The fine structure of the newborn rat adrenal cortex

Summary The structural changes of the zona juxtamedullaris of the rat adrenal cortex at birth, have been examined by the light and the electron microscope. In this zone clusters of medullary cells lying among the strands of cortical tissue were observed. In the inner portion of the zona juxtamedullaris two types of adrenocortical cells were found: light and very-dark cells. The latter are smaller than the light cells and are always in close connection with the medullary tissue. The ultrastructural features of the very-dark cells suggest that these elements are in degeneration. This finding supports the hypothesis that at birth there is a partial degeneration of the rat zona juxtamedullaris, i.e. the zone corresponding to the fetal zone of some mammalian species.It is proposed that in all mammalian species at birth there is a partial regression of the zona juxtamedullaris and that the regression of the fetal zone is only the quantitative increase of this phenomenon. This hypothesis is discussed in relation to numerous data demonstrating that there are enzymatic conditions in the rat during fetal life, which permit a discrete hypertrophy of the adrenal cortex.The author wishes to express his appreciation to Dr. A. Gambino and to Mr. G. Gottardo for technical assistance.     

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.     

The relationship between serum and cell type on the development of rat and pig cultured preadipocytes

Stromal-vascular cells from rats and pigs were isolated from adipose tissue and used to measure preadipocyte proliferation and differentiation. Cells from rats and pigs were grown in either 2.5% pig serum or 2.5% rat serum. Cells were either supplemented or unsupplemented with insulin after five days of growth in culture. In these cultures, pig fat cells developed as discrete clusters while rat fat cells developed as loose clusters or as individual cells. Rat cells had greater levels of sn-glycerol phosphate dehydrogenase activity compared to pig cells. Rat serum increased soluble protein in plated cells when compared to cells grown in pig serum. Pig serum increased glycerol phosphate dehydrogenase specific activity when compared to rat serum. In this system, there was no response to insulin. The cells grown in rat serum did not resemble adipocytes in regard to the presence of large lipid droplets (oil red 0 staining). These results demonstrate that rat and pig stromal-vascular cells in culture are morphologically different. Cells from both species, however, responded similarly to sera from either species showing that cells from rats and pigs responded to the growth and differentiation factors present in these sera.Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Image analysis of rat satellite cell proliferation in vitro

Myogenic cells were isolated from adult rat skeletal muscles and cultured in vitro. Cell proliferation was analyzed between days 1 and 14. The cell cycle phases were determined by examining Feulgen-stained cultures with a cell image processor. The nuclei were automatically analyzed by calculating 18 parameters relating to the texture and densitometry of chromatin and the shape of each nucleus. Cell cycle phases were characterized (Moustafa and Brugal, 1984). The recognition methods made it possible to analyse the nuclei of the myogenic cell populations which were either involved in each phase of the mitotic cycle, or left out of the cycle after fusion into myotubes.After 3 hr of culture 10% of the cell population was involved in the cell cycle. In the presence of foetal calf serum, this percentage increased until day 3 after plating. At that time, the DNA content of 28.2% of the cell population was higher than 3C, whereas it is 2C in G1 or G0 nuclei; image analysis showed that 42% of these cells were in S or G2 phase. From day 4, the proliferation rate gradually slowed down until day 8. After day 8, when numerous myotubes differentiated, the percentage of S and G2 phase cells had diminished to between 3 and 8%. The percentage of nuclei in G0 increased when the first myotubes differentiated around day 5. Myotube nuclei were largely in G0. When horse serum was added to the culture medium on day 4 to enhance myotube differentiation, significant cell proliferation was observed before cell fusion.These methods of analysis give the first daily pattern of myogenic cell proliferation and fusion in a cell population isolated from adult muscles.     

Purification of the microsomal Ca2+-ATPase from rat liver

Summary The Ca²⁺-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficollsucrose treatment, column chromatography with agarose-hexane adenosine 5-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca²⁺-ATPase was stable for at least two weeks when stored at –70°C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca²⁺-ATPase. Further characterization of the ER Ca²⁺-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca²⁺-ATPase cross-reacted with the purified Ca²⁺-ATPase from rat liver ER membranes.     

Single chloride channels in endosomal vesicle preparations from rat kidney cortex

Summary Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H⁺ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl^– channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of –60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than –50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl^–=Br^–=I^–>SO ₄ ^2– F^–. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl^–-flux measurements and studies on the H⁺ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H⁺ uptake into the endosomes.     

Systematics of theLactobacillus population on rat intestinal mucosa with special reference toLactobacillus reuteri

The systematics of theLactobacillus population of the intestines of 88 different rats was studied; 80 rats had been fed on fermented oat-meal soup (Molin et al. 1992). One-hundred-twenty-twoLactobacillus strains from the intestinal mucosa were phenotypically classified together with twenty-eight reference strains ofLactobacillus andLeuconostoc, using 49 unit characters. Data were examined using Jaccard coefficient, and unweighted pair group algorithm with arithmetic averages. Two major and eleven minor clusters were defined at the 76% S_J-similarity level: Cluster 1 included thirty isolates which could not be identified further, but had resemblance to the type strains ofL. jensenii, L. gasseri, L. crispatus, and to some extent toL. acidophilus. Cluster 12 including fifty-four intestinal isolates was identified asL. reuteri; and so was cluster 13 (five isolates). Isolates of the major clusters were found in all parts of the intestines. The genomic homogeneity of theL. reuteri isolates was scrutinized by endonuclease restriction analysis of the chromosomal DNA, and the isolates could be divided into six genomic strains.     

Comparison of linoleate,palmitate and acetate metabolism in rat ventral prostate

Rat ventral prostate incorporated (1-¹⁴C)acetate, (1-¹⁴C)palmitate and (1-¹⁴C)linoleate into different phospholipids in a time-dependent process. The rate of incorporation into total phospholipids was higher with linoleate (10.0 nmol/g) than with either palmitate (5.8 nmol/g) or acetate (4.7 nmol/g). Predominant labelling with all the radioactive substrates assayed was found in choline glycerophospholipids (PC). The radioactive profiles for linoleate in the other ventral prostate phospholipids differed from those obtained with palmitate and acetate. Specifically linoleate was incorporated into inositol glycerophospholipids plus lysoethanolamine glycerophospholipids (PI+LPE) and not into sphingomyelin (SM), while palmitate and acetate incorporated into SM but not into PI+LPE. Acetate showed the highest oxidation to CO₂ whereas no differences were observed in the radioactivity incorporated into CO₂ from a saturated (palmitate) or an essential unsaturated fatty acid (linoleate). These studies also show zinc-dependence by the acetate to CO₂ oxidation.Abbreviations PL total phospholipids - PC choline glycerophospholipids - PE ethanolamine glycerophospholipids - PI+LPE inositol glycerophospholipids plus lysoethanolamine glycerophospholipids - PS serine glycerophospholipids - SM sphingomyelin     

Effects of copper aspirinate and aspirin on tissue copper,zinc, and iron concentrations following chronic oral treatment in the adjuvant arthritic rat

Concentrations of copper, zinc, and iron were analyzed and compared in a number of tissues of adjuvant arthritic rats following 22 d of chronic treatment (per os) with either vehicle, aspirin or copper aspirinate, at doses of 100 mg/kg, 200 mg/kg, or 400 mg/kg. Such chronic treatment resulted in a negative balance in copper, zinc, and iron in many tissues. Among the tissues examined, liver and kidney exhibited the greatest changes in metal concentrations; brain and skeletal muscle exhibited the least. Arthritis-induced changes in the concentrations of all three metals in the liver were reversed upon treatment with aspirin. Treatment with copper aspirinate, on the other hand, resulted in an extremely high accumulation of copper in the liver. Arthritis-induced changes in copper, zinc, and iron concentrations in the pancreas and copper concentration in the plasma were generally not reversed upon treatment with either aspirin or copper aspirinate. Among the three metals examined, the degree of change observed as a result of drug treatments was greatest for iron and least for zinc. Finally, it appeared that the effects of aspirin and copper aspirinate on tissue metal concentrations were independent of the antiarthritic effects of these compounds.