females spawn fertilized eggs after copulation. The eggs are demersal and adhesive, released as a clump forming a thin layer on the bottom of tank. There was no significant difference in embryonic development between this species and other oviparous teleostean species. Hatching occurred between 17 and 18 days after spawning at a mean water temperature of 8.5?C. The newly hatched larvae averaged 4.44 mm in body length (BL). The larvae attained to post-larval stage at 5.80 mm BL, and juvenile stage at 10.2 mm BL. A specific feature of the post-larvae was the appearance of three lines of the melanophores on the caudal part of fin fold. Carotenoid first appeared on the nape at 8.70 mm BL, heavily emerged beyond 12.9 mm BL, and turned up on the back also beyond 15.2 mm BL. Scales on the lateral line were completed by 18.5 mm BL. Three pairs of flaps were observed on the dorsal surface of the head at 37.0 mm BL. External features of adult specimens are almost completed by 52.0 mm BL, yet the tip of the first preopercular was not branched but remained simple.     

On the degradation of dermorphin and D-Arg2-dermorphin analogs by a soluble rat brain extract

Degradation of dermorphin, [D-Arg2]dermorphin and [D-Arg2, Gly3, Phe4]dermorphin in a soluble rat brain extract was examined. The former two heptapeptides were degraded in a similar fashion to produce corresponding N-terminal tetrapeptide as the main degradation product along with the parallel release of Tyr5, Pro6 and Ser7-NH2. Tyr-D-Arg-Phe-Gly showed a good enzymatic stability. When captopril, an angiotensin-converting enzyme inhibitor, was present in the incubation mixture, hydrolysis of the Gly4-Tyr5 bond was markedly suppressed and resulted in release of the corresponding N-terminal hexapeptide as the main degradation product. Combined use of captopril and amastatin, an aminopeptidase inhibitor, markedly suppressed the hydrolysis of these peptides. On the other hand, [D-Arg2, Gly3, Phe4]dermorphin was hydrolyzed easier than the other two heptapeptides and considerable amounts of Tyr1 and Phe4 were released after 20 hr incubation while the N-terminal tetrapeptide, Tyr-D-Arg-Gly-Phe, showed a good enzymatic stability. On the basis of these results, possible degradation pathways of these heptapeptides were discussed.     

Cell-matrix interrelation and cell-to-cell connection in the secretory ameloblast layer of kitten teeth

To investigate the cell-matrix interrelation and the structure and permeability of the junctional complexes of secretory ameloblasts, molar tooth germs from kittens were examined by means of scanning electron microscopy, routine thin sections and freeze-fracture replication. Scanning electron microscopy showed remarkably dissolved growth fronts of enamel in materials that had been fixed with glutaraldehyde and then subjected to EDTA perfusion for 10 min. By the action of EDTA, intercrystallite spaces in rod and interrod enamel were prominently widened, and their longitudinal ends of crystallites displayed irregular and extremely sparse structures. In enamel rods surrounded entirely by interrod enamel, and in enamel rods of the typical key hole shape with successive interrod enamel participation, the most striking dissolution of crystallites occurred at the boundaries between rod and interrod enamel, where broad expanses of rod-sheath spaces were observed. In thin sections, the Tomes processes of secretory ameloblasts occupying the above rods were rectangular or variations of a rectangular shape, respectively; and interameloblast spaces opened to the enamel growth fronts, which corresponded to the junction between rod and interrod enamel. In enamel rods standing in regular rows and showing the typical arcade shape, the centers of the rods were drastically dissolved and exhibited single and deep slits, whereas the boundaries between rod and interrod enamel showed no wide furrows. The Tomes processes occupying such arcade-shaped rods were typically triangular, and the interameloblast space always joined the type-1 face of process, which is responsible for enamel rod formation. Secretory ameloblast possessed two sets of junctional complexes at the proximal and distal ends of the cell body. The distal one was situated proximally to the Tomes process. Freeze-fracture replication demonstrated the functional structures of these junctions: the proximal junction was fascia occludens, and the distal one incomplete zonula occludens with many free-ending tight junctional strands and interstrand spaces or a less developed irregular junction.     

Amidase-like activity of calpain I and calpain II on substance P and its related peptides

Porcine calpains (Ca2+-dependent cysteine proteinases) I and II, which had been purified each to a homogeneous state, were found to hydrolyze specifically carboxyl-terminal amide of substance P and several other biologically active peptidyl amides. This amidase-like activity was demonstrated both by determining released ammonia and by separating products on high-performance liquid chromatography followed by amino acid analysis. The calpain-catalyzed deamidation of substance P occurred exclusively at the carboxyl-terminal amide, leaving the side-chain glutamine intact. Enkepharinamide and MSH-release inhibiting factor were scarcely deamidated. Calpains I and II showed similar specificities for these amide substances and similar profiles of inhibitions by various protease inhibitors, but distinctly different Ca2+ requirements. The specificity constants, kcat/Km, for substance P were found to be three to four orders of magnitude higher than those for the synthetic substrates.     

Activity of recombinant human alpha interferon against influenza virus infection in mice

The in vivo antiviral activity of recombinant human leukocyte hybrid interferon, HuIFN-alpha AD, was examined. Results showed that this material in highly purified form did not protect mice against a lethal dose of influenza virus, although administration of natural MuIFN-alpha/beta to mice infected with a lethal dose of influenza virus had a marked protective effect. The effect of alveolar macrophages treated with IFN on influenza virus replication was examined in vitro. The antiviral activity of alveolar macrophages treated with HuIFN-alpha AD was lower than that of MuIFN-alpha/beta. It is concluded that HuIFN-alpha AD is effective in direct inhibition of influenza virus, but not in indirect inhibition mediated by alveolar macrophages or in protection of mice from influenza virus infection.     

Purification of Ribulose 5-phosphate Kinase and Minor Polypeptides of Pyrenoid from the Green Alga Bryopsis maxima

Ribulose 5-phosphate (Ru5P) kinase (ATP:D-ribulose 5-phosphate1-phosphotrans- ferase; EC 2.7.1.19 [EC] ), an enzyme in the reductivepentose phosphate cycle, was purified from the green alga Bryopsismaxima and its activity and peptide composition were studied.The specific activity of purified Ru5P kinase was 20 µmoleRuBP formed (mg protein)^–1 min^–1 corresponding toa 490-fold purification from the supernatant of chloroplasts.The K_m values of Ru5P kinase for ATP and Ru5P were 69 µMand 330 µM, respectively. The molecular size of Ru5P kinase was estimated as 90 kDa bygel filtration and that of its polypeptide as 41 kDa by SDS-polyacrylamidegel electrophoresis. A small portion of the Ru5P kinase wasfound in a large molecular state (500 kDa) which was consideredto be an inactive form of the enzyme. Ru5P kinase activity has been reported in the pyrenoid of Eremosphaeraviridis as well as ribulose 1,5-bisphosphate carboxylase-oxygenase(RuBisCO) and ribose 5-phosphate isomerase activity (Holdsworth1971). In Bryopsis maxima, among the pyrenoid polypeptides otherthan that of RuBisCO, we found a polypeptide of 42 kDa, similarto that of Ru5P kinase in molecular size and ratio to RuBisCO.A peptide map of the 42 kDa pyrenoid polypeptide, however, showedthat it differed from that of Ru5P kinase. In conclusion, Ru5Pkinase may be not involved in the pyrenoid of this alga. (Received January 19, 1985; Accepted May 15, 1985)