Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study

Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.     

Plant regeneration from isolated microspore cultures of Chinese cabbage (Brassica campestris spp. pekinensis)

Isolated microspores of Chinese cabbage (Brassica campestris ssp. pekinensis) were incubated in modified NN medium containing 10% sucrose in darkness at 33°C for one day followed by culture at 25°C. After 14 days of culture, microspores developed into embryos ranging from globular to cotyledonary stage. Plants were regenerated after transfer of embryos to medium containing 3% sucrose and no plant growth regulators.Abbreviations NN Nitsch and Nitsch - MS Murashige and Skoog - NAA naphthaleneacetic acid - BA 6-benzylaminopurine     

A mutation causing reduced biological activity and stability of thyroxine-binding globulin probably as a result of abnormal glycosylation of the molecule

T4-binding globulin (TBG), a 54-kilodalton glycoprotein, is the major thyroid hormone transport protein in man. The exact nature of the mutations causing X chromosome-linked TBG deficiency, which affect about 1 in 2,500 newborn males, is unknown. Here we report the sequence of a unique variant TBG (TBG-Gary) encoding a protein with severely impaired T4 binding as well as decreased stability at 37 C, resulting in its rapid in vivo denaturation. A single nucleotide substitution in the codon for residue 96 of the mature protein replaces isoleucine with asparagine; this replacement creates an additional site for N-linked glycosylation. The anodal shift of TBG-Gary on isoelectric focusing gel electrophoresis suggests that this new site is likely glycosylated. Since glycosylated is required for TBG to assume its correct tertiary structure, but is not subsequently necessary for maintenance of the biological properties or stability of the molecule, we believe that the likely presence of additional carbohydrate probably affects a higher order structure of the molecule and is thus responsible for the reduced stability and hormone binding activity of TBG-Gary (TBGASN-96).     

Tumor cytotoxicity of human monocyte membrane-bound interleukin-1α induced by synergistic actions of interferon-γ and synthetic acyltripeptide,FK-565

Summary Human blood monocytes were isolated by counter-flow centrifugal elutriation from healthy donors and these noncytotoxic monocytes were rendered tumoricidal to allogeneic melanoma (A375) cells by activation with a synthetic acyltripeptide (FK-565), as assessed by measuring release of [¹²⁵I]iododeoxyuridine in 72 h. When monocytes were treated with FK-565 for 16 h, and then fixed with paraformaldehyde, they showed cytotoxicity to A375 melanoma cells. The fixed-monocyte-mediated cytotoxicity to A375 cells was induced by the synergistic actions of FK-565 and recombinant interferon- (rIFN-), but not other cytokines [rIFN-A, rIFN-, tumor necrosis factor (TNF), interleukin (IL)-2, -3 and -6]. For synergistic activation of monocytes with induction of a membrane-associated antitumor monokine, the monocytes had to be incubated first with rIFN- and then with FK-565. FK-565 also acted synergistically with rIFN- to stimulate monocytes to produce membrane-associated IL-1 activity, which induced C3H/HeJ thymocyte blastogenesis in response to phytohemagglutinin P. The tumoricidal and thymocytestimulating activities of the fixed monocytes were almost completely inhibited by a specific anti-(IL-1) antiserum, but not by a specific anti-(IL-1) antiserum or monoclonal anti-TNF antibody. These results suggest that membrane-associated IL-1 of human blood monocytes can be induced by two activation signals (rIFN- then FK-565) at their suboptimal concentrations.Abbreviations IL interleukin - IFN interferon - TNF tumor necrosis factor     

Intravesical treatment of bladder cancer with recombinant human interferon-β

Summary In order to examine its clinical efficacy, recombinant human interferon- (rIFN-) was instilled intravesically into 51 patients with superficial bladder cancer. Ten patients, who received intermittent intravesical instillation at a dose of (3–36) × 10⁶ U rIFN- on days 1–3 every week, showed no response. Thirty-two patients received intravesical instillation at a dose of (3–36) × 10⁶ U every day for 10–20 days. Eight patients showed partial response, indicating an efficacy rate of 25%. Nine patients received divided doses of 18 × 10⁶ U twice a day every day for 10–20 days. Six patients showed partial response, indicating an efficacy rate of 67%. This value was significantly higher than that obtained by administering divided doses. The response to intravesical instillation therapy with rIFN- varies with treatment protocol. Frequent and longer exposure to rIFN- may induce better regression of superficial bladder cancer. Six incidences of side-effects were found in five cases (9.8%): pollakiuria in one, pain on micturition in two, fever in two, and eruption in one case. All of these side-effects were slight and reversible after drug withdrawal. Laboratory tests showed only a few changes with low severity. Thus, rIFN- is potentially a new drug for instillation therapy of superficial bladder cancer, in view of the absence of adverse effects.     

Antimetastatic effect of endogenous tumor necrosis factor induced by the treatment of recombinant interferon γ followed by an analogue (GLA-60) to synthetic lipid A subunit

Summary We have investigated the effect of endogenous production of tumor necrosis factor (TNF) induced by the combination of recombinant interferon (rIFN) as a primer followed by GLA-60 as a trigger (rIFN/GLA-60) on murine lung metastases caused by B16-BL6 melanoma. In order to examine the therapeutic effect of endogenous TNF on tumor metastasis, the ability of multiple administrations of rIFN/GLA-60 to induce TNF production was also tested. The multiple administrations of rIFN/GLA-60 at intervals of 2 days were effective for the induction of endogenous TNF in mice but continuous multiple administrations of them for 2–4 days were not. In tumor-bearing mice, the production of endogenous TNF by rIFN/GLA-60 was less than that of normal mice, but treatment 3 days after the surgical excision of primary tumors showed the endogenous TNF production to be similar to that in normal mice. In the experimental lung metastasis model, intravenous administration of rIFN followed by intravenous or intranasal administration of GLA-60 showed potent inhibition of lung metastases of B16-BL6 melanoma, whereas the reverse sequence of administration (GLA-60/rIFN) or administration of a mixture of rIFN and GLA-60, which cannot induce the production of TNF, caused no inhibition of lung metastases. These results indicated that the regression of tumor metastases by rIFN/GLA-60 was mediated by the production of endogenous TNF in addition to the direct effects of both immunostimulants. Furthermore, the administration of rIFN and GLA-60 significantly inhibited the tumor metastases in spontaneous lung metastasis model. These results may provide a promising approach for the treatment of cancer metastasis as a result of its ability to induce endogenous TNF.     

A mutant hemolysin with lower biological activity produced by a mutant Vibrio parahaemolyticus

Abstract A mutant toxin (m-TDH) of thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus w was isolated from the culture of a strain of this organism mutagenized with N -methyl- N '-nitro- N -nitrosoguanidine. Although the m-TDH had a molecular structure similar to the native Vp-TDH, the m-TDH retained only about 7% residual hemolytic activity of the native toxin. Furthermore, other biological activities of m-TDH, such as lethality in mice and enterotoxicity in rabbit ileal loops, were also weakened. The m-TDH was immunologically indistinguishable from the native Vp-TDH. These results suggest that the m-TDH is only slightly different in structure from the native Vp-TDH. Also, the mutagenized site in m-TDH, which is not immunogenic, seems to be involved in expressing not only hemolytic activity but also lethal and enterotoxic activity.