Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

HLA-DQ system and insulin-dependent diabetes mellitus in Japanese: does it contribute to the development of IDDM as it does in Caucasians?

Fifty-six unrelated Japanese patients with insulin-dependent diabetes mellitus (IDDM) were HLA-typed, and restriction fragment length polymorphism (RFLP) analysis was performed after enzyme digestion with Bam HI and Taq I by using both DR and DQ probes. As previously reported, increased frequencies of Bw54, Cw1, DR4, and DRw53, which are in strong linkage disequilibrium in the Japanese population and make the characteristic Japanese haplotype, were confirmed. DQw4, a new allele of the DQ system recognized by the monoclonal antibody HU-46 and in linkage disequilibrium with this haplotype, presented the highest IDDM association. The RFLP analysis also showed the strongest correlation to IDDM when the DQ probe was applied. These results indicate that HLA-DQ might play the most important role in the development of IDDM in Japanese as well as in Caucasians. The correlation of DQ amino acid sequences strongly associated with IDDM in Japanese are discussed in this study, and contrasting results were found when such sequences were compared with those of Caucasians.Abbreviations used in this paper IDDM insulin-dependent diabetes mellitus - RFLP restriction fragment length polymorphism - Asp aspartic acid - Asp-57 aspartic acid at the 57th residue of the DQ chain - non-Asp-57 nonaspartic acid at the 57th residue of the DQ chain - R.R. relative risk of Woolf and Haldane     

Unique rearrangement of ergocalciferol side chain in vitro: production of a biologically highly active homologue of 1,25-dihydroxyvitamin D3

In vitro incubation of 24-epi-25-hydroxyvitamin D2 with chicken kidney homogenate produced several compounds, one of which had an affinity equal to that of 1,25-dihydroxyvitamin D2 for the chick intestinal receptor. The affinity of 24-epi-1,25-dihydroxyvitamin D2 for the same receptor was found to be half that of 1,25-dihydroxyvitamin D2. The unknown compound was produced only when homogenate was prepared from pooled kidneys taken from both vitamin D deficient and replete chickens. The compound has been tentatively identified as 1,25-dihydroxy-22-dehydro-26-homovitamin D3 by ultraviolet absorption spectrophotometry and mass spectrometry. Chemical synthesis of 1,25-dihydroxy-22-dehydro-26-homovitamin D3 provided additional evidence for the structure. Administration of this 26-homologue of 1,25-dihydroxyvitamin D3 at the dose level of 650 pmol/rat stimulated bone calcium mobilization in the hypocalcemic rat equal to that of 1,25-dihydroxyvitamin D3. Thus, this paper demonstrates unique methyl migration on the side chain of 24-epi-1,25-dihydroxyvitamin D3 to form a more biologically potent analogue.     

A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.     

Basic studies of cryochemotherapy in a murine tumor system

The combined effect of cryosurgery and anticancer drugs (cryochemotherapy) was studied in an experimental B16 melanoma/BDF1 tumor system. Vascular volume and vascular permeability after cryosurgery of normal skin and the tumor were measured by using 51Cr-labeled red blood cells and 125I-labeled serum albumin. The vascular volume and vascular permeability of both the normal vessels and the tumor vessels greatly increased immediately after cryosurgery, and their vascular volume decreased to less than the normal level within a few hours. However, the tumor vessels showed less dilatation and increase in permeability than the vessels of normal tissue. There was a difference in functional characteristics in response to cryoinjury between the normal vessels and the tumor vessels. The anticancer drugs, peplomycin and adriamycin, were administered intraperitoneally in combination with cryosurgery. When peplomycin was administered 5 min, 1 hr, and 3 hr after cryosurgery, the drug concentration in the frozen tumor was higher than that in the untreated tumor. But when administered 1 hr before cryosurgery, peplomycin was not trapped in the tumor. Trapping of adriamycin was not observed after the same treatment. In cryochemotherapy, it is necessary to administer the appropriate drug at the appropriate time. However, the trapping of the anticancer drug results in a high concentration and lasts for a long time, so that cryochemotherapy is expected to be a new mode of cancer therapy, particularly as a multidisciplinary treatment for cancer.     

Fibrinolysis: Its initiation and regulation

Fibrinolytic system is one of the major proteolytic pathways in vivo and primarily responsible for dissolution of thrombi. Two enzymes are primarily involved in this proteolytic system; plasminogen activator (PA) and plasmin. Plasmin is formed by a limited proteolysis of plasminogen by PA, which is mainly synthesized by and secreted from vascular endothelial cells. This proteolytic process proceeds physiologically only on the surface of fibrin. Thus, initiation and progression of the fibrinolytic process depend on the function of endothelial cells and fibrin formation. Endothelial cells may also synthesize and excrete PA inhibitor (PAI) which inhibits immediately, PA once released. The rates of synthesis and excretion of PA and PAI by endothelial cells are regulated by various factors. Among them, thrombin stimulates the release of PA whereas activated protein C may decrease the release of PAI. Thus, both enzymes enhance fibrinolytic potential. PA which has escaped from inhibition by PAI binds to fibrin. ₂-Plasmin inhibitor (₂PI) inhibits the binding of plasminogen to fibrin, thereby suppressing this fibrin-associated plasminogen activation. A part of ₂PI is cross-linked to fibrin by activated factor XIII when fibrin is formed, and the ₂PI thus cross-linked to fibrin inhibits in situ plasmin formed on fibrin. Thus, ₂PI as well as PAI plays a central role in inhibition of fibrinolysis.     

Dormancy inDioscorea: Different temperature adaptation of seeds,bulbils and subterranean organs in relation to north-south distribution

Temperature dependencies of sprouting and germination were compared for subterranean perennating organs and seeds of ten closely related species of the genusDioscorea (Dioscoreaceae), a group of monocotyledonous summer perennials which are distributed from the tropics to the subarctic. The species used wereD. nipponica Makino,D. tokoro Makino,D. japonica Thunb.,D. tenuipes Franch. et Savat.,D. septemloba Thunb.,D. quinqueloba Thunb.,D. izuensis Akahori,D. bulbifera L. f.spontanea (Makino) Makino et Nemoto,D. pentaphylla. L. andD. alata L.; they are distributed from cold northern areas to warmer southern areas approximately in this order in and around Japan. Bulbil sprouting was also studied in those forming bulbils. Subterranean organs of the tropical species sprouted faster without any prior temperature treatment, whereas those of species from the more northern areas sprouted after prechilling. Northern species required longer, periods of prechilling for sprouting. On the other hand, with seeds or bulbils, the southern species required longer periods of prior temperature treatment for dormancy breaking. This difference in the length of dormant periods between seeds or bulbils and subterranean organs among the ten species may be related to their size and position of shedding; seeds or bulbils are small and are shed on the ground surface, whereas subterranean organs are large and are located below the surface. It is important to determine in other perennials whether the above relation between dormant features of seeds or bulbils and subterranean organs are common properties or not.     

Support of embryonic chick survival by vitamin D metabolites

The provision of 1,25-dihydroxyvitamin D3 as the only source of dietary vitamin D3 to laying hens failed to support normal embryonic development in their fertile eggs. Significant (P less than .001) improvement in embryonic survival to hatching in these eggs resulted from injections of 1,25-dihydroxyvitamin D3, 24,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3, or 24,24-difluoro-25-hydroxyvitamin D3 prior to incubation. Maximum embryonic survival with lowest embryonic mortality was observed when 0.20 micrograms/egg of 1,25-dihydroxyvitamin D3 or 0.60 micrograms/egg 25-hydroxyvitamin D3 was injected. These results indicate that several forms of vitamin D, two of which cannot be converted to 24,25-dihydroxyvitamin D3, can provide this activity; and of the vitamin D compounds tested, 1,25-dihydroxyvitamin D3 may be the most active in supporting embryonic survival in the chick when delivered directly by injection.     

Nutritional effect of possible intermediates of phytosterol dealkylation in the silkworm, Bombyx mori

Several possible substrates and intermediates of phytosterol dealkylation were tested for their ability to support growth and development in the silkworm, $\text{Bombyx mori}$. These compounds were classified into “effective” (fully substitutive for cholesterol), “partially effective” (partially supporting growth) and “ineffective” These results are discussed in relation to the mechanism of phytosterol dealkylation in insects.