EFFECTS OF COLCHICINE, CYTOCHALASIN B, AND 2-DEOXYGLUCOSE ON THE TOPOGRAPHICAL ORGANIZATION OF SURFACE-BOUND CONCANAVALIN A IN NORMAL AND TRANSFORMED FIBROBLASTS

The distribution of surface-bound concanavalin A on the membranes of 3T3, and simian virus 40-transformed 3T3 cultured mouse fibroblasts was examined using a shadow-cast replica technique with a hemocyanin marker. When cells were prefixed in paraformaldehyde, the binding site distribution was always random on both cell types. On the other hand, labeling of transformed cells with concanavalin A (Con A) and hemocyanin at 37°C resulted in the organization of Con A binding sites (CABS) into clusters (primary organization) which were not present on the pseudopodia and other peripheral areas of the membrane (secondary organization). Treatment of transformed cells with colchicine, cytochalasin B, or 2-deoxyglucose did not alter the inherent random distribution of binding sites as determined by fixation before labeling. However, these drugs produced marked changes in the secondary (but not the primary) organization of CABS on transformed cells labeled at 37°C. Colchicine treatment resulted in the formation of a caplike aggregation of binding site clusters near the center of the cell, whereas cytochalasin B and 2-deoxyglucose led to the formation of patches of CABS over the entire membrane, eliminating the inward displacement of patches observed on untreated cells. The distribution of bound Con A on normal cells (3T3) at 37°C was always random, in both control and drug-treated preparations. Pretreatment of cells with Con A enhanced the effect of colchicine on cell morphology, but inhibited the morphological effects of cytochalasin B. The mechanisms that determine receptor movement and disposition are discussed.     

CELL—CELL CONTACTS ACCELERATE CELL COLONY GROWTH IN SPARSE CULTURES OF CHICK EMBRYO FIBROBLASTS

There is no cell proliferation in very sparcely plated chick embryo cell cultures. Substituting conditioned medium or adding of ethanol-fixed homologous cells to the cultures accelerates cell colony growth. The mechanism for the mitogenic action of fixed cells is considered to be the contact stimulation of cell proliferation, and addition of extra cells to sparse culture is believed to mimic the cell micro-environment existing in subconfluent cultures. The role of diverse cell—cell contacts in cultured cell growth regulation is discussed. The procedure used (addition of ethanol-fixed cells) may improve normal cell cloning techniques.     

ON THE ROLE OF THE CYTOSKELETON IN COCCOLITH MORPHOGENESIS: THE EFFECT OF CYTOSKELETON INHIBITORS1

The coccolithophore Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler was cultured in natural seawater with the addition of either the microtubule‐inhibitor colchicine, the actin‐inhibitor cytochalasin B, or the photosynthesis inhibitor 3‐(3,4 dichlorophenyl)‐1,1‐dimethyl‐urea (DCMU). Additionally, E. huxleyi was cultured at different light intensities and temperatures. Growth rate was monitored, and coccolith morphology analyzed. While every treatment affected growth rate, the percentage of malformed coccoliths increased with colchicine, cytochalasin B, and at higher than optimal temperature. These results represent the first experimental evidence for the role of microtubules and actin microfilaments in coccolith morphogenesis.     

STIMULATION OF COLLAGEN SYNTHESIS IN RAT CARDIAC FIBROBLASTS BY EXPOSURE TO HYPOXIC CULTURE CONDITIONS AND SUPPRESSION OF THE EFFECT BY NATRIURETIC PEPTIDES

Synthesis of type I and type III collagens by rat cardiac fibroblasts was stimulated when the cells were cultured under 95% N₂/5% CO₂for one hour followed by incubation under normoxic conditions for 24 hours. The stimulative effect was attenutated by the presence of atrial natriuretic peptide (ANP, 10⁻⁶m) or brain natriuretic peptide (BNP, 10⁻⁶m) in the culture medium. Northern blot analysis indicated that α1(I) and α1(III) collagen mRNA levels were also increased by hypoxia, and decreased with the addition of ANP or BNP in a dose-dependent manner. These results indicate interaction between intracellular signals of a physical stimulus (hypoxic stress) and those of a chemical one (ANP or BNP) and demonstrate that both signals regulate collagen synthesis by cardiac fibroblasts at the levels of the mRNAs. The results also suggest that natriuretic peptides produced by cardiomyocytesin vivomay function as paracrine factors that play a role in the prevention of cardiac fibrosis in ischaemic heart diseases.     

INFLUENCE OF SUCCESSIVE AND COMBINED ULTRAVIOLET A AND B IRRADIATIONS ON MATRIX METALLOELASTASES PRODUCED BY HUMAN DERMAL FIBROBLASTS IN CULTURE

To study the cumulative influence of UV irradiations on skin matrix alterations, human skin fibroblasts were irradiated successively three-fold, at 24h intervals, with UVA (3×5J/cm²), UVB (3×8mJ/cm²), UVA plus UVB (3×5J/cm²and 3×8mJ/cm²) and the levels of 92kDa gelatinase (pro-MMP₉), 72kDa gelatinase (pro-MMP₂) and plasma-membrane elastase type protease were determined, following subsequent 24-h culture in 10% serum-containing medium. UV irradiations had only minor influence (1.4-fold increase for UVB) on secreted levels of pro-MMP₂and decreased the amount of plasma membrane elastase produced by cells. It did however, for UVA and UVB alone, induce a significant increase of 66kDa activated MMP₂production: 2.5- and 1.7-fold respectively. Such enhancement was not observed when combined irradiations were administered. UV exposure possessed a much higher influence on pro-MMP₉secretion by dermal fibroblast enhancing enzyme levels by 2.5-, 6.5- and 5-fold for UVA, UVB and UVA+UVB, respectively.     

BASAL LEVELS OF METALLOTHIONEIN I AND II EXPRESSION IN MOUSE EMBRYO FIBROBLASTS ENHANCE GROWTH IN LOW FOLATE THROUGH A CELL CYCLE MEDIATED PATHWAY

The impact of basal (non-induced) expression levels of metallothionein I and II on the growth of mouse embryo fibroblasts in standard DMEM/F-12 containing 8.8 microm folic acid, and in DMEM/F12 without hypoxanthine, thymidine or folic acid, containing 15 nm or 15 pm[6S]-folinic acid, was assessed by comparing wild-type MT (+/+) and homozygous null MT (-/-) cell lines. No difference in growth rate was observed between the two in DMEM/F12, although MT (-/-) cells displayed a 6-fold decrease in p27(Kip1), a two fold increase in p53 and a slight increase in p21(Waf1). After 6 days in culture, the growth rate for MT (-/-) cells in 15 nm or 15 pm[6S]-folinic acid was half that of MT (+/+). After an additional 6 days in 15 n m folate, both MT (+/+) and (-/-) cells maintained their respective growth rates, while those in 15 pm had ceased to grow. During the initial 6 days in 15 nm folate, neither cell population displayed an increase in apoptosis or a change in cell cycle distribution, even though MT (-/-) cells sustained an additional 4-fold increase in p21(Waf1)and a 6-fold decrease in cyclin E expression. At day 12, however, the MT (-/-) population, but not MT (+/+), underwent a 7-fold increase in apoptosis coupled with a 3 fold increase in S phase cells. Hence, the basal level of MT I and II constitutively expressed in MT (+/+) cells enhances growth in 15nM [6S]-folinic acid by preventing S phase arrest and apoptosis.     

SUGAR-CONTAINING COMPOUNDS ON THE CELL SURFACES OF MARINE DIATOMS MEASURED USING CONCANAVALIN A AND FLOW CYTOMETRY

Some diatom exudates may remain attached to the exterior cell surface, potentially altering cell stickiness and affecting important aspects of the diatom's ecology such as aggregation rates and grazing rates. We measured the accumulation of cell-surface sugar-containing compounds by labeling cultured marine diatoms with fluorescent-tagged sugar-binding lectins and measuring the fluorescence associated with each cell using flow cytometry. The binding of FITC-labeled concanavalin A (FITC-ConA), a lectin that binds to glucose and mannose residues, varied 5-fold among different diatom species in exponential growth (on a per-cell basis) and 2–3-fold within a given species in different physiological states. Although transparent exopolymer particles followed a simple accumulation curve over time in batch culture, FITC-ConA. cell^-1 did not follow the same pattern, suggesting that surface sugar accumulation is not driven simply by the accumulation of such particles in the medium. For Thalassiosira pseudonana (Hustedt) Hasle and Heimdal (3H clone), the amount of sugar-containing compounds on the cell surface increased transiently as growth rate slowed in early stationary phase under both N and Si limitation. For Chaetoceros neogracile (Schuett) van Landingham, FITC-ConA. cell^-1had a strong inverse relationship with growth rate across several Si-limited batch culture experiments. Both results suggest some biological mediation of cell-surface sugar-containing compounds. Our study reveals the great potential for using lectin binding to investigate cell-surface sugars on diatoms. Lectins allow us to investigate noninvasively the role of cell-surface sugar-containing compounds in modifying cell stickiness and aggregation, as well as the partitioning of exuded phytoplankton carbon. We suggest that cell-surface sugar accumulation may be related to diatom stickiness, based on a correlation between our FITC-ConA measurements and stickiness estimates in the literature on several of the species we studied.     

SEPTUM FORMATION IN THE DESMID XANTHIDIUM (CHLOROPHYTA): EFFECTS OF CYTOCHALASIN D AND LATRUNCULIN B SUGGEST THE INVOLVEMENT OF ACTIN MICROFILAMENTS

Septum formation and mitosis were investigated by light and electron microscopical techniques as well as through the completion of inhibitor experiments in the unicellular desmid Xanthidium armatum (Bréb.) Rabenh. In untreated cells, numerous endoplasmic reticulum (ER) cisternae permeated the mitotic apparatus and secretory vesicles, and ER formed a band in front of the linearly growing septum, indicating the predetermined direction of septum growth. Under the influence of cytochalasin D (CD), the vesicle/ER band lost its proper orientation, which led to a malformed septum wall; moreover, abnormal septum branches could potentially have developed. Whereas the septum of an untreated cell only grew at its edge, the CD-induced branches (also with a vesicle/ER band in front) represented additional growing zones. These observations indicated that actin filaments were involved in establishing, maintaining, and orienting the "preforming" vesicle/ER band and, thus, the later septum. Latrunculin B (LB) had more severe effects on septum formation than did CD. Only small accumulations of septum material were found at the septum edge, and no aberrant growth of the septum occurred in LB-treated as in CD-treated cells. This could be explained by the more rapid disturbance of all actin-driven processes after LB treatment, which was assumed, because even low concentrations of the drug rapidly inhibited cytoplasmic streaming.     

慢性肺心病肺动脉平滑肌细胞的胶原合成：体内及体外观察

肺动脉构形重建（ｓｔｒｕｃｔｒｕｒａｌｒｅｍｏｄｅｌｉｎｇ）是慢性肺心病的重要血管病变,但其发生机制至今未完全明了。病变以血管中膜平滑肌细胞肥大、增生和细胞外基质（包括纤维性与非纤维性成分）增多导致的血管壁增厚、血管腔狭窄为特征。本文用天狼星红－偏振光显微镜观察,真彩色全自动图像分析法,测量１０例慢性肺心病尸检肺小动脉中膜厚度及中膜内Ⅰ、Ⅲ两型胶原的含量和所占的百分比。用３Ｈ－胸腺嘧院核苷和３Ｈ－脯氨酸掺入法,观察缺氧内皮细胞条件培养液（ＨＥＣＣＭ）对培养的肺动脉平滑肌细胞（ＰＡＳＭＣＳ）ＤＮＡ及胶原合成的影响。结果：（１）肺心病组４３７支肌型肺动脉平均中膜厚占血管直径的百分值高于对照组５±１．０８％;（２）肺心病组的Ⅰ型和Ⅲ型胶原面积分别占中膜面积的５４．６２％和５１９％,而对照组两型胶原占中膜面积小于２％。（３）ＨＥＣＣＭ组平滑肌细胞的３Ｈ－ＴｄＲ和３Ｈ－脯氨酸掺入量（ｃｐｍ值）,均明显高于常氧对照组（ＮＥＣＣＭ）,两组相比,有显著性差异（Ｐ＜０．０１）。（４）细胞周期分析,ＨＥＣＣＭ组平滑肌细胞的Ｇ０／Ｇ１期细胞数百分值比ＮＥＣＣＭ组少２８％,Ｇ２＋Ｍ期细胞百分值则比ＮＥＣＣＭ组高３０％。可以认为,缺     

人胰岛素A、B链基因的合成、克隆及其在大肠杆菌中的表达

根据人胰岛素A、B链的多肽顺序,设计并用一种简便快速的多肽基因合成了A、B链基因。合成的A 链和B 链基因分别克隆到pWR590质粒上,构建了表达型质粒pWR590-HIA和pWR 590-HIB,它们能够表达由β-半乳糖苷酶N-端约590个氨基酸残基与A 链或B 链组成的融合蛋白(两者之间由Met 连接)。A 链或B 链融合蛋白经BrCN 降解,磺化及分离纯化等步骤,得到了磺化A、B 链。磺化A、B链体外重组得到人胰岛素。     

INDUCTION OF CELLULAR PROCESSES CONTAINING COLLAGENASE AND RETINOID BY INTEGRIN-BINDING TO INTERSTITIAL COLLAGEN IN HEPATIC STELLATE CELL CULTURE

Cultered hepatic stellate cells were induced to elongate long, multipolar cellular processes by interstitial collagen gel used as a substratum, as compared to flattened or round cell shapes on polystyrene surface or on Matrigel containing the basement membrane components, respectively. The process induction was inhibited by several reagents as follows: (1) anti-integrin α2 antibody; (2) an oligopeptide, DGEA, an integrin-binding sequence in type I collagen molecule; (3) wortmannin, a phosphatidylinositol 3-kinase inhibitor. Protein tyrosine phosphorylation was enhanced throughout cells including cellular processes by culturing on type I collagen gel. Dual fluorescence staining showed that the core of the processes contained microtubules, whereas the periphery of the processes comprised fibrillar actin. Thus, the process extension was found to depend on integrin-binding to type I collagen fibres, followed by signal transduction and cytoskeleton assembly. The cellular processes included interstitial collagenase and vitamin A-containing lipid droplets. The lipid droplets and vitamin A-autofluorescence were increased by retinyl acetate addition to the culture medium, suggesting an important role of processes in hepatic stellate cell function.     

THE ROLE OF CDC2, CDC25 AND CYCLIN A GENES IN THE MAINTENANCE OF IMMORTALIZATION AND GROWTH ARREST IN A RAT EMBRYONIC FIBROBLAST CONDITIONAL CELL LINE

Immortalization of rodent cells by oncogenes is a complex biological process which involves the abnormal regulation of genes who control cellular proliferation. The role of the cell cycle control genes cdc2, cdc25 and cyclin A in the maintenance of immortalization and in growth arrest was examined in the tsa14, a SV40 T antigen rat embryonic fibroblast conditional for growth cell line. Analysis of RNA expression showed minimal levels of cdc2 mRNA in both proliferating and growth-arrested tsa14 cells. In contrast, cyclin A mRNA was found downregulated in growth-arrested tsa14 cells, as well as in senescent primary rat embryonic fibroblasts (REFS). The ability of cdc2, or cdc25, or cyclin A genes to maintain the tsa14 immortal phenotype was also examined by electroporations of these genes into the tsa14 cells. Clones over-expressing the electroporated cdc2, or cdc25, or cyclin A, or combinations of these genes growth arrested at the non-permissive conditions similar to controls, thereby suggesting that the expression of these genes alone is insufficient for tsa14 maintenance of immortalization.     

EFFECTS OF EXPOSURE TO ELECTROMAGNETIC RADIATION AT 835 MHz ON GROWTH,MORPHOLOGY AND SECRETORY CHARACTERISTICS OF A MAST CELL ANALOGUE,RBL-2H3

A mast cell line, RBL-2H3, was exposed to 835MHz for 20 minutes, three times per day for 7 days at a power density of 8.1±3mW/cm²From day 4 onwards, it was observed that the rate of DNA synthesis and cell replication increased, that actin distribution and cell morphology became altered, and the amount of β-hexosaminidase (a marker of granule secretion) released in response to a calcium ionophore was significantly enhanced, in comparison to unexposed cultures. There were no effects seen on levels of cytoskeletal protein synthesis or of beta-actin mRNA. Morphological changes persisted following subculture for at least 7 days in the absence of further exposure. It is hypothesized that effects of exposure to an electromagnetic field at 835MHz may be mediated via a signal transduction pathway.     

清香桂碱D和矮陀陀胺碱A,B的结构

本文报道从国产清香桂(Sarcococca ruscifolta)和金丝矮陀陀(Pachysandra axillaria)植物中分得的三个胺碱型新甾体生物碱清香桂碱 D 和矮陀陀胺碱 A、B 的化学结构,并首次归属了它们的~(13)C NMR 数据。     

白首乌甙A,B和C的结构

从著名中药白首乌(Cynanchum auricutatum Royle ex Wight)根中分离得到7个C_2(?)体甙。其小4个已知物——wilfosidc C3N (Ⅰ), C1N (Ⅱ), C1G (Ⅲ), K1N (Ⅴ),和另外3个新C_(21)甾体甙,命名为白首乌甙A (Ⅵ),B (Ⅶ),C(Ⅳ) (cynauricusidc A, B,C)。经光谱数据分析和化学反应,证明其结构分别为:开德甙元 3-氧-β D-葡萄糖吡喃基-(1→4)-α-L-加拿大麻糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃基-(1→4)-α-L-迪吉糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃甙(kidjoranin 3-O-β-D-glucopyranosyl-(1→4)-α-L-cymaropyranosy-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside, Ⅵ);萝藦甙元 3-氧-α-L-加拿大麻糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃基-α-L-迪吉糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃甙(metaplexigenin -O-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-cymaropyranoside,Ⅶ);告达庭 3-氧-β-D-葡萄糖吡喃基-(1→4)-β-L-葡萄糖吡喃基-(1→4)-α-L-加拿大麻糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃基-(1→4-α-L-迪吉糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃基(caudatin 3-O-β-D-glucopyranosy-(1→4)-β-D-glucopyr     

链脲佐菌素诱导糖尿病大鼠胰腺外分泌部A、B细胞密度的变化

本文应用A蛋白金银—过氧化物酶抗过氧化物酶(PAGS-PAP)双重染色法,观察了链脲佐菌素(streptozotocin,STZ)诱导的糖尿病大鼠胰腺外分泌部含高血糖素的单个A细胞(单A细胞)及合胰岛素的单个B细胞(单B细胞)密度的变化.在一次大剂量腹腔注射STZ后第5天和第10天,大鼠胰腺外分泌部单A细胞密度较对照组大鼠增加,而单B细胞密度在注射STZ后第5天较对照组大鼠减少,但在第15天与对照组大鼠接近.在第15天,一些单B细胞分布在靠近胰岛的腺泡中,岛周腺泡含单B细胞的胰岛百分率明显高于对照组.由于胰腺外分泌部的单A、单B细胞在分布特征上与中间细胞相似,在糖尿病时其数量变化也与中间细胞一致,因此,本研究所观察到的单A、单B细胞与前人报道的中间细胞有密切关系.上述单A、单B细胞密度的变化提示,在STZ诱导的糖尿病大鼠,胰腺中的B细胞被STZ破坏后,其外分泌部可能有某些细胞通过中间细胞向单A、单B细胞发生了转化或者单A、单B细胞即此转化过程中的中间细胞.     

乙型肝炎病毒基因重组疫苗免疫儿童成人效果及阻断母婴传播的研究

以中试连续生产的6批乙肝基因重组疫苗(R—Hepavac—B)免疫儿童和成人,每剂10μg,3批苗按0、1、6月,另3批苗按0、1、2月方案接种。每批分别接种8～10岁儿童51～66人,6批共363人,18～20岁成人46～52人,6批共287人。同时用两批血源苗作对照,分别接种儿童共85人,成人84人。又用其中89—10批HBsHg含量为20μg/ml的疫苗,按20μg×3,0、1、6月方案接种HBsAg和HBeAg双阳性母亲所生婴儿36名,HBsAg阳性母亲的婴儿19名。结果显示,10μg重组疫苗3针后,无论儿童和成人其抗HBs抗体阳转率均为100％,而对照,儿童成人各有1例未阳转。儿童按0、1、6方案接种者,其抗体GMT为363.8～470.5mIU,按0、1、2方案接种者,为150.8～195.5mIU。前者高于血源苗对照,而后者持平。成人0、1、6方案GMT为189.4～247.2mIU,0、1、2方案为87.9～96.3mIU,均略低于血源苗对照.经复测,此批血源苗含量为15μg/ml,高于基因苗含量,可能是造成以上结果的原因。母婴阻断结果显示,双阳性母亲子女36人,3针免后86.1％(32/36)的婴儿获得保护。单阳性母亲子女19人全部获得保护。     

EFFECT OF LATRUNCULIN B AND BREFELDIN A ON CYTOKINESIS IN THE BROWN ALGA SCYTOSIPHON LOMENTARIA (SCYTOSIPHONALES,PHAEOPHYCEAE)1

In zygotes of the brown alga Scytosiphon lomentaria (Lyngb.) Link, cytokinesis proceeds by growth of membranous sacs, which are formed by fusion of Golgi vesicles and flat cisternae accumulated at the future cytokinetic plane. It has been reported that depolymerization of actin filaments by latrunculin B does not inhibit mitosis. However, this molecule prevents the formation of the actin plate, which appears at the region of intermingled microtubules from each centrosome just before and during cytokinesis. In this study, zygotes treated with latrunculin B were observed using EM. Remarkably, this reagent inhibited the formation of flat cisternae. Golgi vesicles gathered around the midzone between the two daughter nuclei and fused with the plasma membrane there. As a result, the plasma membrane invaginated, in a complicated manner, into the cytoplasm. However, these invaginations of the plasma membrane never produced a continuous partition membrane. The ultrastructure of zygotes treated with brefeldin A, which prevents Golgi‐mediated secretion, was also examined. Flat cisternae appeared at the future cytokinetic plane, and a new cell partition membrane was formed. However, the partition membrane became thick, because it was filled with amorphous material rather than the normal rigid fibrous material. These results suggested that actin is involved in the formation of flat cisternae, where it is necessary for completion of the new cell partition membrane, and that Golgi vesicles may play an important role in the deposition of cell wall material.     

THALLUS DIFFERENTIATION OF PHOTOSYNTHESIS,GROWTH, REPRODUCTION,AND UV‐B SENSITIVITY IN THE GREEN ALGA ULVA PERTUSA (CHLOROPHYCEAE)1

The chlorophyte Ulva is perceived as a simple and uniform algal form, with little functional differentiation within a thallus. We compared morphology, pigmentation, photosynthesis, growth, reproduction, and UV‐B sensitivity between different thallus regions of Ulva pertusa Kjellman. Thallus thickness and cell size were significantly greater, whereas cell number was less in the basal region than in other regions. Photosynthetic pigment contents were lowest in the basal region and increased toward the marginal region. Photosynthetic capacity and photosynthetic efficiency normalized to fresh weight, area, volume, and cell number showed a progressive increase from the basal to marginal parts; however, on a chl basis those values were equal regardless of thallus part. Values of light saturation point were not statistically different between regions. Growth rates increased from marginal to basal and to middle parts of the thallus, whereas sporulation was highest in marginal (100%) followed by middle (30%) and basal parts (0%). Daily observation over 9 days showed that 56% of the basal cells divided once and did not produce spores, whereas every marginal cell went through its first division and 89% of the primary daughter cells also divided, resulting in 100% sporulation. A 7‐day treatment with PAR and PAR + UV‐A caused a significant decrease in the effective quantum yield of all thallus regions, followed by a recovery toward the initial values, whereas PAR + UV‐A + UV‐B irradiation led to greater photoinhibition and less recovery. Marked differences in the UV‐B sensitivity were observed, with marginal parts being more sensitive and basal parts most resistant.