Partial Purification from Chick Embryos of a Factor which Promotes Myoblast Proliferation and Delays Fusion

Chick embryo extract (EE) contained an activity which promoted myoblast proliferation and delayed fusion. Various tissue extracts prepared from 12-day embryos and adult chicken also showed the activity. We partially purified this active substance from 12-day embryos, following procedures which included extraction at pH 3.5, CM-Sephadex C–50 ion exchange and Sephadex G-75 gel filtration. Judging from the dose-response analyses, the factor was purified by some hundred-fold when EE was used as the starting material. The activity was associated with a macro-molecular substance (MW > 300K daltons) at first, but the apparent molecular weight of the active substance was estimated to be between 16 and 20K daltons at the final step of the preparation. It promoted myoblast proliferation and delayed myotube formation, and was active for both avian and rat myoblasts.
Since bovine pituitary gland fibroblast growth factor (FGF) showed the same activity, the factor may be FGF-related.     

Anticholinesterase Action of a Bromine Compound Isolated from Human Cerebrospinal Fluid

Abstract: L-l-Methylheptyl-γ-bromoacetoacetate was found to be a competitive inhibitor of the acetylcholines-terases (electric eel, K_i= 17.2 μM; rat brain, K_i= 32.6 μM) and of butyrylcholinesterase (horse serum, K_i= 1.2 μM). The L-isomer was a more effective inhibitor than the D-isomer. The bromine atom at the γ-position of the acidic moiety, the specific length of the carbon chain constituting the secondary alcohol moiety, and the presence of the ketone radical at the acidic moiety of the ester were necessary for the anticholinesterase action. 1-Methyl-heptyl-γ-bromoacetoacetate formed a complex with acetylcholinesterase or butyrylcholinesterase without hydrolysis of its own molecule.     

Effect of purine supplementation on the growth of salmonid cell lines in different mammalian sera

The Chinook salmon embryo cell line, CHSE-214, grew well in fetal bovine serum (FBS) but poorly in dialyzed (d) FBS. Purines restored most but not all growth-promoting activity to dFBS, which suggests that purines account for a large portion of the dialyzable fraction's growth-promoting activity. CHSE-214 died in newborn calf serum (NCS) but grew slightly in dNCS, which suggests that the dialyzable fraction of NCS contains a toxic component(s). Little or no proliferation occurred in calf serum (CS); some took place in horse serum (HS). Porcine serum (PS) was very toxic. In all these sera except PS and HS, the purine nucleoside, inosine, significantly enhanced growth, whereas the pyrimidine nucleoside, uridine, was without effect. The other purines, hypoxanthine, adenine, adenosine and guanosine also stimulated proliferation but not as well as inosine. Inosine also enhanced the growth of the rainbow trout gonadal cell line, RTG-2. Although their morphology underwent minor alterations in medium with inosine, CHSE-214 cells could be grown indefinitely in CS and inosine as effectively as in the more expensive FBS.     

Profiles of purine metabolism in leaves and roots of Camellia sinensis seedlings

To determine the metabolic profiles of purine nucleotides and related compounds in leaves and roots of tea (Camellia sinensis), we studied the in situ metabolic fate of 10 different (14)C-labeled precursors in segments from tea seedlings. The activities of key enzymes in tea leaf extracts were also investigated. The rates of uptake of purine precursors were greater in leaf segments than in root segments. Adenine and adenosine were taken up more rapidly than other purine bases and nucleosides. Xanthosine was slowest. Some adenosine, guanosine and inosine was converted to nucleotides by adenosine kinase and inosine/guanosine kinase, but these compounds were easily hydrolyzed, and adenine, guanine and hypoxanthine were generated. These purine bases were salvaged by adenine phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase. Salvage activity of adenine and adenosine was high, and they were converted exclusively to nucleotides. Inosine and hypoxanthine were salvaged to a lesser extent. In situ (14)C-tracer experiments revealed that xanthosine and xanthine were not salvaged, although xanthine phosphoribosyltransferase activity was found in tea extracts. Only some deoxyadenosine and deoxyguanosine was salvaged and utilized for DNA synthesis. However, most of these deoxynucleosides were hydrolyzed to adenine and guanine and then utilized for RNA synthesis. Purine alkaloid biosynthesis in leaves is much greater than in roots. In situ experiments indicate that adenosine, adenine, guanosine, guanine and inosine are better precursors than xanthosine, which is a direct precursor of a major pathway of caffeine biosynthesis. Based on these results, possible routes of purine metabolism are discussed.     

Fusicoccin, an inhibitor of brassinosteroid-induced ethylene production

Fusicoccin was evaluated for its effects on brassinosteroid (BR), indole-3-acetic acid (IAA) and BR + IAA-induced ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC-synthase production by etiolated mung bean ( Vigna radiata L. Rwilez cv. Berken) hypocotyl segments. Fusicoccin inhibition of ethylene and ACC production induced by 2 μ M BR started at concentrations as low as 0.05 μ M . Maximum inhibition occurred at a 1 μ M concentration with no further inhibition at higher concentrations tested. Fusicoccin (1 μ M ) was effective in the inhibition of BR-induced ethylene, ACC and ACC-synthase production at low and high concentrations of BR.
Fusicoccin at concentrations as high as 2 μ M had no effect on ethylene and ACC production promoted by low concentrations of IAA (1 to 10 μ M ). When higher concentrations (100–1000 μ M ) of IAA were used, fusicoccin (1 μ M ) had an inhibitory effect on ethylene and ACC production. Interestingly, fusicoccin (1 μ M ) had little or no effect on ACC-synthase promoted by high concentrations of IAA (1000 μ M ).
When BR and IAA were used in combination, fusicoccin inhibited ethylene and ACC production at concentrations as low as 0.05 μ M with maximum inhibition occurring at 0.5 μ M . At a 1 μ M concentration, fusicoccin was effective in inhibiting the synergistic stimulation of ACC-synthase promoted by BR and IAA.     

CHANGES IN RESPONSE TO PURINE AND PYRIMIDINE DERIVATIVES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURING

1. The growth of the carrot root callus which had been subculturedfor a long period (CCL) was promoted by the addition of 5l0^–8and 5l0^–7 M kinetin, whereas in the callus subculturedfor a short period (CCS) no growth promotion was observed atany concentrations of kinetin tested.
2. CCL showed an increasedgrowth in response to the applicationof kinetin, guanine, adenine,hypoxanthine, uracil, thymine,and cytosine in the presenceof fractions A and C of carrotroot extract, whereas no suchresponse was observed in CCS.CCL required fraction C to respondto uracil and probably purineand pyrimidine derivatives ingeneral.
3. The growth of CCL was promoted by kinetin, guanine,adenine,or hypoxanthine in the medium containing inositol andaminoacids mixture. In this case the growth-promoting actionof guanine,adenine, or hypoxanthine was nullified by kinetin.

(Received December 24, 1964; )     

Fibronectin-independent myoblast fusion in suspension cultures.

Myoblasts cultivated in suspension in serum-free medium were used to examine whether fibronectin influences myoblast fusion. No effect on cell fusion was observed when the medium was supplemented with antibodies against fibronectin (at a concentration effective in inhibiting the myoblast attachment to gelatinized dishes mediated by 1 % horse serum). Purified horse serum fibronectin (70 μg/ml) also had no effect. The assay did, however, detect both inhibition of fusion in low-calcium medium and stimulation of fusion with added embryo extract, horse serum, and fibronectin-depleted horse serum. Thus, although fibronectin may influence cell motility or other processes necessary for fusion in monolayer cultures, it does not affect the fusion process itself.     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.     

Growth-promoting factors for yeast cells ofParacoccidioides brasiliensis

Low-density seedings of yeast cells ofParacoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of sixP. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings ofP. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2–4%, moderately or considerably promoted the growth of theseP. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells ofP. brasiliensis on conventional mycological agar media.     

Plantlet production from the male inflorescence tips of <Emphasis Type="Italic">Musa acuminata</Emphasis> cultivars from South India

Inflorescence apices are suitable explants for the rapid in vitro propagation of Musa spp. However, the diploid and triploid banana cultivars showed different in vitro responses with respect to the hormone combinations in Murashige and Skoog medium. The diploid cultivar (Sannachenkadali, AA) induced a maximum number of multiple shoots in 8.9 μM 6-benzyl adenine (BA) whereas the triploid cultivar (Red banana, AAA) exhibited maximum multiplication in 22.2 μM 6-benzyl adenine. MS medium supplemented with 11.4 μM indole acetic acid and 17.8 μM BA was also suitable for shoot proliferation in triploid cultivar but not in the diploid cultivar. The regenerated shoots were rooted in Murashige and Skoog basal medium within 10–15 days. The rooted plantlets were transferred to vermiculite and maintained at a temperature of 25 ± 2°C for 10 days and then at room temperature (30–32°C) for 2 weeks before transferring to potted soil compost mixture. The plantlets showed 100% survival.     

Estrogen induction of progestophilins in rat estrogen-sensitive cells grown in media supplemented with sera from castrated rats and from rats bearing an alpha-fetoprotein-secreting hepatoma

The purpose of this work was to study the effect of alpha-fetoprotein (AFP) over cell multiplication and the induction of an estradiol-17 beta (E2)-dependent marker, i.e., progestophilins in E-sensitive cells C2(9)RAP derived from a W/Fu rat pituitary tumor. These cells proliferate in isogeneic hosts under the influence of E2, while they proliferate in culture regardless of the presence of E2. C2(9)RAP cells were grown in medium supplemented with 10% horse serum. Progestophilin levels were measured 48 h after adding serum (20% horse, or castrated rat, or AFP-secreting tumor-bearing rat) and estrogen to the 10% horse serum-supplemented medium in which the cells were growing. Maximal induction of progestophilins was obtained at 3 X 10(-10) M E2 in cells grown in medium containing horse or castrated rat serum. In contrast, maximal induction of progestophilins required 3 X 10(-8) M E2 in cells grown in medium supplemented with the serum of Morris hepatoma 7777-bearing rats. This serum contained AFP levels comparable to those present at birth in the rat. 11-Methoxy-17 beta ethynylestradiol (R2858), a synthetic estrogen with little affinity for AFP, was also tested for its ability to induce progestophilins. The degree of maximal induction of progestophilins expressed as percentage of the respective control, was similar for all experimental groups, both with E2 and with R2858. In addition, we compared the free E2 levels in the culture medium with the progestophilin levels and the cell proliferation rate. We found that the progestophilin levels were maximal at free E2 concentrations above 11 pg E2/ml, whereas there was no correlation between the free E2 levels and the proliferation rate. Moreover, the proliferation rate of cells in medium supplemented with horse or castrated rat serum was maximal at concentrations of free E2 below 0.4 pg/ml; whereas cell proliferation was inhibited with hepatoma serum even at concentrations of free E2 of 44 pg/ml. We conclude that the effect of hepatoma serum on the E2 induction of progestophilins seems to be mediated by the effect of AFP on the availability of free estrogen, since it is abolished by the addition of both natural and synthetic estrogens. The inhibitory effect of hepatoma serum upon cell proliferation is not reversed by estrogens and thus seems to be mediated by mechanisms other than E2 trapping by AFP.     

Inhibitory Effect of Ascorbic Acid on the Proliferation and Invasion of Hepatoma Cells in Culture

Effect of ascorbic acid (AsA) on the proliferation and invasion of rat ascites hepatoma AH109A cells was investigated by measuring [³H]thymidine incorporation into acid-insoluble fraction of the cells and by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells, respectively. AsA suppressed the invasion of AH109A cells in a dose-dependent manner at concentrations of 62.5–500 μM, while it inhibited the proliferation of the cells at higher concentrations of 250 and 500 μM. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. AsA suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with AsA, HX and XO or with AsA and hydrogen peroxide. Furthermore, AsA reduced the intracellular peroxide levels in AH109A cells. These results suggest that the antioxidative property of AsA may be involved in its anti-invasive action on hepatoma cells.     

Characteristics of adenosine activity on adenine nucleotide metabolism of rat thymocytes

The effects of adenosine on adenine nucleotide metabolism in [14C]adenine-labeled rat thymocytes were studied. It was shown that adenosine increases the intracellular pool of adenine nucleotides, predominantly ATP, which is accompanied by marked acceleration of their catabolism and a release of labeled products (especially inosine, hypoxanthine and adenosine) from the thymocytes. The effect of adenosine depends on its concentration and manifests itself already at 10(-6) M. 2-Deoxycoformycin partly relieves the effect of adenosine on adenine nucleotide metabolism. Exogenous deoxyadenosine, inosine, hypoxanthine and adenine, unlike adenosine, do not significantly affect the adenine nucleotide catabolism and the label release from the cells. All the effectors under study strongly increase inosine transport from the thymocytes, and inhibit, with the exception of adenosine, the hypoxanthine release from the cells.     

Adenosine phosphorylase activity in mycoplasma-free growth media for mammalian cells

Mammalian cells have enzymes that deaminate adenosine to inosine, which can readily be phosphorolysed to hypoxanthine. They do not, however, possess enzymes to form adenine by the cleavage of adenosine. For this reason, the release of adenine from adenosine by mammalian cell cultures has usually been interpreted as indicating the presence of mycoplasma, a frequent microbial contaminant that contains high levels of adenosine phosphorylase. We found that some human lymphoblast cultures free of mycoplasma showed high levels of adenosine cleavage and that this activity resulted from adenosine phosphorylase in the bovine serum used as the culture growth supplement. A survey of 13 serum supplements disclosed that fetal bovine serum (six lots) contains the highest adenosine phosphorylase activity, ranging from 9 to 648 nmol adenine produced per hour per ml serum; newborn calf serum (four lots) has much less activity, ranging from 0 to 5 nmol adenine produced per hour per ml serum; and donor horse serum (three lots) contains no detectable activity. These results suggest that mycoplasma tests dependent on the presence of adenosine phosphorylase or other enzyme activities may give false-positives with cultures containing fetal bovine serum supplements.     

Metabolism and salvage of adenine and hypoxanthine by myocytes isolated from mature rat heart

Adenine and hypoxanthine can be utilised by cardiac muscle cells as substrates for the synthesis of ATP. A possible therapeutic advantage of these compounds as high-energy precursors is their lack of vasoactive properties. Myocytes isolated from mature rat heart have been used to establish in kinetic detail the capacity of the heart to incorporate adenine, hypoxanthine and ribose into cellular nucleotides. Maximum rates of catalysis by enzymes on the salvage pathways have been established. Whilst the rate of incorporation of adenine into the ATP pool appears to depend upon intracellular concentrations of adenine and phosphoribosylpyrophosphate, for hypoxanthine the pattern is more complex. Hypoxanthine is salvaged at a slow rate compared with adenine, and is incorporated into GTP and IMP as well as into adenine nucleotides. The rate of incorporation of hypoxanthine into both IMP and ATP is accelerated in myocytes incubated with ribose. However, the rate-limiting reaction appears to be that catalysed by adenylosuccinate synthetase, for the rate of ATP synthesis is not accelerated when hypoxanthine concentration is increased from 10 to 50 microM, while the rate of IMP synthesis is more than doubled. Adenine and hypoxanthine phosphoribosyl transferases are present in equal catalytic amounts, but rat cardiac myocytes have very little adenylosuccinate synthetase activity. Exogenous ribose is incorporated into adenine nucleotides in amounts equimolar with adenine or hypoxanthine.     

In vitro cloning of Azadirachta indica from root explants

In vitro cultures of Azadirachta indica A. Juss. were raised by first culturing the root segments on modified Murashige and Skoog (MS) medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 9.84 μM N⁶-(2-isopentenyl) adenine (2iP), 5.71 μM indole-3-acetic acid (IAA), 81.43 μM adenine hemisulphate and 2.27 μM putrescine for 2 d followed by their transfer to the same medium except containing one-tenth of the initially used concentrations of BAP, 2iP and IAA. The regenerated shoots sustained proliferation in the basal medium supplemented with 1.11 μM BAP, 1.43 μM IAA and 135.72 μM adenine hemisulphate. The isolated shoots were rooted to produce plantlets in the presence of 2.46 μM indole-3-butyric acid (IBA). The plantlets showed uniform luxuriant growth under field conditions. True-to-type nature of the field-grown root-regenerated plants was ascertained by random amplified polymorphic DNA (RAPD) analysis.     

Purine uptake by Alcaligenes eutrophus H16

The uptake of adenine, guanine, xanthine, hypoxanthine and uric acid by whole cells was studied, using spectrophotometric techniques, ¹⁴C-labelled compounds and metabolic inhibitors. Three different non-constitutive systems were shown to maintain the uptake of adenine and that of the pairs guanine/hypoxanthine and xanthine/uric acid. —Active transport of adenine was induced by adenine only, but passive uptake was also involved. Maximum K _T values of 110–131 M were observed at the pH optimum of 8.0. —Guanine and hypoxanthine were translocated by one single mechanism as indicated by K _T and K _I values. This system was induced by both these substances but its affinity was 51/2-times higher for guanine than for hypoxanthine; it was noncompetitively stimulated by Mg²⁺. — A further system, induced by xanthine and uric acid, catalyzed the uptake of both these compounds. It exhibited two pH optima (at pH 6.6 and 7.9); inactivation by heat and stimulation or inhibition by several compounds indicated that two separate mechanisms might be involved in the uptake of xanthine and uric acid.     

Inosine synthesis and phosphoribosylpyrophosphate availability in rat liver cells in the presence of allopurinol

In the presence of allopurinol, apparent phosphoribosylpyrophosphate (PP-ribose-P) availability as measured by adenine incorporation into ribonucleotides was decreased in rat liver cells, hypoxanthine incorporation into ribonucleotides was increased, and there was a large synthesis of inosine from hypoxanthine. Inosine was formed directly by the reversal of the purine nucleoside phosphorylase reaction which was very rapid in liver cells. We tested the hypothesis that utilization of ribose 1-phosphate for inosine synthesis could decrease PP-ribose-P availability. Our results indicate that the apparent decrease of PP-ribose-P availability in the presence of allopurinol was due to competition between adenine and hypoxanthine salvage pathways into nucleotides, and not to the synthesis of inosine.     

Hypoxanthine phosphoribosyltransferase activity in tissues and hypoxanthine concentrations in plasma and CSF of the horse in comparison with other species

1. Plasma hypoxanthine and xanthine concentrations are very low in the horse and low in rat, mouse and greyhound compared to concentrations in beagles, man, sheep and rabbit. 2. Activities in erythrocytes of the main enzyme metabolizing hypoxanthine, hypoxanthine phosphori-bosyltransferase, show a similar pattern (Tax et al., 1976, Comp. Biochem. Physiol. 54B, 209-212); thus low activities have been found where plasma concentrations were low. 3. Hypoxanthine phosphoribosyltransferase activities in horse tissue other than erythrocytes are similar to those in man and rabbit with high activities in brain; this enzyme may therefore be functionally important in equine brain.