PRODUCTION OF 1-METHYLADENINE INDUCED BY CONCANAVALIN A IN STARFISH FOLLICLE CELLS

Concanavalin A (Con A) was found to induce maturation of oocytes with follicular envelopes in the starfish, Asterina pectinifera . Treating a Con A sample with 85% ethanol and heat revealed that the maturation-inducing activity of the sample was not due to possible contamination with 1-methyladenine, but to Con A itself. However, Con A had little maturation inducing effect on isolated oocytes from which the follicular envelope had been removed, suggesting that its effect is indirect and probably mediated by the follicle cells. When follicle cells were incubated in seawater containing Con A, a maturation-inducing substance was found to have been produced in the incubation medium. This was purified and identified as 1-methyladenine. Therefore it is concluded that Con A has the same capacity as GSS, a gonad-stimulating peptide hormone of neural origin, to induce production of the maturation-inducing substance. Other plant lectins such as phytohemagglutinin P and wheat germ agglutinin had little effect in inducing production of 1-methyladenine in follicle cells.     

BINDING OF CONCANAVALIN A TO THE SURFACE OF STARFISH FOLLICLE CELLS AND PRODUCTION OF 1-METHYLADENINE

Starfish follicle cells, treated with concanavalin A (Con A), continued to produce 1-methyl-adenine (1-MeAde), an inducer of starfish oocyte maturation, after rinsing with artificial seawater (ASW). On the other hand, they ceased to produce the substance if treated with methyl α-Dmannoside (αMM). These cells produced again 1-MeAde when re-stimulated with Con A after removal of αMM. An optical study with fluorescein revealed that Con A bound to the cells was not dissociated by rinsing with ASW, but was removed if the cells were treated with αMM. These results suggest that continuous binding of Con A to the surface of the follicle cells is essential for the production of 1-MeAde.     

METHIONINE-ACTIVATING ENZYME IN STARFISH FOLLICLE CELLS *

In starfish follicle cells 1-methyladenine is produced under the influence of a gonad-stimulating hormonal peptide (GSS). Since such production of the substance is enhanced by the addition of L-methionine or S-adenosylmethionine in vitro, the presence of methionine-activating enzyme in the follicle cells of the starfish, Asterina pectinifera, was investigated. To detect enzyme activity, the enzyme was partially purified from the supernatant of the follicle-cell homogenate by precipitation with ammonium sulfate followed by gel-filtration on a Sephadex G-150 column. Using such a preparation of the enzyme, the production of S-adenosylmethionine from L-methionine and adenosine triphosphate was clearly demonstrated by thin-layer chromatography. GSS was found to exert no effect on the activity of the methionine-activating enzyme. The hormonal peptide, GSS, is therefore considered to take part in some reaction other than this step in the formation of 1-methyladenine.     

INTERACTIONS OF 1-METHYLADENINE AND THE SURFACE OF STARFISH OOCYTE

Three metabolic inhibitors, mycostatin, concanavalin A (Con A) and cytochalasin B (CB) were used to study the interactions between l-methyladenine (1-MA) and the starfish oocyte surface leading to germinal vesicle breakdown (GVB). Mycostatin and Con A had no obvious effects on GVB. CB did not inhibit, but did delay GVB. This delaying effect was interpreted as having multiple 1-MA reactive "sites" on the surface. The results also suggested that not all of them were needed to react with 1-MA to bring about GVB.     

SITE OF 1-METHYLADENINE RECEPTORS IN THE MATURATION OF STARFISH OOCYTES

Maturation of vitelline coat-free (VCF) oocytes of the starfish, Asterina pectinifera , was studied. When the oocytes, the vitelline coats of which were elevated by adding the ionophorc A-23187, were forced through two sheets of copper mesh, the vitelline coats were completely removed from the oocytes. Although some of the VCF oocytes underwent germinal vesicle breakdown following this mechanical treatment, most of them retained the normal germinal vesicles. These VCF immature oocytes underwent breakdown of germinal vesicles after addition of 1-methyladenine (1-MA). Dose-response curves of VCF oocytes to 1-MA were similar to those of normal oocytes. These results indicate that 1-MA reacts with the plasma membrane and that the presence of the vitelline coat is not prerequisite for inducing oocyte maturation.     

MEMBRANE REACTIONS DURING FERTILIZATION AND 1-METHYLADENINE STIMULATION IN IMMATURE STARFISH OOCYTES

Immature oocytes from starfishes were obtained during the breeding and non-breeding seasons. The oocytes were "fertilized" without previous induction of maturation by 1-methyladenine (1-MA) or germinal vesicle breakdown (GVB). Elevation of the fertilization membrane (FM) was observed with eggs obtained during the breeding season, but not observed with eggs obtained during the non-breeding season. Abnormal development was observed when these "fertilized" eggs with a FM were subsequently treated with 1-MA. The results of these experiments indicated that the elevation of FM was independent of GVB or the mixing of nuclear and cytoplasmic components.     

PHYSIOLOGICAL DEPENDENCE OF BRAIN METHIONINE AND S-ADENOSYLMETHIONINE CONCENTRATIONS ON SERUM AMINO ACID PATTERN

Abstract— Animals maintained on rat chow and water ad libitum in quarters illuminated for 12 h/day show diurnal rhythms in serum methionine and brain S -adenosylmethionine (SAM) concentrations. Brain methionine exhibits no such variation, nor does the ratio of serum methionine to the serum concentrations of six neutral amino acids which are believed to compete with methionine for uptake into brain. Administration of methionine to rats in doses that elevate serum methionine, but keep it within the daily physiological range, significantly increases brain concentrations of both methionine and SAM. The acute feeding of either a protein-free or a 40% casein meal also increases brain methionine and SAM, but does not affect serum methionine; however, both diets also increase the ratio of serum methionine to tyrosine, an amino acid whose postprandial concentration is indicative of the concentrations of the other amino acids that compete with methionine for transport into brain. These findings suggest that brain methionine levels increase physiologically after eating as a result of changes in the serum amino acid pattern. Furthermore, such naturally occurring increases in brain methionine appear to be associated with elevations in brain SAM.     

MARKED DECREASE IN THE RIGIDITY OF STARFISH OOCYTES INDUCED BY 1-METHYLADENINE1

Extreme rigidity of immature starfish oocytes as measured by compression method was found to decline during the early phase of their maturation when induced by 1-methyladenine (1-MeAde). The onset of this decrease in stiffness occurred within 5 to 9 min of 1-MeAde treatment, well before the breakdown of the germinal vesicle, progressively declining to reach a minimum stiffness after 20 min. Dithiothreitol, known as an artificial maturation-inducing agent, caused a similar change. The stiffness is thus expected to serve as a quantitative indicator of the early process of cytoplasmic events, which would induce the breakdown of the germinal vesicle. Cytochalasin B (3 μg/ml) also reduced the stiffness, but unlike the former two agents, the effect was reversible, and did not interfere with the process of maturation. Due to the effect of cytochalasin B, it became possible to enucleate immature oocytes by centrifugal force. Non-nucleate fragments thus obtained still maintained their marked stiffness, which was decreased by the action of 1-MeAde, with a time-course similar to that of intact oocytes.     

EFFECT OF LOCAL APPLICATION OF 1-METHYLADENINE ON THE SITE OF POLAR BODY FORMATION IN STARFISH OOCYTE*

Mechanism by which the site of polar body formation is determined in starfish oocytes was investigated in relation to the action of 1-methyladenine (1-MeAde). Local staining with Nile Blue of Asterina pectinifera oocytes revealed that there exists a prospective site of polar body formation (PSPBF) on the nearest surface to the position of germinal vesicle. The site of polar body formation was found to shift to some extent from PSPBF toward the area locally applied with 1-MeAde, suggesting that the actual site of polar body formation is not determined yet at the germinal vesicle stage. Oocytes whose germinal vesicles had been shifted by centrifugation from PSPBF to the opposite surface before the commencement of germinal vesicle breakdown (GVBD) (less than 15 min after 1-MeAde treatment), failed to form polar bodies, whereas oocytes centrifuged after commencement of GVBD (20 min after 1-MeAde treatment) did form polar bodies where their fading germinal vesicles had reached by centrifugation. In the oocytes which failed to form polar bodies by centrifugation, an aster was observed near PSPBF of each oocyte. When inseminated, every oocyte treated with 1-MeAde developed normally irrespectively of the mode of polar body formation including the site and the occurrence, and the animal pole of every larva was derived from PSPBF.     

MATERNAL AND PATERNAL EFFECTS ON FOLLICLE PRODUCTION IN THE MILKWEED ASCLEPIAS SYRIACA (ASCLEPIADACEAE)

We measured follicle production from a diallel cross among ten clones of the common milkweed Asclepias syriaca, to assess the relative contributions of maternal and paternal parents. Specific parental combinations differed in the ability to set fruit, indicated by a significant nuclear specific effect accounting for 28% of the observed variance in follicle production. Several mechanisms might contribute to this effect, including shared incompatibility alleles and expression of zygotic genotypes. The nuclear general effect was not significant, however, suggesting a lack of additive genetic variation for offspring control of fruit maturation. Maternal effects also had an important effect on follicle production, as demonstrated by a significant reciprocal general effect (26% of the variance), almost entirely due to a large maternal component. The small reciprocal general variance component attributable to paternal effects, and nonsignificant reciprocal specific effect, indicating little maternal parent-zygote interaction, suggest that female choice through selective follicle maturation was not important in this experiment. The clones varied in proportion of reproductive output through female function, but a significant tradeoff between male and female success was not detected.     

LACK OF ENHANCEMENT OF DIMETHYLTRYPTAMINE FORMATION IN RAT BRAIN AND RABBIT LUNG IN VIVO BY METHIONINE OR S-ADENOSYLMETHIONINE

In vivo conversion of intracisternally administered [¹⁴C]tryptamine to [¹⁴C]N,N-dimethyl-tryptamine (DMT) in rat brain, even in the presence of an excess of substrate and methyl donor appeared to be insignificant, although enzymatically synthesized [¹⁴C]DMT was recovered readily after intracranial injection. Authenticity of [¹⁴C]DMT was demonstrated by cocrystallization with authentic DMT and oxalic acid to constant specific radioactivity after chromatographic separation of [¹⁴C]DMT. In rabbit lung, the apparent K_m for S-adenosylmcthionine (SAMe) (29 μM) with indolethylamine-N-methyltransferase was found to be close to endogenous levels of SAMe (34 μM) that are not likely to saturate the enzyme normally. Nevertheless. large doses of L-methioninc or SAMe failed to increase the in vivo conversion of [¹⁴C]N-methyltryptamine to [¹⁴C]DMT in this tissue. The production of [¹⁴]DMT was instead markedly irihihitrd by this treatment. possibly due to an effect of S-adeno-sylhomocysteine. Our results fail to support the hypothesis that psychotropic effects of methionine or SAMe are due to increased accumulations of pharmacologically active methylated indoleamines.     

CONTRASTING EFFECTS OF METHIONINE SULFOXIMINE ON UPTAKE AND ASSIMILATION OF AMMONIUM IN ULVA INTESTINALIS (CHLOROPHYCEAE)1

Ammonium is assimilated in algae by the glutamine synthetase (GS)–glutamine:2‐oxoglutarate aminotransferase pathway. In addition to the assimilation of external ammonium taken up across the cell membrane, an alga may have to reassimilate ammonium derived from endogenous sources (i.e. nitrate reduction, photorespiration, and amino acid degradation). Methionine sulfoximine (MSX), an irreversible inhibitor of GS, completely inhibited GS activity in Ulva intestinalis L. after 12 h. However, assimilation of externally derived ammonium was completely inhibited after only 1–2 h in the presence of MSX and was followed by production of endogenous ammonium. However, endogenous ammonium production in U. intestinalis represented only a mean of 4% of total assimilation attributable to GS. The internally controlled rate of ammonium uptake (V_i) was almost completely inhibited in the presence of MSX, suggesting that V_i is a measure of the maximum rate of ammonium assimilation. After complete inhibition of ammonium assimilation in the presence of MSX, the initial or surge (V_s) rate of ammonium uptake in the presence of 400 μM ammonium chloride decreased by only 17%. However, the amount that the rate of ammonium uptake decreased by was very similar to the uninhibited rate of ammonium assimilation. In addition, the decrease in the rate of ammonium uptake in darkness (in the absence of MSX) in the presence of 400 μM ammonium chloride matched the decrease in the rate of ammonium assimilation. However, in the presence of 10 μM ammonium chloride, MSX completely inhibited ammonium assimilation but had no effect on the rate of uptake.     

内皮素—1对大鼠排卵前卵泡颗粒细胞产生孕酮的影响

本文用离体细胞体外孵育法研究了内皮素－１（ＥＴ）对大鼠排卵前卵泡颗粒细胞孕酮生成的影响及其作用机理。结果发现,ＥＴ能显著抑制ｈＣＧ刺激下的孕酮产生,抑制作用在浓度为１０－８ｍｏｌ／Ｌ时,即有显著意义（Ｐ＜０．０５,ｎ＝６）,至１０－７ｍｏｌ／Ｌ时则有非常显著的意义（Ｐ＜０．０１,ｎ＝６）;不同浓度ＥＴ（１０－７—１０－７ｍｏｌ／Ｌ）,对颗粒细胞基础孕酮的产生无明显影响。进一步研究表明,ＥＴ对ｈＣＧ刺激下孕酮生成的抑制作用,在用免抗人内皮素抗血清（ＥＴ－Ａ）１：１０００及ｃＡＭＰ后能明显被逆转。实验中还观察到,ＥＴ使颗粒细胞ＬＨ／ｈＣＧ受体数下降,亲和力降低。本文结果提示,ＥＴ可能为卵巢内的一种局部调节肽,通过作用于ＥＴ受体,干扰ＬＨ／ｈＣＧ受体功能和ｃＡＭＰ生成而抑制颗粒细胞孕酮的产生。     

MESENCHYME CELLS IN STARFISH DEVELOPMENT: EFFECT OF TUNICAMYCIN ON THEIR DIFFERENTIATION, MIGRATION AND FUNCTION

The effect of tunicamycin, an inhibitor of protein glycosylation, on starfish development was investigated. Specific developmental events such as 1) bulging of the archenteron tip, 2) migration of mesenchyme cells, 3) formation of coelomic pouches and 4) mouth formation, are inhibited in the presence of this drug. These events are discussed in connection with differentiation, migration and function of mesenchyme cells. The possibility is discussed that tunicamycin exerts its effect by interfering with de novo synthesis of a cell surface factor(s) supporting dynamic cell surface activities.     

南方鲶卵巢滤泡细胞和卵膜生成的组织学研究

南方鲶的卵巢滤泡细胞源于卵巢基质细胞,从发生到退化分为零散卵泡膜细胞期、单层扁平泡膜细胞期、多层扁平卵泡膜细胞期、立方形颗粒细胞期柱状颗粒细胞期、颗粒细胞分泌期和颗粒细胞退化期。精孔细胞中发育中滤泡细胞分化形成。初级卵精源于卵母细胞,次级卵膜由晚期滤泡细胞分泌形成。本文还对滤泡细胞和卵膜的作用进行了阐述。     

温度和盐度对球形棕囊藻细胞DMSP产量的影响

球形棕囊藻汕头株(Shantou strain,ST)和香港株(Hongkong,HK)是DMSP与DMS的高产株,在20℃、40盐度的培养条件下,二者DMSP产量分别达到161.3 437.60nmol/106cells.细胞内DMSP的积累与释放到细胞外DMS量受盐度、温度等环境因子的影响:在高盐低温条件下,单位藻细胞的DMSP与DMS产量较高.香港株DMSP/DMS的积累和释放与生长时期有关,稳定期细胞内的DMSP含量高达3898.3nmol/106cells,是指数期的12.3倍.       

MNNG对细胞DNA合成及其分布的影响

本文用流式细胞光度术(FCM)等方法研究了MNNG,ENNG和DMS对HeLa细胞DNA含量分布的影响。经MNNG(6.8μmol/L)处理后,细胞分裂减少,DNA合成速率下降,S期细胞的比例随处理时间的延长而增加。DMS显示有类似的现象而ENNG的效应则较小。     

甘丙肽及其受体在嗅成鞘细胞中的表达及对细胞增殖的影响

本实验从新生大鼠嗅球中分离出嗅成鞘细胞,进行体外培养。运用RT-PCR方法检测甘丙肽及其受体在体外培养的嗅成鞘细胞中的表达;运用MTT法检测甘丙肽及其受体激动剂、拮抗剂对嗅成鞘细胞增殖的影响。结果显示:嗅成鞘细胞表达甘丙肽(GAL)及其受体GalR2,而不表达其他两种受体GalR1和GalR3;甘西肽及两种受体激动剂GAL1-11和GAL2-11能够明显地抑制体外培养的嗅成鞘细胞的增殖,这一效应可被非特异性甘丙肽受体拮抗剂M35所阻断。     

Ⅰ型胶原、内皮素对肾小球内皮细胞及系膜细胞产生细胞外基质的影响

本文观察了Ⅰ型胶原、内皮素对肾小球内皮细胞、系膜细胞增殖的影响,同时探讨了Ⅰ型胶原和内皮素对培养的内皮细胞及系膜细胞产生层粘连蛋白、纤连蛋白和Ⅳ型胶原的影响。结果提示:Ⅰ型胶原可以明显促进内皮细胞的增殖(P<0.01),内皮素对系膜细胞的增殖有一定作用:Ⅰ型胶原可以促进内皮细胞产生层粘连蛋白和纤连蛋白,并可以促进系膜细胞产生Ⅳ型胶原;内皮素可以促进系膜细胞产生FN增多(P<0.05)。