Modification of a method of cell enucleation using cytochalasin B and the study of cytoplast viability

The modification of Prescott's (Prescott et al., 1972) method of enucleation in vitro was described. A special teflon chamber faciliatating the enucleation of monolayer cultured cells to produce cytoplasts and karyoplasts was constructed. Mouse L-cells were enucleated by exposing to cytochalasine B (10 gamma/ml) followed by centrifugation. The fraction of cells enucleated in the chamber was about 98%. The life time of cytoplasts in cultural medium after their enucleation was 48 hours (sometimes 56-72 hours) as tested by vital neutral red staining. The cytoplasts that survived were shown to accumulate large lysosomes, and the evidence of appearing ring-like fibrillar structures was provided using a simple technique of cytoskeleton observation under light microscope.     

Heat shock modulates the ability of A-549 cell line from human lung adenocarcinoma to regulate the intensity of DNA and protein biosynthesis by an autocrine mechanism]

A-549 cells of human lung adenocarcinoma were subjected to heat shock (30 min, 44 degrees C) which caused substantial decreases in the rates of biosynthesis of the great bulk of cellular proteins with simultaneous increases in the synthesis rates of the 70 kDa protein predominantly localized in cell cytosol. By the 6th hour after the heat shock cessation this protein synthesis reached its maximum; by the 18th hour it was no longer detectable, while the protein itself was not denatured. During the recovery after the heat shock the ability of the serum-free culture medium conditioned by A-549 cells in autocrine regulation of [3H]thymidine incorporation into DNA and [3H]leucine incorporation into proteins changed also. The conditioned medium obtained within 1-3 hours after the heat shock did not influence the intensity of DNA synthesis, while the medium obtained 4-48 hours after the heat shock stimulated this process, the maximal effect (3.3-fold stimulation) being observed in the case of the 48-hour conditioned medium. Temporary (1 hour) acidification of the conditioned media down to pH 2.0 resulted in complete inhibition of the stimulating activity. Besides, these media acquired an ability to inhibit [3H]thymidine incorporation into the DNA of tracer cells. Study of effects of conditioned media on the rate of [3H]leucine incorporation into A-549 cell proteins revealed that the media obtained 1-4 hours after the heat shock inhibited this process, while the media obtained 6-18 hours thereafter stimulated it 1.2-2.1-fold. In the test systems under study temporary acidification of the media increased their stimulating influence on [3H]leucine incorporation into cellular proteins.     

In vitro fertilization of horse follicular oocytes matured in vitro

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 x 10(6) sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophore-treated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.     

Kinetics of cellular proliferation and hormonal receptors in EVSA-T breast cancer cell line.

The hormone-independent human breast cancer cell line EVSA-T, originally described as negative estrogen and progesterone receptors is shown to become positive hormone receptors when the cellular proliferation rate is slowed down. The experimental procedure included the following steps: 1) EVSA-T cells were seeded in minimum essential medium supplemented with 10% fetal bovine serum and kept undisturbed for 2 days; 2) culture medium was replaced with Dulbecco's solution and Ham's F-12 and cells were incubated in serum-free media for another 24 h; 3) then, cells were "rescued" with 10% FBS supplemented medium and estrogen (ER) and progesterone receptors (PgR) were measured immediately, time 0, and 6, 12, 18, 24 and 30 h after the media were changed. Cell yield was quantified at the same times. Experimental data indicate that changing the proliferation kinetics makes it possible to detect estradiol and progesterone receptors on EVSA-T cells. Estrogen receptor appeared at 18 h after rescue, 6 hours before progesterone receptor could be detected. Immunohistochemical analysis of ER content confirmed this observation, showing maximal positive stain at 18 h. Furthermore, ER disappeared when cells recovered their normal proliferation rate.     

Staining of Paneth Cells with Best's Carmine After Methylation

When sections are methylated (cone. HC1, 0.8 ml in absolute methanol, 100 ml; at 58 C) prior to staining with Best's carmine, the granules of Paneth cells of man, rat and mouse stain a bright red, but they do not stain at all with this stain without prior methylation. With paraffin sections after neutral formalin fixation, the required 2-hr methylation did not prevent the staining of neutral mucosubstances and glycogen, but after methylation for 12 hr, these substances no longer stained although the reaction of the granules of Paneth cells became still more intense. The advantages of this staining technique are: (1) There is good contrast because the background stains faintly and, of the structures in the intestinal wall, only eosinophilic leukocytes and a part of the collagen fibrils stain in addition to the granules of Paneth cells. (2) The result is more reliable and the staining easier to perform than with the majority of other techniques, since no differentation is necessary. The method is especially suited for detecting Paneth cells in pathological conditions and in altered tissues or areas in which these cells are scanty.     

氧化低密度脂蛋白对THP-1巨噬细胞吞噬和分泌功能的影响

目的:探讨氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)对巨噬细胞源性泡沫细胞吞噬功能和炎症相关因子分泌功能的影响。方法:利用佛波酯(phorbol ester,PMA)诱导THP-1细胞分化形成巨噬细胞,之后采用ox-LDL处理48小时后,诱导其形成泡沫细胞。利用中性红吞噬实验,分析泡沫细胞形成前后吞噬功能的变化;通过ELISA法,检测细胞培养上清中肿瘤坏死因子α(tumor necrosis factorα,TNF-α)含量,观察ox-LDL对THP-1巨噬细胞功能的影响。结果:细胞形态学结果表明,我们成功利用ox-LDL诱导THP-1巨噬细胞形成泡沫细胞;进一步发现ox-LDL诱导THP-1巨噬细胞表面的清道夫受体CD36表达升高,并促进细胞吞噬功能增加,进一步促进细胞内胆固醇含量显著升高(P0.05);同时,ox-LDL能够刺激巨噬细胞大量分泌TNF-α(P0.05)。结论:ox-LDL通过增强清道夫受体CD36表达,提高巨噬细胞的吞噬功能,引起大量胆固醇聚集,产生细胞毒性损伤,并促进TNF-α炎性因子的大量分泌。     

Characterisation of different morphological features of black tiger shrimp (Penaeus monodon) haemocytes using monoclonal antibodies

Monoclonal antibodies (mabs) specific for Penaeus monodon haemocytes were produced by immunising mice with membrane lysates of shrimp haemocytes. Four mabs (WSH 6, WSH 7, WSH 8 and WSH 16) were characterised using flow cytometry, light microscopy, laser scanning microscopy, electron microscopy and immunoprecipitation. WSH 6 recognised a carbohydrate determinant on an 85 kDa molecule. WSH 7, WSH 8 and WSH 16 recognised 50, 35 and 115 kDa molecules, respectively. For all mabs, differences in amount and intensity of the labelling were found when haemocytes were fixed immediately in 2% formaldehyde in Alsever's Solution (AS), compared with non-fixed haemocytes that were kept in AS (which reduced activation of the haemocytes) or in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of >80% of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mabs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS and in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different size and electron density. Immunoreactive extracellular thread-like material could be observed in cells in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and the lowest for WSH 16. Double labelling revealed that all mabs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis is put forward that immunoreactive molecules recognised by these mabs, are related to haemocyte activation factors.     

Secretion of the intercalated cells of the fibrillar epithelium of the mucous membrane of the maxillary sinus]

Cytological and histochemical methods were used in order to establish that the intercalated cells of the ciliated epithelium of the rabbit's maxillary sinus mucosa performed gland function giving secretion formed in the shape of granules and represented by neutral mucopolysaccharides. Analysis of the morphology of intercalated cells, the amount of secretion in them, the shape and disposition of the Golgi apparatus and the nucleus allowed establishing 4 successive phases in their secretory cycle: the phase of synthesis, accumulation, aquosity and sectering. Under normal conditions of life the secretory activity of intercalated cells was asynchronous. In experiments with synchronization of the secretory activity by administration of pilocarpine it was established that the duration of the intercalated cells secretory cycle was 16-18 hours. Active participation of alkali phosphatase in the synthesis of secretion in the ciliated epithelium was noted.     

Regulation of cyclic nucleotide phosphodiesterase forms by serum and insulin in cultured fibroblasts.

A rapid reduction of cyclic nucleotide phosphodiesterase activity occurs after the replating of confluent cultures of BHK 21 c/13 fibroblasts into fresh medium. This reduction in activity depends on the density to which the cultures are reseeded and the concentration of serum in the medium. Enzyme activity in BHK cells is restored after 24 to 48 hours if cells are diluted into medium containing 10% fetal calf serum or 0.5% fetal calf serum supplemented with insulin (10(-6)M), but not into 0.5% serum alone. The restoration in enzyme activity is blocked by cycloheximide or Actinomycin D. When BHK cells become quiescent by maintanance in 0.5% serum conditions for 48 hours, a rapid (15--60 minutes) increase in cyclic AMP phosphodiesterase activity occurs when 10% serum is added to the cultures. Enzyme activity is increased even further after 24 to 48 hours in the 10% serum. Cycloheximide or Actinomycin D do not affect the rapid increase in enzyme activity in response to serum, but completely inhibit the long term increase. In contrast to serum, insulin (10(-8) to 10(-6)M) has no short term effect, but does increase enzyme activity after 24 to 48 hours to levels comparable to those seen with addition of 10% serum. As is the case with serum, this long term effect of insulin on enzyme activity is prevented by inhibitors of protein and RNA synthesis. Kinetic analyses of cyclic AMP phosphodiesterase activity in homogenates of quiescent BHK cells indicate the presence of only high Km (congruent to 20 muM) enzyme activity. Addition of serum or insulin to quiescent cells results in the appearance of apparent low Km enzyme activity in homogenates. Sucrose gradient analysis of BHK cells displays two forms of cyclic AMP phosphodiesterase enzyme activity: a 3--4 S form and 5--6 S form. In quiescent cells, the 5--6 S form greatly predominates relative to the 3--4 S form. Addition of serum to quiescent cells results in a rapid appearance of increased 3--4 S form enzyme activity. Insulin also increases the activity of this higher affinity 3--4 S enzyme form after 24 to 48 hours in culture. The functional significance of short and long term regulation of cyclic nucleotide phosphodiesterase(s) in cells is discussed.     

Development of a biofilm model for Listeria monocytogenes EGD-e

The ability to form persistent biofilms makes the pathogenic bacterium Listeria monocytogenes a hazardous contaminant in food processing environments. Growth and biofilm formation of L. monocytogenes EGD-e were studied in defined medium (HTM) and in tryptic soy broth (TSB) with different supplements. TSB + 1% glucose gave optimal results. Using this medium, biofilm development on the model surface polystyrene (microtiter plate) was monitored by the standard crystal violet staining for adherent cells after bacterial cultivation for 24 and 48 h at five different temperatures (4, 18, 25, 30 and 37°C). In parallel, the matrix exopolysaccharide formed after 48 h of incubation was quantified by staining with ruthenium red. In both assays incubation at 30°C yielded the highest values. The formation of larger scale biofilms on dialysis membranes, placed on TSB agar with 1% glucose for 48 h, was studied by scanning electron microscopy. Contiguous and multilayered biofilms were observed at 18, 25, 30 and 37°C incubation temperature. The methodology is suitable for quantitative and microscopic studies and, in addition, yields sufficient cell mass for subsequent biochemical and molecular biological analyses.     

Loss of Rna and Protein, And Changes in Dna During A 30-Hour Cold Perchloric Acid Extraction of Cultured Cells

KB cells derived from human carcinoma were fixed in acetic-alcohol (1:3) and extracted with 10% perchloric acid (PCA) at 4 C for 1, 3, 6, 9, 12, 24 and 30 hr. Cells were then washed in water and stained for nucleic acids, proteins, polysaccharides, and lipids. Control cells were kept in water for 30 hr prior to staining. Acridine orange (AO) fluorochroming revealed color changes in residual cytoplasmic and nucleolar RNA as well as DNA during extraction--interpreted as indicative of molecular alterations. All nucleic acid stains (AO, gallocyanin chromalum, and azure B bromide) demonstrated a differential extraction of RNA, with cytoplasmic RNA being removed in about 6 hr and nucleolar RNA requiring 6 more hours for complete extraction. Large granules appeared early in nuclei. These were positive for DNA by azure B, gallocyanin chromalum, Feulgen, and fluorescent-Feulgen. These same granules stained for protein by mercuric bromphenol blue and alkaline Biebrich scarlet. At 24 hr, there was visual and Feulgen-cytophotometric evidence for a slight loss of DNA, which may amount to 10-20%. There was a progressive loss of cytoplasmic and nuclear but not nucleolar protein during PCA treatment. Concurrently, large protein-positive granules appeared in the cytoplasm. Apparently, PCA treatment in combination with an aqueous wash was responsible for some protein loss. Glycogen was gradually lost (fluorescent PAS) and redistributed in cells. Lipids were unaffected (Sudan black B).     

Identification of normal and leukemic granulocytic cells with merocyanine 540

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytes and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in erythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single agent stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.     

Identification of Normal and Leukemic Granulocytic Cells with Merocyanine 540

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytos and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in crythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single a p t stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.     

Vital Staining with Neutral Red and Trypan Blue of 3H-Thymidine-Labeled Cells Prior to Autoradiography

Mouse cells cultivated in vitro were continuously labeled with tritiated thymidine for 48 hr. In representative groups of labeled cells, the percentages of dead cells were obtained by vital staining with either neutral red or trypan blue. After fixation and auto-radiography, the fraction of nonlabeled cells was determined in the same group of cells which had been used for interpretation of viability. Neither neutral red nor trypan blue used prior to autoradiography caused spurious grain formation in the emulsion.     

Normal human epidermal keratinocytes express in vitro specific molecular forms of (pro)filaggrin recognized by rheumatoid arthritis-associated antifilaggrin autoantibodies.

BACKGROUND: The so-called antikeratin antibodies and the antiperinuclear factor are the most specific serological markers of rheumatoid arthritis (RA). They were recently shown to be largely the same autoantibodies and to recognize human epidermal filaggrins and profilaggrin-related proteins of buccal epithelial cells (collectively referred to as (pro)filaggrin). MATERIALS AND METHODS: To further characterize the target antigens, we investigated their expression by normal human epidermal keratinocytes cultured in differentiating conditions, using immunofluorescence and immunoblotting with RA sera and three different monoclonal antibodies to (pro)filaggrin. RESULTS: On the cornified, stratified epithelial sheets obtained in vitro, RA sera with anti(pro)filaggrin autoantibodies (AFA) produced granular staining of the stratum granulosum and diffuse staining of the stratum corneum. The antigens recognized by RA sera strictly colocalized with (pro)filaggrin in keratohyalin granules. Following sequential extraction of the proteins from the epithelial sheets, the RA sera and the three monoclonal antibodies to (pro)filaggrin, recognized a series of low-salt-soluble molecules, including a neutral/acidic isoform of filaggrin and several proteins with sizes and pI intermediates between this isoform and profilaggrin. They also recognized urea-soluble high-molecular-weight profilaggrin-related molecules. CONCLUSIONS: These results show that in vitro epidermal keratinocytes express various molecular forms of (pro) filaggrin that bear epitopes targeted by AFA of RA sera, and that some of these are absent from epidermis. Moreover, these epitopes, which are present on the keratohyalin granules of buccal epithelial cells but not on those of epidermal cells, are present on the granules of the cultured keratinocytes. This work completes the molecular characterization of the proteins targeted by AFA.     

RELATIONSHIPS BETWEEN DISTRIBUTION OF TUBULIN-PARACRYSTALS,GRANULES STAINING WITH NEUTRAL RED AND CLEAVAGE ACTIVITY IN SEA URCHIN EGG-FRAGMENTS1,2

Unfertilized eggs of Japanese sea urchins (Temnopleurus toreumaticus and Hemicentrotus pulcherrimus)were separated by centrifugation into two fractions (nucleated light and enucleated heavy fragments) or three fractions (nucleated light, enucleated middle, and enucleated heavy fragments). These fragments were stained with neutral red and then fertilized. Cleavage took place only in fragments containing cytoplasmic granules staining with neutral red: no cleavage occurred in fragment without these granules. When fragments of unfertilized eggs were incubated in a solution in sea water of 10^?4M vinblastine, a mitotic poison that specifically binds to tubulin, tubulin-paracrystals were found in all kinds of fragments, irrespective of whether they had stained granules and cleavage activity. These results suggest that lack of cleavage activity in the fragments is not due to the absence of polymerizable tubulin molecules in the cytoplasm, but rather to other factors, such as the absence of granules staining with neutral red. In other words, there is no relation between the distribution of these granules and polymerizable tubulin, but a close relation between the number of stainable granules and cleavage activity. Quantitative analysis of tubulin molecules in the egg fragments is necessary for confirmation of this idea.     

NOTE ON THE PENETRATION OF HYDROXYL IONS INTO GELATIN JELLIES

O ne of the characteristic features of living cells is that they are permeable to ammonia, but relatively impermeable to caustic soda or caustic potash. The usual method of demonstrating this fact is by staining cells with neutral red. This dye is readily taken up by living cells and is at first diffused through the cytoplasm, which thereby acquires a distinct red colour. At a later stage the dye is usually aggregated into irregular granules. When exposed to dilute solutions of ammonia the neutral red in living cells rapidly becomes yellow, indicating the penetration of the alkali. In NaOH or K. OH the colour of the dye does not change, however, until the cell begins to show obvious signs of degeneration.     

Control of alkaline phosphatase activity in C3H10T1/2 cells: role of retinoic acid and cell density.

The enzyme alkaline phosphatase (AP) has been shown to be lost or inappropriately expressed during carcinogenesis in some tissues. Because retinoic acid (RA) appears to play a role in the normal regulation of the enzyme (RA up-regulates AP in a variety of cell types) we have suggested that altered AP expression in some cancers may be caused by a defect in the ability of the cells to respond normally to retinoid. We have begun to use the chemically transformable mouse embryo fibroblast cell, C3H10T1/2, to investigate this possibility. In this initial study we characterized AP regulation in normal C3H10T1/2 cells and show that: (1) 10(-7) M RA increases AP activity within 3-4 h in serum-free medium; (2) serum inhibits short-term induction (0-8 h) in a concentration-dependent manner (10% serum causes complete inhibition); (3) during long-term RA exposure (24 h and 48 h), induction can be detected in serum-containing medium; (4) AP induction is dose related at RA concentrations from 10(-10) M to 10(-6) M in serum-free medium; (5) 10(-5) M RA is ineffective at inducing AP in serum-free medium during 8 h but is the most effective concentration in serum-containing medium during 24 h and 48 h exposures; (6) AP inducibility by RA requires near-confluent cell densities; and (7) when cultures become confluent, cells become constitutive for AP and no longer require RA for enzyme expression. The effects of serum and cell density on AP inducibility by RA and implications of the RA up-regulation of AP for teratogenesis are discussed.     

脂肪来源干细胞体外成骨和成脂及成神经的诱导分化研究

目的:探讨脂肪来源干细胞体外成骨和成脂及成神经的诱导分化情况。方法:选取10只SPF级雄性SD大鼠,将其不同部位的脂肪组织取出,分别采用不同方法对其向成骨、成脂及成神经等方向进行诱导分化并对其结果进行鉴定。结果:ADSC表达中,CD29占(99.11±0.13)%,CD44占(95.94±0.71)%,CD45占(0.12±0.09)%。经4周的成骨诱导后,茜素红S染色在细胞团中央发现红色钙化结节存在,碱性磷酸酶染色在细胞的胞质内观察到紫红色颗粒,经7d成脂诱导后,油红"O"染色在细胞质内观察到橙红色脂滴;经过6d的神经干培养基诱导后,通过免疫荧光染色证明诱导的Nestin细胞、神经丝蛋白-200以及GFAP等均出现阳性表达。结论:ADSC具备向脂肪、成骨及神经元等细胞进行多向分化的潜能,具有来源广、易于操作、体外增殖快速等优越性,并且不存在免疫排斥及医学伦理学问题,发展前景广阔。     

Stick-plaque test--an economic method of quantitative determination of viruses]

An economic method for quantitative assay of viruses is presented. In this "canule stick-plaque test" (German abbreviation SPT) samples of viruses, geometrically diluted and taken up by a canule, are inoculated by sticking into monolayer cell cultures overlayed with agar medium. A plaquelike CPE detectable by neutral red staining develops in the area of the inoculation. The frequency of this CPE formation depends on the concentration of viruses in the inoculated dilution. This dose-response allows calculation of the ID50. In this way it is possible to carry out titration involving 6 dilutions and 10 inoculations per dilution using 3 common Petri dishes (6 cm in diameter), only. The sensitivity, accuracy, and reproductibility of this method are described and discussed.