金属硫蛋白与红细胞的相互作用

根据金属硫蛋白(MT)对标记在膜上的马来酰亚胺自旋标记物的ESR波谱的影响,研究了MT与红细胞膜的相互作用,发现不同种属的MT对膜构象的影响不同.体外实验表明,MT可以吸附在红细胞的表面,用CdCl₂诱导家兔,对血浆和红细胞溶血液中的MT组分进行色谱分离,发现血液中的MT主要存在于血细胞中.进而对血液MT的来源、分布及其重要的生物学意义进行了讨论.     

Interaction of mammalian kidney membrane and mitochondria in vitro

1. In many parts of the kidney tubule, there exists a close physical association of membrane and mitochondria apparently related to salt transport. The present study examines the influence of adding rat kidney membrane fragments on mitochondrial respiration in vitro. The results are compared with previous findings on the effect of rat kidney membrane fragments on cytoplasmic glycolysis.

2. The membrane fraction stimulates mitochondrial respiration. The stimulus decreases with higher levels of ADP and is abolished by dinitrophenol. Stimulus of glycolysis by kidney membranes is related to a specific coupling of membrane ATPase activity and phosphoglycerate kinase. The stimulus of respiration appears to be due solely to the enhanced availability of ADP in presence of an ATPase system.     

cAMP levels in proliferating rat mammary epithelial cells in vitro and in vivo

Agents that elevate intracellular cAMP levels are required for growth of many cell types in culture including normal rat mammary epithelial (RME) cells. To determine if the intracellular levels of cAMP that result from stimulation by agents such as cholera toxin (CT) or prostaglandin E-1 (PGE-1) are within the physiological range, cAMP levels were determined in RME cells growing in primary culture and compared to levels measured in freshly isolated mammary epithelium. The results indicate that the cAMP levels of mammary epithelial organoids obtained from 45-day-old virgin rats are 4 to 6 pmol/10⁶ cells. Growth of RME cells in primary culture in the presence of CT results in cAMP levels of approximately 15 to 20 pmol/10⁶ cells early in culture when cells are proliferating rapidly. As cells approach confluence, cAMP concentrations decrease to levels observed in fresh organoids. CT-stimulated cAMP levels appear to be within the range of those found in pregnant mammary epithelium in vivo. Growth of RME cells in medium supplemented with PGE-1 instead of CT results in cAMP levels equivalent to those found in fresh mammary epithelial organoids and under these conditions the growth rate is approximately half that found in CT-stimulated cells. These results indicate cAMP to be a positive regulator of cell growth in vivo at levels that are within the physiological range.     

Interaction of sporozoites of Theileria parva with bovine lymphocytes in vitro. 1. Early events after invasion

Sporozoites of Theileria parva rapidly enter bovine lymphocytes by a mechanism of passive endocytosis that depends upon progressive circumferential binding of ligands on the parasite to receptors on the host-cell membrane. Within 10 min of entry, the micronemes of the sporozoite discharge their content and the enveloping host-cell membrane is lysed. The host cell responds within 30 min of invasion by polymerization of microtubules arrayed tangential to the sporozite and converging upon the cytocentrum. Multivesicular bodies and lysosomes are generated and gather around the parasite but are ineffective in the absence of an endocytotic membrane with which they can fuse. Thus Theileria parva can be added to the category of obligate intracellular parasites that ensure their survival by lysis of the parasitophorous vacuole.     

Immunolocalization of alpha-transforming growth factor in the developing rat mammary gland in vivo, rat mammary cells in vitro and in human breast diseases

Summary Immunoreactive alpha-transforming growth factor (-TGF) was shown by immunocytochemistry to be present in the rat mammary gland at various stages of development, the staining being most intense in mature myoepithelial cells. -TGF was also detected in the secretions of the mammary glands of pregnant and lactating rats. -TGF in the extracts of rat mammary glands at each stage of development, and in several rat mammary cell lines and in culture medium in which they had been grown, was shown by Western blotting to consist primarily of a protein of molecular weight 50 kDa. The amount of this protein was greater in the mammary gland of the lactating rat than in resting or involuting glands. -TGF was also found in some, but not all, human breast carcinomas, and in benign hyperplastic breast diseases.     

Bleb formation in granulosa cells of rat pre-ovulatory follicles in vivo and in vitro

To determine if hormone-induced events leading to ovulation an granulosa cell luteinization might be associated with changes in the surface configuration of granulosa cells we have studied the morphology of granulosa cells from the preovulatory follicles both in vivo and in vitro. In vivo, granulosa cells in follicles from rats primed with estradiol and FSH developed bulbous protrusions termed blebs in response to injected hCG. The blebs were restricted to the adluminal granulosa cells which possess the least number of receptors for hCG. When granulosa cells from follicles of rats primed with estradiol and FSH were cultured in vitro, in the absence of serum, approximately 10% of the cells formed blebs. In the presence of 10% rat or fetal calf serum, nearly 90% of the cells formed blebs by 18 hr. Serum-induced bleb formation was prevented by 1 mM dibutyryl cycle-AMP plus 0.5 mM methyl isobutyl xanthine and by cytochalasin B (25 mug/ml), while 0.1 muM colchicine had no effect. Fibronectin at 25 mug/ml increased bleb formation three-fold over control values in serum-free medium. When hCG was included in serum containing medium, the majority of the cells remained smooth without any blebs. Thus, in contrast to its action in vivo, hCG inhibited the formation of blebs in vitro. When the cells incubated in the presence of dbcAMP plus methyl isobutyl xanthine in serum-containing medium, none of the cells formed blebs. One explanation for the seemingly opposite actions of hCG in vivo and in vitro is that hCG might act to alter the permeability of the pre-ovulatory follicles, and thereby allow the admission of serum. The admitted serum component(s) could then induce the formation of blebs on receptor-deficient adluminal cells that did not have elevated cAMP concentrations. The results suggest that fibronectin and/or other serum components, act to induce microfilament-dependent, cAMP-inhibited bleb formation on granulosa cells in vivo and in vitro.     

睡菜离体快繁技术的研究

以睡菜的幼嫩茎段为外植体,接种到附加不同浓度激素配比(6-BA/NAA)的MS培养基,诱导睡菜愈伤组织、芽及根的生长。研究发现,外植体在1.0mg/L 6-BA+0.1mg/L NAA+MS的培养基上培养10d,可观察到浅绿色的愈伤组织。愈伤组织转接到4.0mg/L 6-BA+0.3mg/L NAA+MS培养基上2周左右可生成芽。对带芽的愈伤组织再进行诱导生根进而形成完整再生植株,最适根诱导培养基为0.3mg/L 6-BA+1.0mg/L NAA+MS培养基。该实验采用植物离体快繁技术成功建立了睡菜再生体系,为睡菜种苗规模化奠定了技术基础。     

Novel in vitro and in vivo inhibitors of prolyl endopeptidase

Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.Inhibition of prolyl endopeptidase by Z-cyclohexyl prolinal and Z-indolinyl prolinal occurs with slow, tight binding inhibition and K_i values of 2 – 3 nM. In vivo enzyme inhibition is also observed with a half time for recovery of enzyme activity of 3 – 4 h.     

Prostaglandin production by human peripheral blood monocytes changes with in vitro differentiation

To investigate the influence of in vitro culture on prostaglandin (PG) production, human monocyte-enriched peripheral blood mononuclear cells were isolated and incubated on gelatin-coated plates. On days zero, five and eleven of culture, the cells were examined microscopically and the production of PGF_2α, PGE₂, PGD₂, F metabolite (PGFM) and E metabolite (PGEM) were measured by radioimmunoassay. Differences in PG output were analyzed using the Wilcoxon and Friedman tests. Freshly isolated human peripheral blood monocytes produced mainly PGE₂. In vitro, however, PGE₂ production decreased from 196 (48–288) fmol/10⁶ cells per 3h on day zero of culture to 28 (6–51) on day eleven (p=0.04); median (range), n=7. Prostaglandin D₂ and PGEM output decreased similarly, but these differences failed to reach significance. Prostaglandin F_2α and PGFM output, on the other hand, increased from 32 and 19 fmol/10⁶ cells per 3h, respectively, on day zero of culture to 127 (p<0.05) and 58 (p=0.01) on day eleven. Changes in PG output were associated with in vitro differentiation as evidenced by changes in cellular morphology. These result suggest that differentiation of human peripheral blood monocytes in vitro is accompanied by a shift in PG output from PGE₂ and PGD₂, towards PGF_2α.     

Phagocytosis by carp granulocytes; in vivo and in vitro observations

The phagocytic ability of carp (Cyprinus carpio L.) granulocytes was evaluated in vivo and in vitro. In suspensions of head kidney cells, neutrophil granulocytes incorporated both latex beads and coccidian merozoites. In intestinal tissues from carp with a Goussia carpelli infection, all granulocyte cell types (neutrophils and cells of the basophilic-eosinophilic complex) phagocytosed cell detritus and coccidian developmental stages, mainly merozoites.     

Corticotropin releasing factor stimulates cAMP formation in pituitary corticotropic tumor cells

Addition of corticotropin-releasing factor (CRF) to membranes from two ACTH-secreting pituitary tumors strikingly increased in a dose-dependent fashion adenylate cyclase (AC) activity. Significant stimulation was already apparent at 10(-9)M CRF. Stimulation of AC activity by CRF in membranes from non-tumoral tissue adjacent to tumoral corticotrophs was considerably lower, and was lacking in membranes from a growth hormone secreting tumor. These data correlated well with in vivo pre-surgery and post-surgery ACTH responsiveness to CRF of the tumor bearing patients. Basal AC activity was higher in pituitary adenomas than in non-tumoral adjacent tissue. It is concluded that 1) a CRF-sensitive AC exists in ACTH-secreting tumor cells and, 2) increased sensitivity to CRF, as evidenced by greater stimulation of AC activity, may be responsible for the increased ACTH output of tumoral corticotrophs.     

15-hydroxyeicosatetraenoic acid stimulates migration of human retinal microvessel endothelium in vitro and neovascularization in vivo

We evaluated 15-hydroxyeicosatetraenoic acid (15-HETE), a major arachidonic acid product of vascular endothelium and leukocytes, for its effect on neovascularization. In a modified Boyden chamber assay, 15-HETE (10⁻⁷ M) sitmulated human retinal microvessel endothelial cell migration by 42 ± 10% (mean ± S.E.M., p<0.01). 12-HETE, a major arachidonic acid metabolite of platelets, had no such effect. Further studies in the rabbit corneal pocket assay revealed that 15-HETE stimulated neovascularization . Concentrations at which the effects were observed are within the range generated by several cell types and are achievable in human serum. 15-HETE stimulation of human endothelial cell migration and neovascularization suggests that it may play a role in vasoproliferative disorders.     

Radiosensitization mechanism of riboflavin in vitro

Riboflavin, suggested to be a radiosensitizer, was studied in murine thymocytes and human hepatoma L02 cell line in vitro with MTT method and fluorescence microscopy. When the murine thymocytes treated with 5–400 μmol/L riboflavin were irradiated by 5 Gy ⁶⁰Co γ ionizing radiation, the low concentration groups, i.e. treated with 5–50 μmol/L riboflavin, showed a different surviving fractions-time relating correlation compared with the high concentration groups, i.e. treated with 100–400 μmol/L riboflavin. The former had a high survival level at the end of irradiation, but which, after 4-h incubation, decreased rapidly to a low level. On the contrary, the high concentration groups showed a low survival level at the end of irradiation, and a poor correlation was found between the surviving fraction and the incubation time, after 4 h a little difference was observed. The results of fluorescence microscopy indicated that under low concentration conditions, the riboflavin localized mainly in nucleus (both perinuclear area and inside of nuclear membrane), while under high concentration conditions, intensive riboflavin also localized around cytoplasmic membranes. Thus we can conclude: the riboflavin had radiosensitivity effect on DNA under low concentration conditions, and enhanced the damage to cytoplasmic membrane under high concentration conditions. Also the most effective concentration of riboflavin can be evaluated to be approximate 100 μmol/L.     

Dietary phytoestrogens and cancer: in vitro and in vivo studies

Thirty postmenopausal women (11 omnivores, 10 vegetarians and 9 apparently healthy women with surgically removed breast cancer) were investigated with regard to the association of their urinary excretion of estrogens, lignans and isoflavonoids (all diphenols) with plasma sex hormone binding globulin (SHBG). A statistically significant positive correlation between urinary total diphenol excretion and plasma SHBG was found which remained statistically significant after elimination of the confounding effect of body mass determined by body mass index (BMI). Furthermore we found a statistically significant negative correlation between plasma SHBG and urinary excretion of 16α-hydroxyestrone and estriol which also remained significant after eliminating the effect of BMI. Furthermore we observed that enterolactone (Enl) stimulates the synthesis of SHBG by HepG2 liver cancer cells in culture acting synergistically with estradiol and at physiological concentrations. Enl was rapidly conjugated by the liver cells, mainly to its monosulfate. Several lignans and the isoflavonoids daidzein and equol were found to compete with estradiol for binding to the rat uterine type II estrogen binding site (the s.c. bioflavonoid receptor). It is suggested that lignans and isoflavonoids may affect uptake and metabolism of sex hormones by participating in the regulation of plasma SHBG levels and in this way influence their biological activity and that they may inhibit cancer cell growth like some flavonoids by competing with estradiol for the type II estrogen binding sites.     

Cell-mediated lymphocytotoxicity following in vitro sensitization of frozen stored human mononuclear cells in heterologous serum

Human peripheral mononuclear cells were subjected to controlled-rate freezing and stored at−196 °C. Following rapid thawing and slow removal of DMSO, the cells exhibited normal immune responsiveness to phytohemagglutinin and in mixed lymphocyte culture. After in vitro sensitization to frozen allogeneic cells in heterologous serum they developed cell-mediated lymphocytotoxicity demonstrated by ⁵¹Crrelease from specific targets isogeneic with the sensitizing cells.