In Vitro Binding of Wheat-Germ Proteins to the 5'-Upstream Region of the rolC Gene of Ri Plasmid

In vitro binding of nuclear proteins from wheat germ to the5'-upstream region of the rolC gene of Ri plasmid was investigated.The specific DNA sequences interacting with proteins were detectedby DNase I footprinting. (Received October 8, 1990; Accepted November 30, 1990)     

Identification and genetic analysis of semidwarfism-related proteins in rice (Oryza sativa L.)

Summary By transferring a semidwarf gene (sd-1) from Taichung Native 1 into a tall Japanese cultivar, Norin 29, through seven backcrosses, a semidwarf near-isogenic line SC-TN1 was obtained. The proteins of the embryo in Norin 29 and SC-TN1 were separated by two-dimensional electrophoresis. Most of the proteins showed the same electrophoretic pattern. However, it was found that there was a difference in the appearance of two basic glycoproteins designated as SRP-1 and SRP-2. These proteins exhibited the same molecular mass, but different isoelectric points. Hybridization results indicated that a single locus controls SRP-1 and SRP-2 with codominant alleles. The gene symbol Srp was given to this locus, with alleles Srp-1 and Srp-2 responsible for SRP-1 and SRP-2, respectively. Srp-2 was found in all of the semidwarf cultivars and lines having sd-1, except a tall cultivar Tsaiyuan-chung. This finding suggests that Srp-2 may be closely linked with sd-1. The amounts of these proteins markedly increased after water absorption of the seed, suggesting that these proteins may be related to the early development of the plant.     

Morphological effects of cadmium on proximal tubular cells in rats

Twenty-four male rats of the Wistar strain divided into four groups were injected sc with a dose of 0.8, 1.5, and 3.0 mg Cd/kg body wt as CdCl₂ in saline, and saline alone to the control rats, three times a week for 3 wk. Cadmium levels of whole kidney homogenate, supernatant (cytosol), precipitate, and metallothionein (MT) fraction were measured. Histological changes of the renal proximal tubules were investigated by optical and electron microscopy. In the kidneys, Cd levels were increased with the increment of Cd dosage; 80–90% of Cd was contained in cytosol, and 55–75% was in MT fraction. Non-MT-Cd reached a maximum in the 1.5 mg Cd group, whereas that of the 3.0 mg Cd group showed some decline. With increasing Cd doses, the size of nuclei and nucleoli in the cells of proximal tubule showed significant enlargement and also an increase in the number of nucleoli on light microscopy. At higher doses, chromatin condensation of the tubular nuclei and vacuolar degeneration of the tubular cells were evident. On electron microscopy, perichromatin granules of the proximal tubular nuclei were increased in number, especially in the rats of Cd 0.8 mg and 1.5 mg/kg groups. As the Cd doses increased, ring-shaped nucleoli were increased in number and nucleolar segregation was observed more clearly. Moreover, in the 3.0 mg/kg Cd group, nuclear indentation and nucleoli containing compact dense granules were observed. In the cytoplasm, there was an increase of lysosomes, myelin bodies, ring-shaped mitochondria, and vesiculation; ultimate changes were degeneration and cell necrosis. The injured cells were heterogenously distributed in each nephron and this heterogeneity was attributed in the difference in Cd content and cell cycle in each cell of the nephron.     

An establishment of ELISA for murine IL-6

Summary Murine interleukin-6 (mIL-6) was expressed inEscherichia coli as human growth hormone (hGH) fusion protein. The products were cleaved by thrombin to liberate mIL-6. Monoclonal and polyclonal antibodies specific to mIL-6 were prepared by immunizing rats with mIL-6 thus obtained. ELISA for the quantitation of mIL-6 was also established, which could detect mIL-6 in a quantity as low as 2 ng/ml.     

On some freshwater harpacticois from Japan,closely related to Canthocamptus mirabilis Štěrba

Preliminary comparisons are presented, of certain diagnostic characters of four species of Canthocamptus, all the specimens examined here being collected from freshwater bodies throughout Japan. The geographical distribution of each species is also discussed.Contribution No. 69 from the Itako Hydrobiological Station, Ibaraki University.     

Mode of inhibitory action of cholecystokinin in amylase release from isolated rat pancreatic acini--inhibition of secretory process post to protein kinase C-calcium ion systems

The incubation of isolated rat pancreatic acini with low doses (1 x 10(-11)-1 x 10(-10) M) of cholecystokinin-octapeptide (CCK8) induced amylase release. This CCK8-induced amylase release has been shown to be mediated through the protein kinase C activation and the Ca2+ mobilization which are linked to the phospholipase C-mediated hydrolysis of phosphoinositides. However, the incubation of the acini with high doses (1 x 10(-9)-1 x 10(-7) M) of CCK8 reduced amylase release to the level less than that induced by the maximally effective dose (1 x 10(-10) M) of this secretagogue. Under the same conditions, the high doses of this secretagogue did not inhibit the phospholipase C-mediated hydrolysis of phosphoinositides. The stimulatory action of the maximally effective dose of CCK8 in amylase release was mimicked by the simultaneous addition of protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate (TPA) and Ca2+ ionophore A23187. A high dose (1 x 10(-7) M) of CCK8 reduced the amylase release induced by the combination of TPA and A23187. These results suggest that the high doses of CCK8 inhibit the secretory process post to the protein kinase C-Ca2+ systems and thereby reduce the amylase release induced by the maximally effective dose of CCK8 in rat pancreatic acini.     

Primary structure of non-histone protein HMG1 revealed by the nucleotide sequence

The isolation and sequencing of a cDNA clone coding for the entire sequence of pig thymus non-histone protein HMG1 are described. The sequence analysis reveals a complete 2192-nucleotide sequence with a 5'-terminal untranslated region of 11 nucleotides, 642 nucleotides of an open reading frame that encoded 214 amino acids, and a 3'-terminal untranslated region of 1539 nucleotides. The HMG1 protein, deduced from the nucleotide sequence, has a molecular weight of 24,785 and a C-terminal of a continuous run of 30 acidic amino acids, encoded by a simple repeating sequence of (GAN)30. The predicted amino acid sequence is homologous to HMG1, HMG2, and HMG-T sequences from several sources, suggesting that the protein conformation is under evolutionary constraints. Northern blot analysis reveals that another hybridizable RNA species of smaller size is present. Southern blot analyses suggest that pig genome contains several HMG1 gene equivalents.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. II. Establishment of a T cell hybrid clone constitutively producing killer-helper factor(s) (KHF) and functional analysis of released KHF

T cell hybridoma lines were constructed by fusion of Mycobacterium tuberculosis-primed and boosted BALB/c T cells with the AKR-derived T lymphoma cell line BW5147. Certain of the hybridomas prepared in this manner secreted constitutively into their culture supernatants biologically active molecules that displayed precursors of cytotoxic T cell activating properties characteristic of killer-helper factor (KHF). Cell surface analysis revealed that the hybridomas were indeed somatic cell hybrids between the two respective partner cells used for fusion. KHF properties of these hybridoma supernatants were verified by their capacity to stimulate peanut agglutinin-binding (PNA+) C3H/He thymocytes to respond in vitro to 2,4,6-trinitrophenyl(TNP)-modified syngeneic stimulator cells in conjunction with suboptimal doses (10 U/ml) of interleukin 2 (IL 2) for the generation of H-2-restricted, TNP-reactive cytotoxic T cells. The biologically active molecules secreted by a T cell hybrid clone (2Y4) were, like conventional KHF, distinct from IL 1, IL 2, or immune interferon (IFN-gamma). The partially purified KHF derived from 2Y4 cells shows activity at apparent m.w. range of 34,000 to 60,000 on gel permeation, and is relatively homogeneous with respect to isoelectric point, which was approximately 4.5 to 4.7. The partially purified 2Y4-KHF is able to augment proliferation of as well as the expression of IL 2 receptors on PNA+ thymocytes in conjunction with IL 2. Finally, addition of 2Y4-KHF on day 0, followed by the addition of IL 2 on day 2 for 7 days of culture was effective in generating potent CTL responses, whereas addition of IL 2 on day 0, followed by the addition of 2Y4-KHF on day 2 to the culture was ineffective.     

Hormonal regulation of dopa decarboxylase during a larval molt

Cuticular sclerotization in insects requires dopamine derivatives and thus the presence of dopa decarboxylase (DDC), the enzyme which converts dopa to dopamine. During the last half of the larval molt of the tobacco hornworm, Manduca sexta, beginning at 16 hr after head capsule slippage, the epidermal DDC activity increased fourfold. By contrast, allatectomized larvae which were destined to produce a melanized cuticle showed a sevenfold increase. This increase in DDC activity was prevented by infusion of 20-hydroxyecdysone (20HE) into the larva, indicating that the fall of the ecdysteroid titer is necessary for the increase. In vitro 20HE also prevented the increase in a dose-dependent manner when the epidermis was explanted at 16 hr after head capsule slippage but had less effect on epidermis explanted 3 hr later. Both 5 micrograms/ml alpha-amanitin and 100 micrograms/ml cycloheximide also prevented the increase. Application of juvenile hormone I showed that the critical period for determination of the level of the later increase in DDC activity was about 4 hr after head capsule slippage at the peak of the ecdysteroid titer. Apparently then the rise and fall of ecdysteroid regulate different aspects of DDC synthesis, the rise determining its later appearance and the fall timing this appearance.