Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

13C nuclear magnetic resonance (NMR) of methotrexate, trimethoprim, and pyrimethamine enriched 90% with 13C at C2 has provided a sensitive means of detecting the state of protonation of the heterocyclic rings of these inhibitors. In each case, protonation of N1 causes an upfield movement of the chemical shift of C2 by more than 6 ppm. By this method it has been shown that, at pH values up to 9.2, methotrexate is bound to bovine liver dihydrofolate reductase with N1 of the inhibitor protonated, just as in the case of the complex with reductase from Streptococcus faecium and Lactobacillus casei. Furthermore, trimethoprim bound to reductase from any of the three sources, and pyrimethamine bound to either of the bacterial reductases also have N1 protonated even at pH values up to 10. This implies that in all cases there is a strong interaction between protonated N1 of the inhibitor and the carboxylate group of the active site aspartate or glutamate. In every case pKa of the bound inhibitor is increased by several units, a finding in accord with crystallographic evidence that inhibitor bound to L. casei reductase is in a hydrophobic environment and that N1 is not hydrogen-bonded to water. It was confirmed by titration of protein fluorescence that trimethoprim has greater affinity for bacterial reductase than for vertebrate (bovine) reductase, and that this selectivity is more marked in ternary complexes in which NADPH is also bound to the active site. However, the data cited above indicate that this difference in affinities is not due to a weaker ionic interaction between protonated N1 of trimethoprim and the bovine enzyme. Instead, binding of the trimethoprim side chain to hydrophobic sites on the enzyme must provide less binding energy in the case of the mammalian enzyme.     

Increased incidence of developmental anomalies among descendants of carriers of methylenebisacrylamide-induced balanced reciprocal translocations

N,N'-Methylenebisacrylamide (MBA), a dimer of the monomeric acrylamide, was studied for induction of clastogenic effects in germ cells of male mice. It was found to be effective in inducing dominant-lethal mutations and heritable translocations in maturing sperm. The semisterile translocation carriers and their normal counterparts were used to determine the health impact of transmitted chromosomal rearrangements through anatomical analysis of their immediate descendants in utero. As expected, semisterility resulted primarily from embryonic death during the periimplantation stages presumably caused by sperm segregants with unbalanced chromosome complement fertilizing some of the eggs. Among conceptuses that survived to mid- and late-gestation stages, there was an increased incidence of developmental anomalies including fetal death and phenotypic defects. These anomalies are assumed to be caused by certain types of unbalanced segregants that are compatible with survival beyond the periimplantation period. This class of unbalanced segregants represent in humans a major health problem to the mother and her conceptus.     

Occurrence and activity of iron- and sulfur-oxidizing microorganisms in alkaline coal strip mine spoils

Spoils samples collected from a coal strip mine in southeastern Montana were examined for populations and activities of iron- and sulfur-oxidizing bacteria. Spoils examined were of three types: (a) acidic pyrite-rich waste coal, (b) oxidation halo material, and (c) alkaline material, which was the most widespread type. Bacterial numbers, sulfur oxidation, and¹⁴CO₂ uptake activity declined to low levels in the summer when spoils were dry. Even in wetter spring months pyritic spoils contained relatively low numbers of acidophilic iron- and sulfur-oxidizing bacteria, probably indicative of water stress since the same spoils incubated with excess water or dilute mineral salts showed considerably greater bacterial numbers and activity. Certain wells in coal and spoils aquifers contained substantial populations of iron-oxidizing acidophilic bacteria. However, these wells were always of alkaline or neutral pH, indicating that bacterial pyrite oxidation occurred where groundwaters contacted either replaced spoils or coal that contained pyrite or other metal sulfides. Bacterial activity may contribute to trace metal and sulfate leaching in the area.     

Cloning of cDNA for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on T and natural killer cells.

Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.     

Melphalan, a second chemical for which specific-locus mutation induction in the mouse is maximum in early spermatids.

Melphalan (MLP), a bifunctional alkylating agent structurally related to the highly mutagenic chemical chlorambucil (CHL), was found to induce high frequencies of specific-locus mutations in postspermatogonial germ cells of the mouse, and to be one of only a few chemicals that is also mutagenic in spermatogonial stem cells. Productivity patterns following MLP exposures resembled those that had been found for CHL. Mutation rates in successive male germ-cell stages were measured at three MLP-exposure levels in a total of 95,375 offspring. While the induced (experimental minus historical-control) mutation rate is relatively low in stem-cell spermatogonia (1.2 x 10(-5) per locus at a weighted-mean exposure of 7.3 mg/kg), it is about 5 times higher in poststem-cell stages overall, and peaks at 26.7 x 10(-5) per locus in early spermatids at a weighted-mean exposure of only 5.7 mg/kg. This "type-2 pattern" of mutation yield (Russell et al., 1990), i.e., peak sensitivity in early spermatids, has heretofore been found for only one other chemical, CHL. Mutation-rate data earlier reported for CHL (Russell et al., 1989) were augmented in the present study for comparison with MLP-induced rates. Because of the greater toxicity of MLP, average exposures used for this chemical were only about one-half of those for CHL. When MLP and CHL mutation rates are extrapolated to equimolar doses, they appear very similar for poststem-cell stages overall. However, in the case of CHL, a somewhat higher proportion of the mutations is induced in early spermatids than in the case of MLP.     

Marine macroalgae as foods for fishes: an evaluation of potential food quality

Synopsis A revitalized view of feeding by herbivorous marine fishes is sought through two questions. First, What characteristics of major taxa of algae identify them as predictably high or low quality foods? Second, are marine algae valuable foods for fishes which do not mechanically disrupt cell walls and do not harbor specialized enzymes or microbes capable of lysing cell walls? Energy, ash and nutrient content of 16 species of marine algae were employed to assess food quality of fleshy red, green, brown and calcareous red algae. On the basis of ash, calories, total protein and total lipid content, fleshy algae should be superior to calcareous algae as foods for fishes; in addition, green algae should be superior to brown algae and brown algae superior to red algae. When the probable digestibility of storage and extracellular carbohydrates is considered, green and red algae are predicted superior to brown algae as food. Two species of damselfishes (Pomacentridae) from the Gulf of California,Eupomacentrus rectifraenum andMicrospathodon dorsalis, eat red and green algae and ignore brown and calcareous algae. They feed, therefore, in a fashion consistent with predictions based only on algal chemistry. These fishes absorb at least 20–24% of the biomass, 57–67% of the protein, 46–56% of the lipid and 37–44% of the carbohydrate contained in algae eaten in the wild. Since these damselfishes do not masticate their food, it appears that herbivorous fishes can digest major fractions of algal nutrients without mechanical destruction of algal cells.     

Direct fermentation of cellodextrins to ethanol byCandida wickerhamii andC. Lusitaniae

Summary Production of ethanol from cellodextrins, as large as cellohexose, byCandida lusitaniae andC. wickerhamii was studied.C. lusitaniae fermented only glucose and cellobiose, whereasC. wickerhamii efficiently fermented cellodextrins. Maximum ethanol yields of 29.2 g/liter from 54 g/liter cellodextrins were achieved byC. wickerhamii in 3–4 days.