Effect of amniotic fluid and fetal bovine serum on the morphogenesis of mouse duodenal villi in organ culture

Summary When duodenal explants of 15-day mouse foetuses are cultured in Trowell T8 medium during 72 h, villi do not develop. If mouse amniotic fluid (25%) is added to the same medium, short villi appear after 12 h of culture and medium size villi are seen at 48 h. Bovine amniotic fluid and fetal bovine serum promote cell growth but morphogenesis of intestinal villi is far less stimulated.Supported by grand MA-6069 from the Medical Research Council of Canada.     

Acceleration of the speed of neuronal growth by a new neurotropic substance: N-isopropyl-amino-2-pyrimidine]

16 hrs. after per os administration of 14C N-isopropyl-amino-2-pyrimidine (IAP) in the Rat, radioactivity of the sciatic nerve is significantly higher than in plasma and other organs. In vitro IAP (base, orthophosphate or dichloracetate) accelerates neurite outgrowth of explained spinal ganglion after 48 hrs. of incubation in nutritive medium. Results suggest that IAP incorporated in nerve cell acts on cellular mechanisms controlling nerve growth.     

结肠腺癌中结肠癌干细胞的分离、鉴定

目的从人结肠腺癌组织中分离、鉴定结肠癌干细胞,并初步观察其生物学特性。方法利用新鲜结肠腺癌组织,无血清悬浮成球培养,流式细胞检测ESA、CD44表达情况,体外观察其诱导分化及CK20、Muc表达情况,Balb／C小鼠移植观察其成瘤情况。结果从人原代结肠腺癌中分离、纯化EpCAM^high CD44^＋结肠癌干细胞,结肠癌原代细胞中EpCAM^highCD44^＋细胞比例为1．7％～38％（平均5．4％）。单克隆形成实验证实结肠癌组织中存在肿瘤干细胞。其比例为（2．07±0．11）％,分离获得的EpCAM^highCD44^＋细胞能在无血清培养基中“成球”,在血清诱导下能贴壁分化;将EpCAM^highCD44^＋细胞移植在Balb／C裸鼠体内,表现出很强的致瘤性,移植瘤中EpCAM^highCD44^＋细胞比例为3．6％～43．2％（平均15．2％）,所有的移植瘤经组织学测定,均形成腺管样结构,表达结肠特异性分化标志物CK-20、中性上皮粘蛋白（neutral epithelial mucins,Muc）。结论人结肠腺癌组织中存在EpCAM^highCD44^＋细胞群,具有和普通干细胞相类似的无限增殖、自我更新和分化能力。     

A new substrate for cultures of dissociated primary rat brain

Different substrates were used to coat plastic petri dishes for the cultivation of dissociated fetal rat brain cells. Only on surfaces which were coated with a mixture of serum and non-reconstituted collagen, did the majority of the inoculated cells attach singly or as aggregates within 24 h. The attachment of the cells was followed by the outgrowth of cellular processes either from single cells or from aggregates in the same time period. This did not occur on collagen or serum treated or on regular plastic dishes. Under the latter conditions a similar outgrowth was observed only after 3–5 days.     

Über die Beeinträchtigung der Wirkung von Spindelgiften in der Zellkultur durch menschliches Serum sowie durch Seren verschiedener Tierspezies

Summary Chick embryo fibroblasts, cultivatedin vitro, require higher concentrations of podophyllotoxin derivatives for complete mitotic arrest when the medium contains human adult or cord serum than when it contains horse, bovine, guinea pig, rat, mouse, or cock serum. This difference is due to a higher binding capacity of human serum for these compounds. No such difference was found with a colchicine and a peltatin derivative.     

Role of serum components in the binding and phagocytosis of oxidatively damaged erythrocytes by autologous mouse macrophages

To investigate the role of autologous serum components in the recognition of damaged cells by macrophages, we examined the binding and phagocytosis of damage oxidatively damaged red blood cells with Cu2+ and ascorbate (oxRBCs) by autologous resident mouse peritoneal macrophages. The binding of oxRBCs by macrophages was independent of the presence of serum. However, phagocytosis by macrophages increased with serum concentration, and macrophages showed little ingestion of oxRBCs in a serum-free medium. Macrophages neither bound nor appreciably ingested native RBCs (before oxidation) in either the absence or presence of autologous serum. Mouse macrophages ingested significantly more native as well as oxRBCs in the presence of heat-inactivated fetal calf serum than in the presence of heat-inactivated mouse serum. Pretreated oxRBCs with normal serum were rarely ingested by macrophages in a serum-free medium. Phagocytosis of oxRBCs was significantly inhibited by depletion of IgG or calcium from serum, by heat inactivation of complement, or by antiserum against mouse C3. These results demonstrate that serum components such as IgG, C3, and calcium are involved in phagocytosis of oxRBCs by autologous macrophages.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Emblica officinalis reduces serum, aortic and hepatic cholesterol in rabbits

Emblica officinalis reduced serum cholesterol (p less than 0.001), aortic cholesterol (p less than 0.001) and hepatic cholesterol (p less than 0.001) significantly in rabbits. Emblica officinalis did not influence euglobulin clot lysis time, platelet adhesiveness or serum triglyceride levels.     

Effect of cell proliferation on the condensation of the X chromosome in culture medium of the individual karyotype 49 XXXXX]

The influence of cell proliferation on the condensation of the X chromosome was observed in vitro in human fibroblasts with 49 XXXXX karyotype. The frequency of cells with four Barr-bodies is low during the logarithmic growth phase and increases to 80% when the cells are becoming confluent or, independently of cell contact, when cell growth is arrested in a medium with low serum content. The condensation of th X chromosomes is reversible when the cells start growing again in medium with a higher serum content.     

The stoichiometry of peptide-heparan sulfate binding as a determinant of uptake efficiency of cell-penetrating peptides

Binding to negatively charged heparan sulfates (HS) at the cell surface is considered the first step in the internalization of cationic cell-penetrating peptides (CPPs). However, little is known about the relation of the characteristics of the HS-CPP interaction such as affinity, stoichiometry, and clustering with uptake. In this study, we investigated a collection of mutants of a cyclic CPP derived from human lactoferrin with respect to HS binding and uptake. The thermodynamic parameters of HS binding were determined by isothermal titration calorimetry, clustering of HS was investigated by dynamic light scattering, and cellular uptake by flow cytometry and confocal microscopy. Whereas mutations of non-arginine amino acids that are conserved across lactoferrins of different mammalia only had a minor effect on uptake efficiency, changes in the number of arginine residues influenced the uptake significantly. In general, introduction of arginine residues and cyclization improved the HS affinity and the ability to cluster HS. In particular, there was a strong negative correlation between stoichiometry and uptake, indicating that crosslinking of HS is the driving force for the uptake of arginine-rich CPPs. Using glycan microarrays presenting a collection of synthetic HS, we show that a minimal chain length of HS is required for peptide binding.     

Effect of cell proliferation on the condensation of X chromosomes in the form of Barr-bodies, in human 46 XX fibroblast cultures with different serum concentrations]

The frequency of Barr-bodies in cultivated 46 XX human fibroblasts considerably increases when in confluent monolayer cultures the cells no longer divide. Or, independently of cell contact, when the cells are grown in medium with low serum content, which will slow down or entirely arrest further growth. The frequency of Barr-bodies again diminishes when after the administration of culture medium with sufficient serum concentration, the cells start growing again, indicating that the condensation of the X chromosome is reversible under control of the cell proliferation.     

Demonstration of neuronal activities in primary cultures from fetal mouse hypothalamus maintained in a serum-free medium

Fetal neuronal cells dissociated from mouse hypothalamus are able to grow in a serum-free medium for two to four weeks. Several neuronal activities have been measured during the in vitro cell development. Whereas glutamic acid decarboxylase (EC 4.1.1.15) activity disappears, those of thyroliberin (TRH) and tyrosine hydroxylase (EC. 1.14.16.2) remain stable, and acetylcholine transferase one (EC 2.3.1.6) rises earlier than in a serum supplemented medium. These results show that primary cultures grown in serum-free medium offer a promising model for studying neuronal activity differentiation.     

Tubulin pools in human erythrocytes: altered distribution in hypertensive patients affects Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity

The presence of tubulin in human erythrocytes was demonstrated using five different antibodies. Tubulin was distributed among three operationally distinguishable pools: membrane, sedimentable structure and soluble fraction. It is known that in erythrocytes from hypertensive subjects (HS), the Na⁺, K⁺-ATPase (NKA) activity is partially inhibited as compared with erythrocytes from normal subjects (NS). In erythrocytes from HS the membrane tubulin pool is increased by ~150%. NKA was found to be forming a complex with acetylated tubulin that results in inhibition of enzymes. This complex was also increased in erythrocytes from HS. Treatment of erythrocytes from HS with nocodazol caused a decrease of acetylated tubulin in the membrane and stimulation of NKA activity, whereas taxol treatment on erythrocytes from NS had the opposite effect. These results suggest that, in erythrocytes from HS, tubulin was translocated to the membrane, where it associated with NKA with the consequent enzyme inhibition.     

Hormone content of mouse pancreatic islets subjected to different in vivo and in vitro functional demands

Summary Mice treated for 4 days with tolbutamide displayed decreased serum glucose values with a concomitant decrease of their islet insulin content. Mouse islets cultured for 1 week at a low (3 mM) or a high (28 mM) glucose concentration contained less insulin than non-cultured islets and islets cultured at a medium (11 mM) glucose concentration. All groups of cultured islets contained more glucagon than non-cultured islets. The somatostatin content of high- and medium-glucose cultured islets was higher than that of freshly isolated islets.The skilled technical assistance of Astrid Nordin, Ewa Forsbeck and Eva Törnelius is gratefully acknowledged. Financial support was received from the Swedish Medical Research Council (12X-109; 12X-2297), the Nordic Insulin Fund and the Swedish Diabetes Association.     

The collagen substratum influences in vitro hatching and attachment of the mouse blastocyst in a serumless medium

Summary When a serumless medium is used for the in vitro growth and development of post-blastocyst mouse embryos, a collagen substratum causes a delay in the hatching from the zona pellucida. However, the collagen substratum is essential for blastocyst attachment and trophoblast cell outgrowth after hatching has taken place.     

Radioimmunoassay of some hormones simultaneously measured in serum and breast cyst fluid

Blood and breast cyst fluid were drawn simultaneously for hormonal determination. There was no difference between serum and cyst fluid values of PRL and TSH. A significant difference was noted for LH (p less than 0.01) and FSH (p less than 0.05), serum concentrations being higher than cyst fluid concentrations.     

人结直肠癌细胞系的成球培养方法及转移侵袭能力研究

目的探索人结直肠癌细胞系形成肿瘤细胞球的培养方法及球体内间质化标志物的表达检测。方法结直肠癌细胞系SW480、SW620、LOVO、HT29及HCT116在添加了不同细胞生长因子的无血清培养基（SFM）中悬浮培养,传代更新,诱导分化。免疫荧光技术和RT-PCR分别检测细胞球中间质化标志物N—cadherin及Vimentin的表迭情况。结果5种细胞系均能在特制的SFM中形成细胞球,并稳定的传代增殖。在用血清诱导分化后,又重新贴壁生长,且与亲代细胞保持一致的细胞形态。细胞球中间质化标志物N—cadherin、Vimentin表达均上调。结论结直肠癌细胞系在低黏附及添加了细胞生长因子R—Spondin 1、Noggin的无血清环境中更容易形成悬浮生长的肿瘤细胞球,细胞球细胞比亲代细胞更具有转移侵袭能力。     

Activators of serum lipoprotein lipase in alloxan diabetic rats

Summary The amount of free fatty acid (FFA) liberated with intralipid in post-heparin guinea-pig serum, when serum from alloxan diabetic rat is incubated, is higher than that liberated when serum from healthy rats is used. The above effect is probably due to larger quantities of lipoprotein lipase present in the diabetic serum.     

Persistence of added retinoids in organ culture media during induction of mucous metaplasia and glandular morphogenesis in hamster cheek pouches

The retinoid concentration (determined colorimetrically) did not change significantly in retinyl acetate-supplemented (6 micrograms/ml) Eagle's Minimal Essential Medium containing 10% fetal calf serum when stored at -20 or 4 degrees C over 7 days. After the medium was incubated at 37 degrees C for 48 h, 37-49% of the retinoid remained, whether or not tissue (neonatal Syrian hamster cheek pouch) was present, and irrespective of explant age. The normal retinoid level in the tissue was approximately 0.25 micrograms per gram. Therefore, neonatal hamster cheek pouches, incubated in medium with the addition of 6 micrograms of retinyl acetate per ml of medium and undergoing mucous metaplasia and some mucous gland morphogenesis, were continually being exposed to retinoid levels which, though gradually decreasing, remained well above their normal physiological level.