Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture

A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin, transferrin, epidermal growth factor, poly-D-lysine, bovine albumin, oleic acid, and bovine alpha-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetal bovine serum (FBS), and colonies, albeit of smaller sizes, did form. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking alpha-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding alpha-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

The ultrastructural basis of the permeability of arterial endothelium to horseradish peroxidase

The thoracic aorta and basilar artery, in which the incidence of atherosclerosis is known to be different, were examined to elucidate the correlation between the structure of the intercellular cleft junction between adjacent endothelial cells and its permeability to HRP. Tannic acid or HRP in the vessel lumen passed through the intercellular clefts of the thoracic aorta into the subendothelial space, whereas in the basilar artery they were unable to penetrate beyond the tight junction of the intercellular clefts. Freeze-fracture replicas revealed that the tight junctions of the thoracic aorta consisted of one to two junctional strands in most areas of the cleaved planes, with discontinuities in some places, whereas those of the basilar artery consisted of a continuous belt-like meshwork of six anastomosing junctional strands on average. These observations confirm that the structure of endothelial junctions in arteries has a close correlation with the permeability of the intercellular clefts to HRP.     

Elektronenmikroskopische Untersuchungen am Nucleus praeopticus der Kröte (Bufo vulgaris formosus)

Zusammenfassung Die Feinstruktur der neurosekretorischen Nervenzellen des Nucleus praeopticus magnocellularis der Kröte (Bufo vulgaris formosus) und ihre Umgebung wurde untersucht.Die neurosekretorischen Zellen enthalten drei Arten von osmiophilen Gebilden: die neurosekretorischen Elementargranula, die neurosekretorischen Kügelchen und die Einschlußkörper.Die neurosekretorischen Elementargranula besitzen einen Durchmesser von 1000–3000 Å, durchschnittlich von 1300–1500 Å. Sie entstehen im Golgi-Apparat (Perikaryon) der betreffenden Zellen wie bei den schon beschriebenen anderen Tierarten.Die neurosekretorischen Kügelchen haben einen Durchmesser von 4000 Å bis zu mehreren . Sie kommen zuerst in den Ergastoplasmacisternen des Perikaryons vor und wandern dann innerhalb des Axons caudalwärts ab, ebenso wie die Elementargraunla, verlieren sich aber vor dem Erreichen der Neurohypophyse. Nach Lage und Gestalt entsprechen sie den Kolloidtropfen, die von vielen Lichtmikroskopikern für die neurosekretorischen Zellen niederer Vertebraten beschrieben wurden.Die Einschlußkörper treten vornehmlich im zentralen Bezirk des Perikaryons in Erscheinung. Sie sind so groß wie die Mitochondrien und besitzen verschiedene Innenstrukturen. Auf Grund der Struktur und der histochemischen Reaktion möchten wir diese Einschlußkörper den Lipofuscingranula mit saurer Phosphatase zuordnen.Die neurosekretorischen Nervenzellen schmiegen sich an den die Kapillare umgebenden Perivaskularraum unmittelbar an, innerhalb dessen die Basalmembran unvollkommen ausgebildet ist oder ganz fehlt.Stellenweise dehnt sich ein Abschnitt des Endothels durch den Perivaskularraum hindurch entlang der Außenfläche des Perivaskularraums aus, wobei sich die Endothelzellen der Kapillare und die neurosekretorischen Nervenzellen direkt berühren können. Eine poröse Bauweise des Endothels wurde nicht nachgewiesen. Zwischen den Ependymzellen des III. Ventrikels und den darunterliegenden neurosekretorischen Nervenzellen sind oftmals auffallend große Extrazellularräume zu beobachten, die durch den Spaltraum der benachbarten Ependymzellen mit dem Ventrikellumen kommunizieren. Sie enthalten mikrovilliartige Ausläufer der Ependymzellen und die geschilderten, neurosekretorische Bildungen führenden Axone. Eine Ausstoßung dieser Axone in den Ventrikel wurde nicht festgestellt.Diese Untersuchung wurde zum Teil mit finanzieller Unterstützung durch das Japanische Unterrichtsministerium im Jahre 1963 durchgeführt.Der kurze Inhalt dieser Arbeit wurde unter dem Thema 'Electron microscopic studies on the praeoptic nucleus in the toad am 5. und 6. September 1963 auf dem Kongreß für Endokrinologie in Gunma, Japan, vorgetragen.     

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.     

p-nitrophenyl butyrate hydrolyzing activity of hormone-sensitive lipase from bovine adipose tissue

The "esterase" activity of hormone-sensitive lipase (HSL) was studied using water-soluble p-nitrophenyl butyrate (PNPB) as a substrate. Bovine adipose tissue HSL was purified to near homogeneity by precipitation at pH 5.0, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, and high performance ion-exchange columns on Mono Q and Mono S. The purified preparation hydrolyzed emulsified triolein and cholesteryl oleate (CO), and water-soluble PNPB. In the two last steps of purification, the elution profile of the CO-hydrolyzing activity coincided with that of PNPB-hydrolyzing activity. The HSL was adsorbed to heparin-Sepharose and the CO- and PNPB-hydrolyzing activities were eluted together in the same peak. Diisopropylfluorophosphate (DFP) strongly inhibited the HSL activities and the inhibition profiles of the triolein-; CO-, and PNPB-hydrolyzing activities were essentially identical. Only one polypeptide of Mr 84,000 in partial purified HSL fraction was labeled by affinity labeling with [3H]DFP. On digestion of the enzyme with trypsin, the triolein- and CO-hydrolyzing activities were lost more rapidly than the PNPB-hydrolyzing activity. Phosphorylation increased the triolein-hydrolyzing activity to 40% more than that of the control, but did not affect the CO- and PNPB-hydrolyzing activities.     

Effect of rice fiber on human fecal microflora

The effects of brown rice, containing fourfold as much dietary fiber as polished rice, on the human fecal microflora were determined. Significantly increased numbers of Bifidobacterium adolescentis and Enterococcus faecalis were observed during the brown rice intake, whereas the total counts and the numbers of bacteroides, Eubacterium aerofaciens, and Escherichia coli during the intake were lower than those before and after the intake. Lower numbers of clostridia and low incidences of Clostridium paraputrificum and C. perfringens were observed during the brown rice intake.     

Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase.

The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences. The pattern of identity of amino acid sequences was quite evident in the regions that include structurally and functionally important residues of Bacillus subtilis thermolysin.