De novo synthesis of peroxidase in cotton ovule culture medium

The biosynthesis of cotton ( Gossypium hirsutum L. 'Stoneville 208') peroxidase (EC 1.11.1.7) has been investigated in an organ culture system, since this enzyme may play a role in cell wall biogenesis or host defense mechanisms. Electrophoretic analysis of proteins from cotton ovule cultures indicated relatively few proteins being released into the surrounding medium. De novo synthesis of released peroxidase and other medium proteins was determined by in vivo labeling of ovule cultures with [³⁵S]-methionine. Analysis of labeled culture medium by denatured gel electrophoresis followed by fluorography showed incorporation of isotope into 2 major proteins with molecular weights of 30 kD and 56 kD, as well as a limited number of minor proteins. Similar analysis of native isoelectric focusing gels coupled with autoradiography demonstrated [³⁵S]-methionine incorporation into 2 major proteins with pI values of 4.3 and 5.0. The pI 5.0 protein was shown to have a molecular weight of 30 kD. The pI 4.3 protein had a molecular weight of 56 kD and was shown to be peroxidase by activity staining. Minor radiolabeled proteins were observed in the cationic region of the isoelectric focusing gels.     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

近年来,因病毒侵害人类每年都要蒙受巨大的经济损失和社会损失。犬肾细胞MDCK以其具有的培养容易、增殖快、流感病毒感染效率高等特点,成为适用于流感病毒疫苗生产的重要细胞系之一。以MDCK细胞为研究对象,在自制无血清培养基中成功实现了MDCK细胞从有血清培养到无血清培养的驯化;并通过单细胞悬浮培养驯化过程实现了MDCK细胞的无血清单细胞悬浮培养,获得了适于无血清单细胞悬浮生长的ssf-MDCK细胞株,无血清单细胞悬浮批培养最大活细胞密度可达3.81×106 cells/mL,最大比生长速率可达0.056 h?     

类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

旨在克隆、表达与纯化类弹性蛋白多肽,并测定类弹性蛋白的相变温度对不同的盐敏感程度。从头设计了类弹性蛋白多肽的序列并人工合成其编码基因片段,克隆至改造后的表达载体pET-22b中,构建重组表达载体,转化至大肠杆菌BL21(DE3) 中并诱导表达,采用可逆相变循环经高速离心对其进行纯化,并考察了盐类型及浓度对类弹性蛋白相变温度的影响。结果表明：0.4 mmol/L的Na2CO3能使25 μmol/L类弹性蛋白多肽 [KV8F-20] 相变温度降低至19 ℃,此类弹性蛋白多肽序列有望开发成一新型纯化标签,为今后     

植物组织培养外植体褐变的研究进展

外植体的褐变是组织培养过程中的主要障碍。目前一般认为影响褐变的因素主要有外植体材料、培养基和培养条件。为了防止褐变的发生,主要可以从外植体和培养条件、进行细胞筛选和材料的预处理、抑制剂或吸附剂的加入等方面入手。酚、酚酶的区域性分布假说、自由基伤害假说、保护酶系统假说是褐变机理方面较为成熟的3种假说。     

De novo synthesis of DNA in human platelets

Platelets, incubated with radiolabeled thymidine and purified free of contaminating nucleated cells, were analyzed for their ability to synthesize DNA. The only DNA species isolated from these purified platelets was mitochondrial DNA. The CsCl gradient-purified platelet DNA was treated with the restriction endonucleases EcoRI, HindIII and HpaI yielding the expected pattern for human mitochondrial DNA. Nitrocellulose blots of the electrophoresed, restriction endonuclease-treated DNA were fluorographed. All of the DNA fragments generated by the restriction enzymes were labeled, indicating de novo synthesis. This was further substantiated by inhibition of DNA synthesis by ethidium bromide and 2',3'-dideoxythymidine. Platelet DNA appeared to become greatly fragmented after 4 to 7 days storage while all of the thymidine incorporated was observed in intact mitochondrial DNA. These results indicate a continuous degradation of platelet mitochondrial DNA with no apparent repair mechanism. The ability of platelets to synthesize DNA may be associated with the protein synthetic capacity of platelets previously described.     

De novo synthesis of phytochrome in pumpkin hooks

Phytochrome becomes density labeled in the hook of pumpkin (Cucurbita pepo L.) seedlings grown in the dark on D₂O, indicating that the protein moiety of the pigment is synthesized de novo during development. Red light causes a rapid decline of the total phytochrome level in the hook of etiolated seedlings but upon return to the dark, phytochrome again accumulates. These newly appearing molecules are also synthesized de novo. Newly synthesized phytochrome in both dark-grown and red-irradiated seedlings is in the red-absorbing form. Turnover of the red-absorbing form is indicated by the density labeling of phytochrome during a period when the total phytochrome level in the hook of dark-grown seedlings remains constant. However, it was not possible to determine whether this results from intracellular turnover or turnover of the whole cell population during hook growth.     

Development of porcine embryos from one- and two-cell stages to blastocysts in culture medium supplemented with porcine oviductal fluid

Oviductal fluid (OVF) was harvested chronically from 5 sows beginning on Day 1 of the estrous cycle (Day 0 of estrous cycle = day of detected estrus) and used for embryo culture (Day 3 OVF only). Two experiments were conducted to investigate in vitro development of 1-cell and 2-cell porcine embryos in a modified Kreb's Ringer bicarbonate medium (culture medium, CM), early luteal phase OVF or CM supplemented with OVF (CM-OVF, 25% OVF v/v in CM) with or without transfer to fresh CM. In Experiment 1, 1-cell and 2-cell embryos were harvested from sows (n = 7) approximately 44 h after detected estrus. In Experiment 2, 1-cell embryos were collected from 5 sows treated with altrenogest and gonadotropins, approximately 50 h after injection of human chorionic gonadotropin. The volume of OVF (ml) declined progressively throughout the 4 days of collection (24 h, 8.44 +/- 0.28; 48 h, 6.88 +/- 1.78; 72 h, 4.96 +/- 0.35; 96 h, 4.64 +/- 0.25 after onset of estrus; p less than .01). In both experiments, development to blastocyst stage was lowest among embryos cultured in OVF and highest among those cultured in CM-OVF (Experiment 1: CM, 27.3; OVF, 10; CM-OVF, 63.6; Experiment 2: CM, 26.7; OVF, 0; CM-OVF, 82.4; % blastocyst formation).(ABSTRACT TRUNCATED AT 250 WORDS)     

De novo design and synthesis of HIV-1 integrase inhibitors

Existing AIDS therapies are out of reach for most HIV-infected people in developing countries and, where available, they are limited by their toxicity and their cost. New anti-HIV agents are needed urgently to combat emerging viral resistance and reduce the side effects associated with currently available drugs. Toward this end, LeapFrog, a de novo drug design program was used to design novel, potent, and selective inhibitors of HIV-1 integrase. The designed compounds were synthesized and tested for in vitro inhibition of HIV-1 integrase. Out of the 25 compounds that were designed, and synthesized, four molecules (compounds 23, 26, 43, and 59) showed moderate to low inhibition of HIV-1 integrase for 3'-processing and 3'-strand transfer activities. Nonetheless, these compounds possess structural features not seen in known HIV-1 integrase inhibitors and thus can serve as excellent leads for further optimization of anti-HIV-1 integrase activity.     

De novo synthesis of peroxidase isozymes in sweet potato slices

The peroxidase content of sweet potato slices (Ipomoea batatas Lam.) increased nearly 100-fold following 84 hours incubation in an air atmosphere containing ethylene, 1 microliter per liter. The object of experiments reported here is to determine if this increase in peroxidase activity results from synthesis de novo of the enzyme or from activation of a preexisting inactive form of the enzyme.     

De novo synthesis of 3'-nucleotidase in germinating wheat embryo

The enzyme 3′-AMP nucleotidase was purified 2,500- to 5,000-fold from extracts of an acetone powder of wheat (Triticum aestivum) embryonic axes germinated for 40 hours. Sodium dodecyl sulfate acrylamide gel electrophoresis and chromatography on Biogel-P100 indicate that the enzyme is monomeric with a molecular weight of 39,000. Extracts of embryos germinated up to 6 hours have only 1% of the 40-hour level of enzyme activity. To see if the increase to 40 hours represents de novo synthesis, extracts were compared for their ability to react with a rabbit antibody prepared against the enzyme. In immunodiffusion tests, 40-hour extracts showed a strong precipitin line coincident with that of the purified enzyme, whereas no precipitation was observed with 1-hour extracts. When the enzyme present in 40-hour extracts was partially inactivated by EDTA, it still blocked the ability of the antibody to inhibit enzyme activity. Extracts of 1-hour embryos, in contrast, were not able to block the inhibitory activity of the antibody. Embryos allowed to take up ³⁵SO₄ between 40 and 46 hours of germination synthesized ³⁵S-labeled 3′-nucleotidase. In contrast, no radioactive protein synthesized by embryos during the first 6 hours of germination coincided on gel electrophoresis with the enzyme. These results indicate that the increase in 3′-nucleotidase activity is a consequence of de novo synthesis of the enzyme.     

De novo purine synthesis in vegetative cells and myxospores of Myxococcus xanthus

This study was designed to determine whether vegetative cells and myxospores of Myxococcus xanthus were capable of classical de novo purine biosynthesis. To answer this question, vegetative and myxospore extracts of M. xanthus FBa were tested for their ability to synthesize the second de novo intermediate, 5'-phosphoribosylglycinamide, from beginning precursors either by way of phosphoribosyl-pyrophosphate amido transferase (EC 2.4.2.14) or ribose-5-phosphate amino transferase. Both the amido and amino transferase routes occurred in both types of extracts, and both enzymes appear to be present at about the same level (per milligram of protein) in vegetative cells, myxospores, and in a bacterial prototype, Salmonella typhimurium. The dose response of the vegetative and myxospore forms of both enzymes towards adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) suggests that the allosteric structure of both enzymes is changed little by sporulation. Both enzymes were inhibited to varying degrees by a variety of purine nucleotides besides AMP, GMP, and 3':5' cyclic AMP.     

De novo CDP-choline-dependent glycerophosphorylcholine synthesis and its involvement as an intermediate in phosphatidylcholine synthesis

The activity of CDP-choline-dependent glycerophosphorylcholine synthetase (CDP-choline:sn-3-glycerophosphate cholinetransferase), a newly discovered enzyme involved in the recently proposed pathways of acyl-specific phosphatidylcholine synthesis, is reported in rat liver. Endogenous CDP-choline, synthesized via the CMP-driven back reaction of phosphorylcholine transferase, is also shown to be a choline donor for this glycerophosphorylcholine synthetase. The function of glycerophosphorylcholine as an intermediate in phosphatidylcholine synthesis is demonstrated by specific isotope trapping whereby unlabelled glycerophosphorylcholine inhibited label incorporation from sn-[14C]glycerol-3-phosphate into phosphatidylcholine in mouse gastrocnemius, a tissue that is essentially devoid of the cytidine pathway for phosphatidylcholine synthesis and uses a non-allelic glycerophosphorylcholine synthetase (exogenous PC:sn-3-glycerophosphate cholinetransferase) in the synthesis of glycerophosphorylcholine.     

De novo synthesis and recycling pathways of sphingomyelin in rat Sertoli cells

Sertoli cells from 19-day-old rats have two molecular species of sphingomyelin (SM1 and SM2) with different kinetic characteristics and fatty acid composition. Here, we have studied the incorporation of [14C]-choline and [14C]-palmitic acid into SM in presence or absence of fumonisin B1, an inhibitor of ceramide synthesis, and beta-chloroalanine, an inhibitor of sphinganine synthesis. The contributions of de novo synthesis and recycling pathways were estimated by analysis of the inhibition caused by these drugs. SM1 was synthesized more by sphingosine recycling, and SM2 was synthesized principally by ceramide recycling than SM1. De novo synthesis seems to be important for the two SM types, but our results showed that this pathway is more extensively utilized by SM2. In conclusion, using Sertoli cell cultures, we have shown for the first time that in the same cell different molecular species of SM are synthesized by different pathways.     

Disparate effects of estradiol on egg transport and oviductal protein synthesis in mated and cyclic rats

Previously, we found that the dose of estradiol (E2) required to accelerate egg transport increases 5- to 10-fold, in mated compared to cyclic rats. Here we examined protein synthesis in the oviduct of mated and cyclic rats following a single injection of E2 known to accelerate oviductal egg transport or after concomitant treatment with progesterone (P4) known to block this acceleration. On Day 1 of the cycle or pregnancy, E2, P4, or E2 + P4 were injected s.c., and 4 h later oviducts were removed and incubated for 8 h in medium with 35S-methionine. Tissue proteins were separated by SDS-PAGE, and protein bands were quantitated by fluorography and densitometry. In mated rats, E2 and P4 increased different protein bands and P4 did not affect the fluorographic pattern induced by E2. In contrast with mated rats, none of these treatments changed the fluorographic pattern of the oviductal proteins in cyclic rats. Estradiol-induced egg transport acceleration was then compared under conditions in which oviductal protein synthesis was suppressed. Mated and cyclic rats treated with equipotent doses of E2 for accelerating egg transport also received actinomycin D (Act D) locally. Estradiol-induced oviductal egg loss was partially blocked by Act D in mated but had no effect in cyclic rats. We conclude that the oviduct of mated and cyclic rats differs in that only the former responds with increased protein synthesis to a pulse of exogenous E2 and P4 and requires an intact protein synthesis machinery in order to accelerate egg transport in response to E2.     

De novo synthesis of steroids (from acetate) by isolated rat Sertoli cells.

Sertoli cells from 17 day old rats were shown to convert [¹⁴C]acetate to [¹⁴C]-labelled cholesterol, pregnenolone and 17α-hydroxypregnenolone$\text{in}$$\text{vitro}$. Identification was by several systems of thin layer and gas chromatography of the extracted steroids and their sylil and acetyl derivatives and by recrystallizations with authentic and acetylated unlabelled steroids. Several other steroids formed from acetate were tentatively identified. No androstenedione or testosterone were formed. That the Sertoli cell cultures were free of Leydig cells was established by the absence of histochemically detectable 3β-hydroxysteroid dehydrogenase activity and the inability of the cultures to oxidize the 3β-hydroxyl group of [¹⁴C]pregnenolone. This is the first direct evidence that Sertoli cells have the capacity to synthesize steroids $\text{de}$$\text{novo}$ from acetate.