Transfer of reducing equivalents into mitochondria by the interconversions of proline and Δ1-pyrroline-5-carboxylate

Direct evidence is presented for a proline cycle using a cell-free experimental system which sequentially transfers ³H from [1-³H]glucose to NADP⁺ to Δ¹-pyrroline-5-carboxylate and yields [³H]proline. The formation of [³H]proline depends on the presence of NADP, Δ¹-pyrroline-5-carboxylate, and the enzymes glucose-6-phosphate dehydrogenase and Δ¹-pyrroline-5-carboxylate reductase. The production of [³H]proline from unlabeled proline in the presence of mitochondria provides direct evidence for one complete turn of a proline cycle which transfers reducing equivalents produced by glucose oxidation in the pentose pathway into mitochondria. In this cycle, proline is oxidized to Δ¹-pyrroline-5-carboxylate by mitochondrial proline oxidase. Δ¹-pyrroline-5-carboxylate is released from mitochondria and is recycled back to proline by Δ¹-pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH. At the maximal rate observed, 60% of Δ¹-pyrroline-5-carboxylate produced is recycled back to proline. This cycle provides a mechanism for transferring reducing equivalents from NADPH into mitochondria and is linked to glucose oxidation in the pentose pathway by NADPH turnover.     

Vererbungsstudien

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Hermaphroditismus im Tierreich vom genetischen Standpunkt

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DNA unwinding and inhibition of mouse leukemia L1210 DNA topoisomerase I by intercalators.

The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.     

DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells

Mitomycin C (MMC), a quinone-containing antitumor drug, has been shown to alkylate DNA and to form DNA cross-links. The ability of MMC to alkylate O6-guanine and to form interstrand cross-links (ISC) has been studied using Mer+ and Mer- human embryonic cells. Mer+ (IMR-90) cells have been reported to contain an O6-alkylguanine transferase enzyme and are, in general, more resistant to alkylating agents than the Mer- (VA-13) cell line, which is deficient in the repair of O6-lesions in DNA. Studies reported here show that MMC is more cytotoxic to VA-13 cells compared to IMR-90 cells. The alkaline elution technique was used to quantify MMC-induced ISC, and double strand breaks (DSB) in these cells. The drug-dependent formation of DSB was significantly lower in IMR-90 cells than in VA-13 cells. In contrast, no significant difference in cross-linking could be detected at the end of 2-h drug treatment. Although a small increase in cross-link frequency was observed in the VA-13 cell line relative to the IMR-90 cell line 6 h post drug treatment, it is not clear whether monoalkylated adducts at the O6-position are formed, and contribute to cross-link formation for differential cytotoxicity in VA-13 cells. Electron spin resonance and spin-trapping technique were used to detect the formation of hydroxyl radical from MMC-treated cells. Our studies show that MMC significantly stimulated the formation of hydroxyl radical in VA-13 cells, but not in the IMR-90 cells. The formation of the hydroxyl radical was inhibited by superoxide dismutase (SOD) and catalase. In addition, the presence of these enzymes partially protected VA-13 cells from MMC toxicity but not IMR-90 cells. Further studies indicated that the decreased free radical formation and resistance to MMC may be due to the increased activities of catalase and glutathione transferase in the IMR-90 cell line. These results suggest that MMC-dependent DNA damage (alkylation and DNA DSB) and the stimulation of oxy-radical formation may play critical roles in the determination of MMC-induced cell killing.     

Characterization of pregnant mare's serum gonadotropin-stimulated rat ovarian aromatase and its inhibition by 10-propargylestr-4-ene-3,17-dione

Aromatase, the important regulatory enzyme that converts androgens to estrogens, is found in relatively high levels in the human placenta. However, since the ovary is the major source of the estrogens in females, we undertook studies to compare the rodent ovarian enzyme with that from human placenta. Pregnant mare's serum gonadotropin (PMSG) markedly increases aromatase activity in the ovaries of immature rats, and this model was used in order to reproducibly obtain high enzyme levels. An injection of PMSG resulted in a specific stimulation of aromatase activity 12 times the increase in ovarian weight in 48 h. Kinetic studies demonstrated that, although the PMSG-stimulated ovarian microsomes had one-tenth the specific activity of the human placenta, the Km values were similar (about 33 and 44 nM, respectively). The potent inhibitor of placenta aromatase, 10-propargylestr-4-ene-3,17-dione, was used to further characterize the enzyme. It inhibited the rat aromatase with an I50 of 36 nM and exhibited time-dependent inhibition with a half-life of inactivation of 16 min and a Ki of 15 nM. These values are similar to those we obtained with the human enzyme (10 nM, 12 min, and 5 nM, respectively). The enzyme parameters in the presence and absence of the inhibitor suggest that the enzymes from the two sources are kinetically quite similar.     

Experiences with reducing point sources of phosphorus to lakes

Experiences over the last 25 years have demonstrated that eutrophication can be reversed, and that phosphorus is most often the nutrient through which control should be exerted.The reduction of the external load of phosphorus to a eutrophic lake is a necessary condition for lake restoration, but may not in itself be sufficient. Three main response patterns to a reduction in external load are identified. These include reduction in lake phosphorus that leads to sufficient reduction in chlorophyll to change the trophic category, to make the lakes less eutrophic or have small or no effect. The factors that determine the actual response are discussed.It is clear that interventions to restore eutrophic lakes have not always given the results expected. Limnological studies are necessary for well-grounded predictions.     

Survival ofPseudomonas syringae pv.lachrymans with cucumber roots

Summary Survival ofPseudomonas syringae pv.lachrymans with seedling cucumber roots, root washings, rhizosphere soil, and nonrhizosphere soil was determined 7–8 days after the soil surface was watered with a cell suspension of the bacterium. Plants were in pots in the green-house and soil was not sterilized. Survival was best with roots and root washings, next best in rhizosphere soil, and poor in nonrhizosphere soil.     

The 5-hydroxyl of myo-inositol is essential for uptake into HSDM1C1 mouse fibrosarcoma cells

We attempted to replace the myo-inositol in cellular inositol phosphatides with 5-deoxy-myo-inositol to evaluate the role of inositol 1,4,5-trisphosphate as a second messenger. This analog, lacking a 5-hydroxyl, might be incorporated into 5-deoxyphosphatidylinositol and converted to the corresponding phosphatidylcyclitol 4-phosphate but could not be converted to phosphatidylinositol 4,5-diphosphate, the precursor of the second messenger molecule inositol 1,4,5-trisphosphate. We synthesized 5-deoxy-myo-inositol and found that this analog does not replace myo-inositol as an essential growth factor for essential fatty acid deficient HSDM1C1 mouse fibrosarcoma cells. Furthermore, [5-3H]-5-deoxy-myo-inositol was neither incorporated into the phospholipids nor accumulated in the cytoplasm of these cells. It appears that this cell line has a specific myo-inositol uptake system that excludes a potentially harmful analog of inositol.     

Coprophagy in larval Culiseta bergrothi (Diptera: Culicidae)

Two experimental groups of Culiseta bergrothi Edw. larvae were kept under laboratory conditions, but in water from the breeding locality. The ‘donors’ were fed on a suspension of charcoal powder and baker's yeast; the ‘receivers’ had access to the faecal pellets of the donors. After 1.5 hours the gut of almost all receivers was more or less filled with charcoal particles. The possible consequences of the added yeast suspension to food choice and feeding behaviour are discussed.