Slalom chromatography: size-dependent separation of DNA molecules by a hydrodynamic phenomenon

Slalom chromatography, a size-dependent DNA fractionation method based on a new principle [Hirabayashi, J., & Kasai, K. (1989) Anal. Biochem. 178, 336-341], was systematically studied in detail. In this method, larger DNA fragments are eluted much later than smaller ones from columns packed with spherical microbeads. Elution of a series of DNA fragments was systematically examined by using columns packed with polymer-based packings of different diameter and different pore size for high-performance gel permeation chromatography. Packings of smaller diameter proved to be superior for resolving the smaller size range of DNA, while the reverse was the case for larger DNAs. Application of a faster flow rate led to larger retardation of every DNA fragment, while at the lowest flow rate applied (0.067 cm/min), all the fragments were eluted almost at the void volume. When the column temperature was lowered, retardation of DNA became larger. On the other hand, differences in the chemical nature and the pore size of packings, or in the hydrophobicity of the eluting solvent, had little effect on DNA retardation. Size-dependent fractionation of DNA was also achieved even on columns packed with nonporous packings having anionic groups (cation exchangers). In conclusion, these results confirmed the previous conclusion that slalom chromatography is not based on an adsorption or equilibrium phenomenon but should be attributed to a hydrodynamic phenomenon.     

Selective Degradation of Specific Components of Fertilization Coat and Differentiation of Hatching Gland Cells during the Two Phase Hatching of Bufo japonicus Embryos

The embryonic hatching process in the toad, Bufo japonicus , consists of two phases: rupture of the outer jelly strings at stage 20 (neural tube) and an escape from the inner jelly layers and fertilization coat (FC) of individual embryos at stage 23 (tailbud). SDS-PAGE analyses of FCs revealed that, of the eight major protein bands, two components with 58 K and 62 K in molecular weight gradually decreased from stage 18–19 on and totally disappeared at stage 22. When the FCs were treated with a hatching medium prepared by culturing denuded prehatching embryos, both 58 K and 62 K components of the FCs were solubilized, and in the solubilized materials 18 K and 31 K components appeared. Electron microscopy showed that a meshwork of filament bundles present in the FCs before stage 17 became dissociated at stage 19–20, and completely disappeared at stage 23, just before the hatching of embryos. Hatching gland cells (HGCs), an epidermal cell with numerous secretory granules, were first identified at stage 19, and underwent active secretion of the granules during stage 19–23. These results indicate that the hydrolytic degradation of 58K and 62 K components in FCs effected by the hatching enzyme constitutes the basic mechanism of embryonic hatching during both the first and second phases.     

Expression in Escherichia coli of chemically synthesized gene for a novel opiate peptide alpha-neo-endorphin

Chemically synthesized alpha-neo-endorphin gene was fused to the Escherichia coli beta-galactosidase gene on the plasmid pKO13. The resulting recombinant DNA was used to transform E. coli cells. Radioimmunoassay for alpha-neo-endorphin in CNBr-treated bacterial cells showed that alpha-neo-endorphin was synthesized at approximately 5 x 10(5) molecules per single E. coli cell. One of the transformants, WA802/p alpha NE2, was used for alpha-neo-endorphin purification. From 10.9 g of wet cells, we isolated 4 mg of chemically pure and biologically active alpha-neo-endorphin.     

Acrosome reaction in sperm of the toad,bufo bufo japonicus

The acrosome in the sperm of the toad, Bufo bufo japonicus, consists of a membrane-limited acrosomal cap and a fibrous perforatorium. When sperm are incubated with the oviducal pars recta extract (PRE) for 30–60 min, the outer acrosomal membrane fuses with the overlying plasma membrane at several points with concomitant loss of the contents of the acrosomal cap. The inner acrosomal membrane thus exposed fuses with the plasma membrane at the caudal end of the acrosomal region. This PRE-induced acrosome reaction is completely inhibited by soybean trypsin inhibitor. Sperm found in the innermost jelly layer of inseminated eggs possess an intact acrosome, but those either passing through the vitelline coat or localizing in the perivitelline space are acrosome-reacted in the same manner as when treated with PRE. These observations, combined with recent evidence showing involvement of the pars recta substance in fertilization, indicate that the acrosome reaction occurring in a fertilizing sperm at or near the surface of the vitelline coat is a response to a substance that is derived from the pars recta and deposited in the vitelline coat.     

Evidence for a dual effect of dibutyryl cyclic AMP on the synthesis of tyrosine aminotransferase in rat liver

A single injection of dibutyryl cyclic AMP (Bt2cAMP) into adrenalectomized rats results in rapid and proportionate increases in hepatic tyrosine aminotransferase catalytic activity and in the amount of functional mRNA coding for this enzyme. This effect is transient in that mRNATAT peaks at 0.065% of total poly(A)+RNA activity at 1 h and is back to the basal level of 0.012% in 2.5 h. Enzyme activity peaks at 2.5 h and is back to the basal level by 5 h. If Bt2cAMP is repeatedly injected (0, 1, 2.5, and 4 h), enzyme activity remains at maximal levels for 4 to 5 h, whereas changes in mRNATAT activity are identical with those observed in the single injected rats. The rate of tyrosine aminotransferase synthesis at 5.5 h in the multiply injected rats, a time when mRNATAT has already returned to the basal level, is 3 to 4 times greater than that in either control or singly injected rats at the same time (0.3% of total protein versus 0.07%) and is equivalent to the maximal rate seen 1 h after the initial injection of the cyclic nucleotide. Since the rate of synthesis is increased in proportion to the increase in enzyme catalytic activity, stabilization of the enzyme against degradation is excluded as an induction mechanism at this late time point. These responses are not due to differences in the metabolism of Bt2cAMP, and the effect depends on the presence of metabolically active derivatives of this nucleotide. It thus appears that Bt2cAMP induces the synthesis of tyrosine aminotransferase in rat liver in two distinct ways. One is pretranslational and involves a transient and rapid increase in mRNATAT activity. The second appears to involve a delayed but sustained increase in translation of a basal level of mRNATAT.     

Factors Contributing to the Poor Myelination in the Brain of the Snell Dwarf Mouse

Abstract: Conventional histological examination of the pituitary does not distinguish Snell dwarf mutants (dw/dw) from their normal littermates (+/?) in the neonatal stage. However, immunohistochemical examination of pituitaries of litters born to heterozygous Snell parents revealed that in approximately 25% of the glands examined, the number of positive cells was very low in the neonatal stage. We attempted to delineate the events resulting in the poor myelination in the brain of the Snell dwarf mouse, and to devise an immunohistochemical method for identifying the mutant neonate. Differences in the brain weights of the dw/dw and +/? mice first became apparent on the 10th day of age, and from this time on no further increase in the weight of the dwarf mouse brain was recorded. Increase in CNPase activity was found to be suppressed in the cerebrum and brain stem throughout the developmental stage, but not in the other parts of the brain. The yield of isolated myelin decreased by 58% in the mutant mouse, but CNPase activity was equivalent to that of control myelin. Differences in DNA content per cerebrum from the dw/dw and +/? mice first became apparent on the 10th day of age. Henceforth, the dw/dw mice showed no further increase, although the +/? mice continued to increase. [³H]Thymidine incorporation into the DNA fraction in vivo on the 7th day of age, when glial cell proliferation in the cerebrum is most active, was suppressed to about 50% of the control level in all parts of the dwarf brain. These findings indicate that the poor myelination found in the mutant cerebrum is a hypomyelination due to reduced oligodendroglial proliferation caused by lack of circulating growth hormone.     

Co-operative response of chemically excitable membrane III. Three-state model

The general three-state model is formulated first, which is the direct extension of the unified two-state model previously formulated (Kijima & Kijima, 1978). In this model, each protomer in a symmetrically interacting system (oligomers or lattices) can take three states, S, R and Q, where S and R states are the same as in the two-state model and Q state is another state either corresponding to a different open-state of ionophore from R open-state or corresponding to another closed state of ionophore. The model has no restriction on the value of Hill coefficient at the midpoint of the dose-response curves in contrast to two-state models. It is applied on GABA sensitive inhibitory synapse of crayfish muscle to account for anomalous behaviour of the membrane in I^? solution.The simplified versions of the above general three-state model are also formulated (simplified three-state model), in which it is assumed that R and Q state are equivalent in regard to the nearest neighbor interaction. By this assumption, R and Q state are collectively treated as state A and mathematical formula obtained on Ising model are applicable on this model. This model is applied on the insect sugar receptor which was shown to be incompatible with the two-state models (Kijima & Kijima, 1980). Further simplification of the above simplified model results in two convenient models: three-state KNF model and three-state MWC model, which have minimum parameters but sufficient to account for most experiments. They give plausible physico-chemical base on the “classical model” in which the existence of both inactive and active ligand-receptor complex is assumed.     

Inhibition of thyroxine-induced metamorphosis by prolactin in tadpoles, Rana catesbeiana.

Thyroxine (T4)-prolactin interactions on hepatic arginase and ornithine transcarbamylase (OTC) as well as hind legs, tail, digestive tract and median eminence were investigated in tadpoles, Rana catesbeiana. Prolactin completely blocked T4-induced tail resorption, but failed to suppress hind-leg growth, shortening of digestive tract and promotion by T4 of the median eminence development. Prolactin blocked T4-induced increase in hepatic arginase activity but not in hepatic OTC activity. A possibility that T4 and prolactin are regulating the hepatic arginase indirectly is discussed.     

Correlation between circadian rhythms of polyamine synthesis and cell proliferation in rat liver.

In rats fed ad libitum, a marked circadian rhythm with a peak at night was observed in the hepatic level of ornithine decarboxylase (ODC) [EC 4.1.1.17], the enzyme for the first step of polyamine synthesis. A similar rhythm was found in the hepatic content of putrescine, but not of spermidine or spermine. The mitotic activity of the liver also exhibited a clear rhythm with a peak in the daytime. The rhythms of both ODC and mitosis were generated by cyclic ingestion of proteinous food, since the peaks shifted when rats were meal-fed and both activities disappeared on starvation or protein deprivation. The close parallel between the rhythms suggested that synthesis of polyamine, especially that of putrescine, was a prerequisite for the rhythmic growth of liver. The dietary induction of hepatic ODC depended on the nutritive value of dietary protein; zein or gelatin was effective only when supplemented with limiting amino acids and there was a good correlation between the hepatic ODC level and the relative growth rate.     

Characterization of the WIDR: a human colon carcinoma cell line.

We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.