Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates

Summary For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-suppelemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates. This work was supported by a grant no. 61771923 from the Ministry of Education, Science and Culture of Japan.     

Spatio-temporal distribution patterns of two tachinid flies,Epicampocera succincta andCompsilura concinnata parasitizingPieris

Larvae of genus Pieris in the northern part of Kyoto City are parasitized by two tachinid flies:Epicampocera succincta, a specialist on genus Pieris, and Compsilura concinnata, a generalist with very wide host-range. We surveyed the parasitism rates of Pieris by both flies for two years at six study areas. In these study areas, there lived three host species in the genus Pieris: P. rapae, P. melete, and P. napi, but neither tachinid parasitized P. napi to any significant extent. In the mountainous district, P. rapae and P. melete coexisted and their populations were relatively continuous, while in the lowland, only P. rapae larvae were abundant in spring and autumn, but even they disappeared in summer. Parasitisms by E. succincta occurred mainly in mountainous district and never in the lowland. C. concinnata parasitized Pieris in all the areas, but its parasitisms occurred mainly in autumn. We analyzed the factors affecting the spatial and temporal patterns of parasitism rates and presumed that the temporal discontinuity of host population restricted the distribution of the specialist parasitoid.     

Changes of lung surfactant and pressure-volume curve in bleomycin-induced pulmonary fibrosis.

We investigated whether alveolar surface force increased and participated in the lung pressure-volume relationship in bleomycin-induced pulmonary fibrosis in hamsters and, if so, whether lung surfactant was hampered in the lungs. On the air-filled pressure-volume curve, decreases of lung volume from control level were significantly higher at 3-8 cmH2O pressure on day 10 than on day 30. Because the change of lung tissue elasticity evaluated from the saline-filled pressure-volume curve was equal for the 2 days, the higher decrease of air volume on day 10 was due primarily to contribution of alveolar surface force. Pressure differences between deflation limbs of air-filled and saline-filled pressure-volume curves, which represented net alveolar surface force, were significantly higher at any lung volume between 50 and 90% total lung capacity on day 10, but almost no significance was observed on day 30. Phospholipid concentration in bronchoalveolar lavage fluid significantly decreased on day 10 but had improved by day 30. Analysis of phospholipid species in purified lung surfactant showed decreased fractions of disaturated phosphatidylcholine and phosphatidylglycerol on day 10. Surface-active properties of the surfactant, measured by a modified Wilhelmy balance, were remarkably hampered on day 10, but most of them had improved by day 30. We consider that the quantitative and functional abnormalities of lung surfactant have a part in the aggravation of lung mechanics in the acute phase of pulmonary fibrosis.     

Plant regeneration from isolated microspore cultures of Chinese cabbage (Brassica campestris spp. pekinensis)

Isolated microspores of Chinese cabbage (Brassica campestris ssp. pekinensis) were incubated in modified NN medium containing 10% sucrose in darkness at 33°C for one day followed by culture at 25°C. After 14 days of culture, microspores developed into embryos ranging from globular to cotyledonary stage. Plants were regenerated after transfer of embryos to medium containing 3% sucrose and no plant growth regulators.Abbreviations NN Nitsch and Nitsch - MS Murashige and Skoog - NAA naphthaleneacetic acid - BA 6-benzylaminopurine     

Detection of multiple forms of human ceruloplasmin. A novel Mr 200,000 form

Three polypeptides with apparent Mr = 200,000, 135,000, and 115,000, reacting with antibody to human ceruloplasmin (Cp), were consistently found in sera of normal adult and newborn subjects, patients with Wilson's disease, as well as in the oxidase-active fraction of purified human Cp, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentrations of the three Cp polypeptides were proportional to the total Cp oxidase activity measured in whole serum. Peptide mapping revealed that the three Cp polypeptides were closely related. Cross-linking of Cp135 resulted in dimers with electrophoretic mobility similar to that of Cp200. A common shift in electrophoretic mobility following N-glycanase treatment indicated that all three polypeptides were N-glycosylated, and that the apparent differences in molecular mass could not be related to the carbohydrate moiety. Immunoprecipitates of cell lysates of [35S]cysteine labeled HepG2 cells revealed the presence of two species of newly synthesized Cp polypeptides, Mr 200,000 and 135,000, which were secreted into the media. Secretion of Cp200 by the human liver appears to be physiologic and may be the result of posttranslational modification of Cp135.     

Effects of infection with Angiostrongylus cantonensis on the circulating haemocyte population and the haematopoietic organ of the host snail M-line Biomphalaria glabrata

The number of circulating haemocytes, the size of the haematopoietic organ, and the size of haemocyte capsules around the parasite were studied in M-line Biomphalaria glabrata snails exposed to 100 or 400 first-stage larvae of Angiostrongylus cantonensis. The number of haemocytes in exposed snails increased significantly at 1 day post-exposure, decreased to control value, and then increased again. The decrease in number of circulating haemocytes is probably due to the removal of cells from the circulation to participate in encapsulation of larvae. The majority of circulating haemocytes in M-line B. glabrata are fully-spread granulocytes, which increase significantly in number in snails following exposure to A. cantonensis larvae. However, populations of partially-spread granulocytes, round cells, hyalinocytes and miscellaneous haemocytes were relatively constant. The size of capsules around the parasite increased during the 42-day interval of the experiment. The haematopoietic organ increased in size in response to infection.     

Effective pretreatment by cysteine proteinase inhibitor for improved analysis of protein components of trematodes on SDS-PAGE

Since Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinesis, when their extracts were treated with E-64.     

The role of lipocortin I in macrophage-mediated immunosuppression in tumor-bearing mice

The soluble mediators and/or mechanisms involved in immunosuppression in tumor-bearing hosts are not well characterized, although macrophages have long been recognized as major participants. We have investigated the role of lipocortin I, a phospholipid-binding protein, in macrophage-mediated immunosuppression in tumor-bearing mice. Proliferation of splenic lymphocytes in response to the mitogens (PHA, Con A, LPS, and PWM) was severely suppressed in tumor (Sqc-NH-1 carcinoma)-bearing mice. This immunosuppression was associated with a decrease in T and B lymphocytes and an increase in macrophages in these spleens. Mac-2+ macrophages were found only in spleens from tumor-bearing mice. Splenic macrophages from tumor-bearing, but not normal, mice were responsible for this immunosuppression, as revealed by negative and positive selection experiments. The levels of lipocortin I mRNA expression were markedly increased in peripheral blood cells from tumor-bearing mice as compared with those from normal mice. Lipocortin I mRNA was strongly induced in splenic mononuclear cells from tumor-bearing mice. Furthermore, these cells displayed increased expression of lipocortin I protein, as judged by Western blot analysis with polyclonal anti-lipocortin I serum. Some nonimmune organs such as the heart, submaxillary gland, muscle, and bladder also displayed increased levels of lipocortin I mRNA expression in tumor-bearing mice. Mac-2+ macrophages among the splenic mononuclear cells in tumor-bearing mice expressed lipocortin I mRNA, as judged by negative and positive selection experiments. Most of these Mac-2+ macrophages also had Mac-1 and Mac-3 Ag. Lipocortin I protein was increased in the serum of tumor-bearing mice as compared with normal mice. The culture supernatants of splenic cells from tumor-bearing mice suppressed the mitogenic responses of splenic cells from normal mice, and addition of anti-lipocortin I antiserum inhibited this suppression. Furthermore, recombinant mouse lipocortin I suppressed mitogenic responses of splenic cells from normal mice. In summary, Mac-2+ macrophage-derived lipocortin I was largely involved in immunosuppression in tumor-bearing mice.