Zofia M. Zielińska

The transfer of one-carbon units in insect metabolism. Pathways of folate coenzyme synthesis

Abstract Enzymes involved in the reduction of folate to tetrahydrofolate and in binding the one carbon-units to tetrahydrofolate have been investigated in preparations of insect tissues. In the two-step reduction of folate to tetrahydrofolate, dihydrofolate appears to be the preferred substrate for the oxidoreductases extracted from Galleria mellonella . The enzyme using formate as a C 1 source is much more active than the enzyme system using the hydroxymethyl group of serine. No enzyme system which would catalyse the binding of the formimino group either from formiminoglutamate or formiminoglycine was found.

The origin of phospholipid ethanolamine and choline in a sawfly, Acantholyda nemoralis

Abstract The origin of phospholipid ethanolamine and choline was investigated in Acantholyda nemoralis by injecting the larvae with 14 C formate as a precursor of 3-C of serine. Phospholipids were then extracted and chromatographed on silicic acid and alumina columns. The homogeneity of each of the fractions was checked using thin-layer chromatography, and their nature was proved by means of paper chromatography of the products of the mild alkaline hydrolysis. The predominant phospholipids are stated to be those containing choline (64 per cent) and ethanolamine (23 per cent). The radiocarbon was detected in all phospholipids including the cardiolipin-like acidic phosphatides. The highest specific radioactivity was found in the phosphatidylethanolamine fraction, containing labelled phosphatidylmonomethyl- and phosphatidyldimethylethanolamine. The probable pathways of the biosynthesis of phosphatidylethanolamine and phosphatidylcholine in A. nemoralis are discussed.

Variability in response of dihydrofolate reductase from Galleria mellonella towards urea, urea-like compounds and salts

Abstract 1. 1. The dihydrofolate reductase m the post-mitochondrial supernatant from Galleria mellonella fat body was found to be activated by urea, urea-like compounds and salts. 2. 2. The urea activated enzyme as well as guanidine-HCl or salt activated enzyme exhibited only one p H optimum in contrast to the two p H optima,characteristic of the non-activated enzyme. 3. 3. Dihydrofolate reductase partially purified on Sephadex G-100 was also activated by guanidine-HCl and NH 4 C1, but only immediately after dution, when it exhibited an activity profile with two p H optima. 4. 4. During storage the enzyme spontaneously underwent an activation correlated with the changes in its p H. activity profile. Such an enzyme was inhibited by guanidine-HCL and NH 4 Cl.

Effects of juvenile hormone and related insect growth regulators on mammalian cell proliferation: A comparative study

Abstract The effects of the synthetic C18 juvenile hormone, methoprene (ZR-0515), and of the compound ZR-0619 on morphology, mitotic activity, and pattern of growth of a subline of L-929 cells were compared. The cells grew as monolayers for 24 hr in a standard medium, then for another 24 or 48 hr under one of the following conditions: (1) in the standard medium, (2) in standard medium with the addition of one of the compounds tested (20, 50, 100 μg/ml), or (3) in the presence of solvent at the appropriate concentrations (0.1, 0.25, 0.5%). The susceptibility of the cells to the compounds under examination was compared by analysis of variance of the data for mitotic indices and for the frequency of occurrence of monokaryocytes and polykaryocyts in the cultures. We found that the solvent itself did not influence either cell morphology or mitotic activity of the cells until it was at 0.5% concentration whereas juvenile hormone and either of its analogs suppressed this activity significantly at all three concentrations. At both lower concentrations applied, their officacy was similar, since the fall of the mitotic indices for the cells of the media with the hormone or its analogs did not differ significantly until their concentration was 100 μg/ml, when most of the cells died out in the media with the hormone, but grow in the media with either of the analogs, although their morphology and growth patterns were more or less affected. The number of monokaryocytes and polykaryocytes did not change in cultures of the cells under our experimental conditions.

Interference of a synthetic C18 juvenile hormone and related insect growth regulators with macromolecular biosynthesis in mammalian cells.

Two juvenile hormone analogs (JHA), methoprene (ZR-0515), and ZR-0619 and a synthetic C18 juvenile hormone (JH) itself were compared in their effects on protein and nucleic acid biosynthesis and on the viability of mouse L cells. A short-time exposure of the cells, suspended in phosphate-buffered saline, to the labeled precursors of protein, DNA or RNA, and to juvenile hormone, methoprene, or ZR-0619 (2–100 μg/ml) considerably depressed the synthesis of these macromolecules. The effects of the juvenile hormone were evidently stronger than those of methoprene or ZR-0619. RNA synthesis was affected to a lesser extent than protein or DNA synthesis. Under the same experimental conditions, both the JHA were less cytocidal than the juvenile hormone itself. Calf serum added to the incubation mixture protected the cells against the effects of juvenile hormone and the JHA.

Sensitivity of insect ovarian tissues to various pteridines as tested in tissue culture.

Abstract The effect of pteridine derivatives and analogues on the cell outgrowth from the ovarian explants of the waxmoth, Galleria mellonella , was examined in hanging-drop cultures and cytochemical tests were made for succinate and glucose-6-phosphate dehydrogenases. Most of the derivatives and analogues of 2-amino-4-hydroxypteridine injured to a lesser or greater extent insect ovarian tissues in vitro , depending on the drug structure and the concentration applied. Of all the pteridine derivatives and analogues tested only 2-amino-4-mercaptopteridine and its C-6,7-dimethyl derivative (10 μM) promoted cell outgrowth from the explants as did folate.

Folate derivatives in the metabolism of insects—II. Biosynthesis of nucleic acids in the polytrophic ovaries of the diapausing larvae of Acantholyda nemoralis Thoms. as promoted by folic acid and its 4-aminoanalogue

Abstract The influence of folate and aminopterin on nucleic-acid biosynthesis in ovaries of Acantholyda nemoralis was investigated. The substances were injected into the body cavity and 24 hr later ovaries were removed and analysed for nucleic acids by means of a spectrophotometric method. It has been shown that both folate and its 4-aminoanalogue applied at the time of pre-vitellogenesis caused an increase of about 20–40 per cent in RNA content and of 30–40 per cent in DNA content in ovaries of the larvae, which is probably due to the activation of nucleic-acid synthesis in the nurse cells. In contrast, when applied to the pupae at the time of vitellogenesis when degeneration of the nurse cells occurs, neither folate nor its analogue caused any effect on nucleic-acid biosynthesis. The probable causes of these results are discussed.

FOLATE DERIVATIVES IN THE METABOLISM OF INSECTS. I. MITOSES IN CELLS OF THE FOLLICULAR EPITHELIUM AS EVOKED IN ACANTHOLYDA NEMORALIS THOMS. BY FOLATE AND ITS 4-AMINOANALOGUE.

Abstract Experiments with injection of folic acid and aminopterin were performed on the diapausing larvae of Acantholyda nemoralis (Hymenoptera) at the time of advanced pre-vitellogenesis. A few hours after injection the ovaries were removed and examined cytochemically. Doses from 0·05 to 5 μ g of folic acid per larva caused mitoses in a large number of epithelial cells. Aminopterin (2 and 20 μ g) evoked a similar effect, but all the mitoses were blocked on metaphase. When folic acid treatment was followed by that of aminopterin the mitotic index was very high and all the mitotic stages occurred; this suggests that in Acantholyda nemoralis folate offers some protection against the inhibition of mitoses by its analogue. It seems also that the promoting effect of folate and aminopterin on mitotic activity in the follicular cells could be assumed as an experimental interruption of the diapause when the larvae are physiologically ready for further development.

Dihydrofolate reductase and formyltetrahydrofolate synthetase of mammalian cells of different characteristics

Abstract 1. 1. The enzyme which provides tetrahydrofolate and the enzyme which joins this coenzyme with one-carbon fragment at the oxidation level of formate were investigated in cell extracts. 2. 2. The activity of the former enzyme, but not the latter one, was found to be readily influenced by external and internal factors. In normal embryo cells this enzyme was active only at the exponential phase of cell growth. 3. 3. In transformed cells and in L-cells its activity was higher, persisted in aged cultures, and also exhibited altered properties in comparison with those in their normal counterparts.

One-carbon pathways in amino acid interconversion in the sawfly, Acantholyda nemoralis

Abstract Experiments were based on the injection of 14 C-formate or 3- 14 C-serine into diapausing last instar larvae followed by radioassays of the blood amino acids. The bulk of the radioactivity was always found in serine. Thus the radiocarbon of formate after incorporation into serine became a real source of the label in further metabolic processes. Methionine present in sawfly blood at a concentration of a few mg per cent contained some detectable radiocarbon. This is indirect evidence for the possibility of methionine biosynthesis in this insect. The probable pathways of amino acid interconversions in the sawfly are discussed.