Anti Rat Research Articles

リポ蛋白代謝: 肝細胞

Culture system of adult rat hepatocytes provides a useful experimental model for studies of various metabolism in liver cells. In order to investigate the effects of various hormones on the lipoprotein metabolism in the liver, we have established primary monolayer culture of rat hepatocytes utilizing serum free culture medium. Synthesis of apolipoprotein (Apo) A-I and alkaline triglyceride lipase (H-TGL) was examined in cultured hepatocytes. Adult rat hepatocytes were cultured in serum free culture medium, HI/WO5/BA2000 (International Scientific Industries Inc., (ISI), Cary, I11.) or in Leibovitz-15 (L-15). Liver cells were isolated according to the modified method of Berry and Friend as described previously. Approximately 2.5-3.0×106 isolated liver cells in a final volume of 2.5ml of culture media supplemented with Gentamicin were inoculated into plastic culture dishes (diameter=60mm) (Falcon Plastics, Oxnard, Ca.) which had been coated with calf skin collagen (type IV, or III) or calf skin collagen type III with fibronectin (Collaborative Res.). The culture dishes were placed in a humidified incubator 5% CO2/95% air at 37°C. After 4 hours of culture, the inoculated cells adhered to the bottom of the culture dishes and the culture medium was changed to remove cell debris and loosely attached cells. Metabolic and morphological characteristics of the hepatocytes cultured in either medium were compared. The coarsely granular cells were less in HI/WO5/BA2000 than L-15. Plating efficiencies were 66±6% and 52±7% in HI/WO5/BA2000 and L-15, respectively. Approximately 85-90% of total cells cultured in HI/WO5/BA2000 excluded trypan blue dye. On the other hand, 80-85% of cells cultured in L-15 excluded trypan blue. Rate of albumin synthesis and α-aminoisobutyric acid uptake in hepatocytes cultured in HI/WO5/BA2000 were 2.0 and 2.7 times higher than those in the hepatocytes cultured in L-15, respectively. Tyrosine aminotransferase in the hepatocytes cultured in HI/WO5/BA2000 was induced by dexamethasone to approximately the same level as in the hepatocytes cultured in L-15. The data in the present study indicated that HI/WO5/BA2000 was superior to L-15 as culture medium. The pooled culture media in which rat liver parenchymal cells were cultured for 20 hours were concentrated approximately 10 times by ultrafiltration. Single precipitin line was observed by immunodiffusion and immunoelectrophoresis between anti-rat Apo A-I and concentrated culture media. Total delipidized HDL3 fractions were analyzed by 7% polyacrylamide gel electrophoresis (PAGE) with 8M urea. Apo A-I and Apo C peptides were observed on PAGE, and the PAGE pattern of Apo HDL3 from culture media was identical to the patterns of rat plasma Apo HDL3. [14C] leucine incorporation to Apo A-I synthesized by rat hepatocytes in primary monolayer culture was assayed by counting radioactivities of precipitates formed sequentially by anti rat HDL3 (or Apo A-I) rabbit serum and anti rabbit IgG goat serum. Rate of Apo A-I synthesis and secretion by rat hepatocytes into culture medium was constant up to 6 hours and this rate decreased slightly from 6 to 24 hours. Addition of insulin into culture medium of rat hepatocytes prepared from streptozotocin diabetic rats, increased Apo A-I synthesis. A constant rate of H-TGL synthesis was observed up to 24 hours. The effect of insulin on H-TGL synthesis was studied in cultured hepatocytes prepared from streptozotocin diabetic rats. Addition of insulin into the culture media significantly increased H-TGL synthesis for 6 hours in cultured hepatocytes prepared from streptozotocin diabetic rats. Furthermore, a significant positive correlation was observed between [14C] leuc

Immunoradioautographic study of alpha-fetoprotein in hepatoma cells.

Trypsin treatment, which is frequently used in the field of immunocytochemistry to faciliate conjugated antibody penetration through the cell membrane, was applied for the immunoradiographic demonstration of alpha-fetoprotein in ascites hepatoma cells using 125I-labeled anti rat alpha-fetoprotein antibody. No significant number of silver grains were detectable in the group without trypsin treatment, while numerous silver grains were recognizable in the ascites hepatoma cells after trypsin treatment. This indicates that trypsin treatment is effective for the immunoradiographic demonstration of alpha-fetoprotein and contributes to the quantitative immunoradioautographic investigation.

Quantitative determination of specific proteins in rat epididymis

This paper describes the development of a radioimmunoassay for the measurement of specific proteins (SEP) in rat epididymis. Using [125I]-SEP the assay is useful within the range 0.6–30 ng SEP. With this method we confirmed the organ specificity of SEP which could neither be detected in the cytosol of several other organs in the rat nor in rat blood plasma. Furthermore, SEP appear to be species specific since the epididymal cytosol from man, stallion, bull, dog and rabbit did not react with serum anti rat SEP. Castration decreased the SEP content of rat epididymis. Preincubation of anti-SEP with cauda epididymis spermatozoa sharply decreased the binding capacity of the antiserum. The same treatment did not alter the binding capacity of control anti-LH serum. Immunofluorescence experiments show that SEP are attached to spermatozoa obtained from the cauda epididymis.

Suppressor cell influence in selected strains of inbred rats: III. Evidence for nonspecific suppression by a lymphocyte-macrophage cooperation

Lewis (LE) and Brown-Norway (BN) rats treated in vivo with rabbit anti rat thymocyte serum demonstrate a reversal in relative responsiveness when assayed to mitogens and antigens via lymphocyte transformation, i.e., the BN rat now responds to a greater magnitude than the LE rats. Spleen cells, from both strains, that have been eluted from glass wool columns, demonstrate elevated responses to mitogens and antigens when compared to unfractionated spleen cells. The responses by these unfractionated and nonadherent populations can 1) be further enhanced subsequent to the treatment with anti-thymocyte serum and 2) suppressed after addition of macrophages from either strain, but especially by BN derived macrophages. The adherent cells from LE spleens respond to the above treatments in a similar fashion as the other populations. The responsiveness of adherent BN cells is only partially restored following treatment with anti-thymocyte serum, and is not further suppressed upon the addition of macrophages. These data are indicative of a lymphocyte-macrophage cooperation in this mechanism of suppression.

Immunochemical studies of organ and tumor lipids XXI. Sensitivity and specificity of the cytolipin F - sheep erythrocyte system.

The sensitivity and specificity of the sheep erythrocyte - anti-sheep erythrocyte system to inhibition by pure cytolipin F has been studied with 5 antisera, in order to compare it with the rat erythrocyte-anti-rat lymphosarcoma system and its inhibition by pure cytolipin R. The cytolipin F - sheep erythrocyte system is much more sensitive than the cytolipin R - rat erythrocyte system, inhibition of hemolysis of 6 x 10(6) sheep cells being produced by 10 ng of cytolipin F (combined with a four-fold quantity of lecithin) compared with inhibition of hemolysis of 10(6) rat cells by 50 to 100 ng of cytolipin R (also combined with lecithin). Differences in sensitivity are attributed to the larger number of available cytolipin F determinants on sheep erythrocytes compared with cytolipin R determinants on rat erythrocytes. Studies of the auxiliary lipid enhancement of cytolipin F activity by galactocerebroside, lactosyl ceramide (cytolipin H), and lecithin are also reported.

Rabbit anti rat lymphocyte serum: immunosuppression mediated by a pure IgM fraction.

Eleven rabbits were immunized i.v. weekly for 3 weeks with rat thymus cells during azathioprine administration. The antisera were pooled and fractionated on Sephadex G‐200 to obtain IgM and IgG fractions. The lymphocytotoxic activity was stronger in the IgM than in the IgG fraction. Whole serum, the IgM fraction and the IgG fraction prolonged the survival time of skin allografts from 12.0 to 16.5, 19.2 and 14.4 days respectively. It is concluded that IgM antibodies in antilymphocyte sera also can exhibit immunosuppressive activity.

Effect of anti light-chain antibodies on rat leukocytes in vitro.

The effect of the IgG fraction of rabbit anti rat L‐chain antibodies (aL) and their Fab fragments (Fab aL) on rat blood leukocytes cultured in vitro has been examined. Undigested aL increased the thymidine uptake in both mixed and unmixed leukocyte cultures over a very wide dose range. Fab aL had no effect on either mixed or unmixed leukocyte cultures. The nature of T lymphocyte receptors is briefly discussed.

Rabbit anti rat lymphocyte serum: immunosuppression mediated by IgM antibodies.

Rabbit anti rat lymphocyte sera (ALS) were produced in rabbits treated with azathioprine during the immunization period. Such antisera were cytotoxic to lymphocytes with nearly as high litres as have been seen in untreated animals using the same immunization schedule. Treatment of the antisera with 2‐mercaptoethanol followed by alkylation, resulted in marked loss in titre towards lymphocytes. Separation on Sephadex G‐200 confirmed that most of the cytotoxic activity was to be found among the IgM antibodies. Both “thymus‐azathioprine” and “spleen‐azathioprine” ALS significantly prolonged the survival of skin allografts in rats. Thymus antisera were more efficient than spleen antisera. Reduction and alkylation of active thymus antisera resulted in loss of in vivo immunosuppressive effect.

Antiplasmodial precipitins demonstrated by double diffusion in agar gel in the serum of rats infected with Plasmodium berghei

Abstract Among single serum samples collected from 111 rats during primary infection with Plasmodium berghei , 61 (55%) contained precipitin. Twenty-one were positive before parasitemia crisis, and 40 after crisis. There were fewer positives among the serums of younger rats than in mature rats. A series of 8 hyperimmunizations raised the percentage of positive tests from 55 to 75%. Vaccination with a cell-free plasmodial product before challenge with viable parasites raised the percentage of positive tests to 100%. The kinetics of production of precipitin was studied in a series of closely spaced observations on 46 infected rats throughout infection and into latency. In all but one rat with a fulminating fatal infection, precipitin appeared in the serum some days after inoculation, and remained demonstrable for extended periods, with occasional negative observations. Precipitin was regularly observed during latency. The precipitin was antiplasmodial, and no antierythrocytic precipitin was demonstrated. Furthermore, the species-specificity of at least part of the precipitin was attested by the fact that absorption with a cell-free extract of Plasmodium vinckei failed to remove all of the precipitin from anti berghei rat serum; whereas absorption with a similar preparation of P. berghei removed it all.

A specific soluble substance involved in nephrotoxic nephritis.

SummaryA specific soluble substance which will combine with the nephrotoxic antibody of anti—rat kidney serum was obtained in solution. The material was liberated into solution by digesting rat kidney with trypsin at pH 8. This substance was assayed by its ability to absorb antibodies from nephrotoxic serum. It protected a total of 24 rats without failure. Control experiments excluded trypsin activity as being the protective substance. Further characterization should be possible now that this specific material has been obtained in solution.