Reexamination of the reality or artifacts of the microtrabeculae

The polyethylene glycol (PEG) method revealed that model systems such as erythrocytes and protein solutions, which are supposed to lack structured components, exhibit lattice structures not unlike the microtrabeculae. The compactness of the lattice was dependent on the concentration of proteins. The gelated state of gelatin exhibited lattices more compact than those of the solated state at any given concentration. Comparison of images by PEG and rapid-freezing, deep-etching replica methods showed no basic differences in the ultrastructure of the intestinal epithelial cell. This indicates that the PEG method, including chemical fixation, produces little, if any, disorganization of the cytoskeleton. All of the present findings suggest that cytoplasmic protein, nonstructure-bound or structure-forming, might be present in intact cells which could form microtrabecular structures when specimens are fixed by chemical fixatives without any extractions. Therefore, the microtrabeculae should generally be regarded as a simple marker for the presence of proteinaceous macromolecules. It is also suggested that the microtrabecular lattice, as a whole, might represent a gelated state in a given compartment when another, looser lattice is simultaneously present in the same compartment, i.e., within a single cell.     

Does Tn10 transpose via the cointegrate molecule?

Summary It has been well established that Tn3 and its relatives transpose from one replicon to another by two successive reactions: formation of the cointegrate molecule and resolution from it. Whether or not the 9300 base pair tetracycline resistance transposon Tn10 transposes in the same manner as Tn3 was investigated by two methods.In the first method, 55, a lambda phage carrying Tn10 was lysogenized in an Escherichia coli strain carrying a Tn10 insertion; the phage has a deletion in attP, hence it was lysogenized in a Tn10 sequence in the E. coli chromosome by reciprocal recombination. The chromosomal structure in these lysogens is equivalent to the Tn10-mediated cointegrate molecule of lambda and the E. coli chromosomal DNA. The stability of the cointegrate molecule was examined by measuring the rate of excision of lambda from the host chromosome, and was found to be stable, especially in a Rec^- strain. Because of this stability, the cointegrate molecule should be accumulated if Tn10 transposes via the cointegrate molecule. Then, we examined the configuration of products made by transposition of Tn10 from 55 to the E. coli chromosome. The cointegrate molecule was found in products of Tn10 transposition in a Rec⁺ strain at a frequency of 5% per Tn10 transposition, but this molecule could not be found in a Rec^- strain. Since transposition of Tn10 was recA-independent, absence of the cointegrate molecule formed in a RecA^- strain strongly suggested that the cointegrate molecule is not an obligatory intermediate of transposition of Tn10.In the second method, mobilization of pACYC177 by R388 and by R388:: Tn10 was examined. The pACYC177 plasmid was mobilized by R388::Tn10 at a frequency of 10^-4 per donor but not by R388. It occurred, in most cases, by inverse transposition of R388::Tn10 to pACYC177 forming plasmids such as pACYC177::IS10-R388-IS10. Mobilization of pACYC177 by a Tn10-mediated cointegrate in the form of pACYC177::Tn10-R388-Tn10 was not observed in crosses using a Rec^- donor. These observations also suggested that transposition of Tn10 in Rec^- cells does not occur via the cointegrate molecule.     

Binding of cobalamin analogs to intrinsic factor-cobalamin receptor and its prevention by R binder

It is now known that nonphysiological cobalamin analogs exist in the gastrointestinal tract, but their metabolic behavior is unclear. In this study, [57Co]cobinamide was used to study its affinity to hog intrinsic factor-cobalamin (IF-Cbl) receptor which has no species specificity against human IF-Cbl receptor, and its relation to human saliva R binder. Cobinamide was prepared from [57Co]cyanocobalamin and separated by paper chromatography. Human IF-Cbl complex was bound to IF-Cbl receptor but free cyanocobalamin was not. Although R binder-cobinamide was not bound to the IF-Cbl receptor, free cobinamide was bound to the IF-Cbl receptor to a significant extent (about one-half of IF-cyanocobalamin binding to the IF-Cbl receptor). We then investigated the binding of cobinamide to R binder and trypsin-treated R binder. Association constant of cobinamide binding to the IF-Cbl receptor was 1.0 X 10(9) M-1 which was much lower than that of cobinamide binding to trypsin-treated R binder and to untreated R binder. Further study indicated that cobinamide binding to the IF-Cbl receptor was blocked by the addition of R binder and also by trypsin-treated R binder. We conclude that one of the roles of R binder is to prevent binding of free cobalamin analogs to the IF-Cbl receptor in the gut.     

Characterization of newly isolated monoclonal antibodies against MHC of a Japanese wild mouse

We have already developed nine B10.MOL congenic strains carrying H-2 haplotypes derived from Japanese wild mice, Mus musculus molossinus, with the C57BL/10 genetic background. To obtain monoclonal antibodies against the H-2 antigen of the Japanese wild mouse, we carried out cell fusion using spleen cells from the animal immunized with one of the B10.MOL strains, B10.MOL-SGR (H-2 ^wm7). As a result, 19 hybridomas producing monoclonal antibodies were produced. Analysis with the intro-H-2 recombinants derived from B10.MOL-SGR indicated that 8 of them reacted with the class I and II with the class II molecule. The class I antibodies were tested for their cross -reactivities on wild mice and on the panels of standard inbred and B10.MOL strains. Most of the antibodies reacted with both the Japanese wild mice and the other subspecies, including standard inbred, while two antibodies highly specific for the donor H-2K region reacted with only three wild-derived mice, two M. m. molossinus from Anj o and Shizuoka, Japan, and one M. m. domesticus from Pigeon, Canada. In addition, all of the other four antibodies reactive with the K antigen of B10.MOL-SGR also reacted with the same three wild mice. The wild mice belonging to different subspecies might share very similar H-2K antigenic determinants in spite of their genetic and geographical remoteness.     

Behavior of Japanese monkey (Macaca fuscata) mothers and neonates at parturition

Parturitional behavior in 12 caged Macaca fuscatawas analyzed. Wild-caught mothers showed adequate maternal behaviors immediately following the neonate’s expulsion. Parity differences existed in the behaviors; primiparae were more idiosyncratic than were multiparae. Among multiparae, those with two or more offspring were uniformly adequate, but those with a single birth experience varied in the adequacy of the maternal care they provided at parturition. Mothers embraced and licked their neonates and had ventroventral contact with them frequently immediately after parturition but decreased these behaviors after expulsion of the placenta. In contrast, mothers showed allogrooming after consuming the placenta. Placentophagy was correlated with the level of orality represented by maternal licking behaviors. An isolation-reared primipara reacted to her newborn in a basically negative manner, although she showed little actual aggression. She showed a rapid shift in her negative behavior during the immediate postpartum period. This mother’s newborn sought contact with her, indicating the neonate’s active role in establishing a stable mother-neonate bond.     

Crystallographic studies of the chicken gizzard G-actin X DNase I complex at 5A resolution

The structure of the chicken gizzard G-actin X DNase I complex has been determined at 5 A resolution by an X-ray diffraction method. Protein phases were computed by the multiple isomorphous replacement method using four heavy atom derivatives. The mean figure of merit was 0.65. Dimensions of the three molecular species, the complex, G-actin and DNase I, were determined based on the "cypress wood" models derived from the electron density map. The natures of the heavy atom binding sites are discussed in relation to the distinction between the two component molecules. The pattern of successive contacts between actin molecules observed in the present crystal seems unrelated to that found in F-actin.     

Biosynthesis of the common precursor to vasopressin and neurophysin in vitro in transplantable human oat cell carcinoma of the lung with ectopic vasopressin production

Transplantable human oat cell carcinoma cells of the lung with ectopic vasopressin production were incubated with labeled amino acids and immunoreactive neurophysins in cell extracts were analyzed by isoelectric focusing. When the cells were incubated with L-(35S)-cysteine for 20 h, one major peak (isoelectric point; pI=5.3) and several minor peaks (pI=6.1, 5.7, 5.1, 4.9 and 4.7) of labeled proteins were observed. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the relative molecular mass (Mr) of the pI 5.7 protein was estimated to be 20,000 and that of the pI 6.1 species to be 19,000, while the remainder had a Mr of approximately 10,000. The result of the pulse-labeling experiment has clearly shown that the pI 5.7 and 6.1 proteins, which have affinity for concanavalin A, are biosynthetic precursors for the smaller form of neurophysin with a pI 5.3. When subjected to limited proteolysis with trypsin, the pI 5.7 protein generated a Mr 10,000 protein and a smaller peptide. The Mr 10,000 protein thus produced was identified as neurophysin on the basis of its pH-dependent affinity for vasopressin and the migration pattern on isoelectric focusing. The smaller peptide coeluted with synthetic arginine vasopressin and bound to neurophysin suggesting that it possesses a cysteine-tyrosyl sequence at its N-terminus. Similarly, the pI 6.1 protein liberated neurophysin and vasopressin-like peptide after incubation with trypsin. These results suggests that the glycosylated protein with a pI of 5.7 and a Mr of 20,000 is the common precursor to vasopressin and neurophysin in human oat cell carcinoma of the lung with ectopic vasopressin production. The pI 6.1 protein may be an intermediate in the conversion of the precursor to vasopressin and neurophysin.     

Immunological specificity and biological action of biliary glucagon-like substance in the rabbit

A glucagon-like substance named biliary IRG2000 whose molecular weight is approximately 2,000 was isolated by gel filtration from rabbit bile. This substance showed a strong crossreactivity as equivalent to 25.7 +/- 5.1 ng/ml of porcine glucagon in RIA with antiserum 30K specificity. Biliary IRG2000 brought about a significant increase and delayed the response of blood glucose level in coexistence with porcine glucagon, though it has no appreciable effect on the glucose level when administered singly to the mouse intraperitoneally. The response with the coexistence of these materials was far greater than when porcine glucagon was given alone. In Mortimore's type rat liver perfusion, a significant rise in glucose concentration in effluent was also observed when a mixture of biliary IRG2000 and porcine glucagon was perfused. The rate of 125I-glucagon degradation was found delayed in the presence of biliary IRG2000 when examined in the rat. Thus the increase and delayed response of glucose level in coexistence of porcine glucagon with biliary IRG2000 may be explained by a suppressive effect of glucagon degradation due to biliary IRG2000.     

Long-term cultivation of amphibian melanophores. In vitro ageing and spontaneous transformation to a continuous cell line

Pure melanophore populations isolated from the tail skin of the tadpole, Rana catesbeiana, were mass cultured for a period of 2-3 years. All cell lines of amphibian melanophores studied exhibited growth crisis (in vitro ageing) followed by spontaneous transformation to a continuous cell line, as shown by changes in growth characteristics in mass culture and in clone culture, by the appearance of the cells, and by measurements of cell volumes. Even after becoming a continuous cell line, amphibian melanophores continued to have a diploid chromosome number (2n = 26) in three of four cell lines examined. The chromosome mode in one cell line, however, changed to thirty. Measurement of melanin dispersion after the addition of alpha-melanocyte-stimulating hormone suggested that the mechanism for melanin dispersion in melanophores changed during in vitro ageing.     

Phase Shift in the Potassium Uptake Rhythm of the Duckweed Lemna gibba G3 Caused by an Azide Pulse

A 6-hour application (6-hour pulse) of 1 millimolar azide significantly changed the phase of the potassium uptake rhythm of Lemna gibba G3. The phase response curve obtained was type 0 and very similar to that caused by a 6-hour pulse of low temperature (5°C) or darkness. The magnitude of the phase shift and the type of the phase response curve depended on the concentration of azide. However, 6-hour pulses of 3 millimolar cyanide or 10 micromolar (3-(3,4-dichlorophenyl)-1,1-dimethylurea) failed to shift the phase of the rhythm, while these pulses lowered the rate of carbon dioxide uptake or release. Azide, even at 3 micromolar, selectively reduced the amplitude of the rhythm without inhibiting the mean level of potassium uptake.