Comparative study of embryonic thermoresistance of two inbred strains of the medaka (Oryzias latipes)

Summary By using inbred strains (HO4C and HB32C) of the medaka,Oryzias latipes, the involvement of genetic factor(s) in the determination of thermoresistance of fish was investigated. The thermoresistance of embryos of the medaka was quantitated by the fraction of the embryos surviving 1 day after heat treatment. At early stages of development (st. 13 and st. 20–21), the HO4C strain was more resistant than the HB32C strain. At st. 20–21, the HO4C strain was more resistant than the HB32C strain at all temperatures used (42, 43, and 44°C). At later stages of development (st. 27 and st. 32), however, the HB32C strain was more resistant than the HO4C strain.The results of genetic cross experiments raised the following possibilities; the thermoresistance of embryos at early developmental stages can be lowered by some factor(s) inherited in the HO4C strain and/or increased by those in the HB32C strain. By contrast, the sensitivity of embryos at later stages of development was not affected by factor(s) of their parents, but by their own genetic constitution.     

Chromatin diminution and chromosome elimination in four Japanese hagfish species

Cytogenetic examination of four Japanese hagfish species belonging to the order Myxinida (Eptatretus okinoseanus, E. burgeri. Paramyxine atami, and Myxine garmani) revealed differences in chromosome number between germ cells (spermatocytes and spermatogonia) and somatic cells (liver, blood, gill, and kidney). The differences in chromosome number between spermatogonia (54, 52, 48, and 16) and somatic cells (34, 36, 34, and 14) were 20, 16, 14, and 2 in E. okinoseanus, E. burgeri, P. atami, and M. garmani, respectively. The amount of DNA in a somatic cell (2C) relative to that in a germ cell (2C) averaged 54.6% (E. okinoseanus type A), 44.9% (E. okinoseanus type B), 79.1% (E. burgeri), 60.0% (P. atami), and 70.2% (M. garmani). These results clearly indicate that chromosome elimination takes place during early cleavage in the four hagfish species of Myxinida living in Japanese waters, except in the ancestral germline cells. C-banding of metaphase chromosome preparations of germline and somatic cells from each hagfish species revealed that the C-band-positive chromatin in the ancestral somatic cells had been almost completely eliminated. Three patterns of elimination of this chromatin are discussed.     

Morphological studies of the neuromuscular mechanism shifting from sucking to biting of mice

In order to give a neuroanatomical evidence to the mechanism of shifting from sucking to biting, we investigated in prenatal, newborn and postnatal mice whether there is a time difference in the neurogenesis of the neurons relative to sucking and biting or in the histogenesis of their peripheral effector organs by the HRP labeling technique and electron microscopy. The results obtained are as follows. (1) At birth the facial motoneurons exceed the trigeminal motoneurons in cell area and development. (2) After birth, the trigeminal motoneurons grow rapidly and outstrip the growth of the facial motoneurons at the age of 6 days. (3) Thereafter, the cell area of both neuron types continues to increase gradually. (4) The initial sign of the alpha motor end plates is found in the orbicularis oris muscle innervated by the facial nerve in 17-day-old fetuses, while that of the trigeminal nerve is delayed in the masseter muscle of 18-day-old fetuses. (5) The initial sign of the muscle spindle appears with the sensory terminals in the masseter muscle of 17-day-old fetuses and the fundamental structure of the muscle spindle is formed in 4-day-old youngs. (6) Myelination of the facial nerve begins in 3-day-old youngs, while that of the trigeminal nerve becomes apparent in 4- or 5-day-old youngs. From these bases, it is obvious that the facial nerve elements related to sucking are firstly developed at birth and that the differentiation of the trigeminal nerve elements related to biting is rapidly accelerated after birth.     

Cytochrome P-450C-M/F, a new constitutive form of microsomal cytochrome P-450 in male and female rat liver with estrogen 2- and 16 alpha-hydroxylase activity

A new cytochrome P-450 isozyme, P-450C-M/F, has been purified from untreated rat liver microsomes. The purified preparation was electrophoretically homogeneous and contained 12-15 nmol of P450/mg of protein and had a minimum molecular weight of 48,500. The NH2-terminal amino acid sequence of P-450C-M/F was different from that of other P-450's. Immunoblot analysis of microsomes demonstrated that P-450C-M/F was present in the liver of untreated male as well as female rats. Treatment of rats with phenobarbital, 3-methylcholanthrene, or beta-naphthoflavone did not induce P-450C-M/F. Cytochrome P-450C-M/F exhibited little activities of 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation or hydroxylation of arylhydrocarbon, testosterone, androstenedione, and progesterone. In contrast, it was highly active in N-demethylation of ethylmorphine and benzphetamine and in 2- and 16 alpha-hydroxylation of estrogens, particularly that of estradiol. These studies establish that cytochrome P-450C-M/F is constitutively present in both male and female rats and suggest that it may be involved in the oxidative metabolism of estradiol, particularly in the formation of estriol, the uterotropic metabolite of estradiol.     

Purification of natural human interferon-gamma by antibody affinity chromatography: analysis of constituent protein species in the dimers

A simple procedure for purifying human interferon-gamma from leukocytes was established, based on monoclonal antibody affinity chromatography. The recovery of interferon activity was essentially quantitative, and the specific activity of the product was (4-12) x 10(7) international units/mg protein. SDS-polyacrylamide gel electrophoresis reproducibly revealed four components associated with interferon activity (and no other proteins): two major ones with molecular weights (MW) of 24,000-25,000 (25K) and 19,000-20,000 (20K), a minor one with MW 14,000-15,000 (15K) (these three bands were doublets), and a still less prominent one(s) with MV 40,000-48,000. Gel filtration in neutral solution indicated that all the 25K, 20K, and 15K species exist as oligomers, probably dimers. By means of experiments using a cleavable crosslinking reagent, the dimers were shown to comprise both homo-and heterodimers. Gel filtration in alkali (the condition used during purification) indicated that the molecules are largely in a monomeric state. Thus, the molecules once dissociated in alkali appear to reassociate at random upon neutralization; this process takes place without being accompanied by inactivation.     

The target minor H antigen for F1 cytotoxic T lymphocytes induced by Igh-congenic parental spleen cells is coded for by gene linked to H-2

Graft-vs-host reaction (GvHR) induced in (B10.BR X CWB)F1 (BWF1; H-2k/b, Ighb/b) by i.v. injection with CWB (H-2b, Ighb) spleen cells resulted in complete suppression of cytotoxic T lymphocyte (CTL) responsiveness of the F1 host spleen cells (GvHR-associated immunosuppression). In contrast, GvHR induced in BWF1 mice with CSW (H-2b, Ighj; Igh-congenic to CWB) spleen cells did not affect CTL responsiveness of the F1 host spleen cells at all. The BWF1 hosts undergoing the CSW-induced GvHR generated anti-CSW CTL in their spleens, and the subsequent culture of such BWF1 spleen cells with CSW stimulator cells, augmented the CTL activity. The BWF1 anti-CSW CTL lysed both Con A- and LPS-induced splenic blasts from mouse strains carrying the Ighj allele in the context of self H-2Kb. However, determination of the Igh haplotype in the serum IgG and of the susceptibility of the splenic lymphocytes to the BWF1 anti-CSW CTL on backcross mice, which carry either Ighb/j or Ighb/b in the context of H-2b/b or H-2b/k, showed clearly that Ighj and the gene coding for the target antigen for the BWF1 anti-CSW CTL segregated at ratios close to 1:1. The study in which linkage between the gene(s) coding for the target antigen for the BWF1 anti-CSW CTL and H-2 was examined on CWB X (C3H X CWB)F1 backcross mice and (B10.BR X CSW)F1 X B10 mice demonstrated that the gene, most likely a single gene, coding for the target antigen for the BWF1 anti-CSW CTL is located at 8.5 +/- 4.3 cross-over units to the right or left of the H-2 complex. We designated the minor H antigen to be recognized by the BWF1 anti-CSW CTL as H-X+, and we discuss the distinction between the H-X+ locus and the other minor H loci on chromosome 17.     

In vivo priming of mouse CTL precursors directed to product of a newly defined minor H-42 locus is under a novel control of class II MHC gene

In a previous study, we discovered a new mouse minor histocompatibility antigen encoded by a locus at 8.5 cM apart from the H-2 complex, and we have since named the locus H-42. One allele of H-42, which is named H-42a, had been elucidated, but the other alleles, which we tentatively named H-42b, have not been elucidated. In the present study, we explored MHC control on the anti-H-42a cytotoxic T lymphocyte (CTL) responsiveness in H-42b mice. In vivo immunization (i.v. injection) of H-42b mice with 5 to 30 X 10(6) spleen cells (SC) bearing allogeneic H-42a antigen but carrying H-2 complex (mouse MHC) matched with the H-42b mice failed to prime anti-H-42a CTL but induced stable and specific anti-H-42a CTL unresponsiveness, i.e., tolerance, in the H-42b recipient mice. In contrast, H-2 heterozygous H-42b F1 mice injected with SC bearing H-42a alloantigen on either of the parental H-2 haplotypes were effectively primed to generate anti-H-42a CTL. Exploration of the region or subregion in the H-2 complex of H-42a donor SC that should be compatible with H-42b recipient mice for the induction of their anti-H-42a CTL tolerance demonstrated that the compatibility at I region, most probably I-A subregion, but not at K, S, or D region, determined the induction of the tolerance. MHC class II compatible H-42a skin graft (SG) to H-42b mice, however, consistently primed the anti-H-42a CTL in the H-42b recipients. These results were discussed in several aspects, including uniqueness of MHC class II control on the CTL response to minor H-42a antigen, possibility of inactivation of responding anti-H-42a precursor CTL or helper T cells in H-42b mice by encountering the veto cells present in MHC class II-matched H-42a SC population, and significance of the present observations as a mechanism of CTL tolerance to self-components.     

Dynamic light scattering study of suspensions of purple membrane

Purple membrane from Halobacterium halobium in suspensions has been studied by quasielastic light scattering. The intensity correlation functions of polarized scattered light were measured at various K2 values (K being the magnitude of the scattering vector), and the first cumulant Gamma of the field correlation function G1(tau) was obtained by a cumulant expansion method. The apparent diffusion coefficient Gamma /K2 did not increase monotonically with K2 values and showed a distinct anomaly in an intermediate range of K. A theoretical formulation of G1(tau) for a disc and an extremely oblate ellipsoidal shell of revolution (S. Fujime and K. Kubota, Biophys. Chem. 23 (1985) 1) was applied to the analysis of the spectra, and characteristic features of experimental spectra were well reproduced. It was suggested that a strong interference effect between scattered rays on Gamma /K2 should be attributed to a slight noncircular shape of the purple membrane and that a contribution to Gamma /K2 from membrane flexibility should be taken into account. This study will provide experimental evidence of the feasibility of membrane studies by dynamic light scattering.     

Purification and characterization of an antibacterial factor from snail mucus

The antibacterial factor from the body surface of the African giant snail, Achatina fulica Férussac, was isolated by DEAE-Toyopearl 650M ion exchange chromatography. The isolated preparation exhibited highly positive antibacterial activity both for the Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus and for the Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, but it lost such activity when heated at 75 degrees C for 5 min. The antibacterial factor of the snail mucus was a glycoprotein whose molecular weight (MW) was about 160,000. It was composed of two subunits of MW 70,000-80,000.     

Structural changes of alpha2- and ovomacroglobulins

The plasma α₂-macroglobulin and its egg white homologue ovomacroglobulin were purified from several different species and their structure before and after the reaction with proteinases studied by electron microscopy. The negatively stained specimens showed either a ringlike structure or a flowerlike one before the reaction with proteinses, but their structures changed into open rectangular ones after the reaction. The translational frictional ratio f/f ₀ of human α2-macroglobulin and crocodilian ovomacroglobulin given in the literature is between 1.5 and 1.6 before and after the reaction with proteinases. The value reflects asymmetry due not to a high axial ratio, but rather to an openness of the structure resulting in a partially free draining character of the molecules. The computational method developed by Bloomfield and his co-workers based on the formalism of Kirkwood is used to calculate the frictional ratio of several models constructed from small spheres. The overall shape of the models is derived from electron micrographs. Although the degree of hydration is an unknown parameter in the calculation, reasonable agreement is obtained between the experimental values of f/f ₀ and the calculated ones. Combination of electron microscopic and hydrodynamic methods would be fruitful in the structural study of giant proteins such as α₂-macroglobulin.