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Federally funded university water programs have had limited success in halting the degradation of water resources in agricultural, rural, and urbanizing watersheds for the past five decades.USDA-funded university water programs have advanced our understanding of watershed processes and the development of best management practices (BMPs; e.g., conservation tillage, nutrient management, alternative and innovative septic systems, and riparian buffers) to mitigate environmental risks from anthropogenic activities, in particular from agriculture, to our water resources; yet water degradation persists and has worsened in many watersheds (Howarth et al. 2000; Mueller and Spahr 2006). The National Research Council (2012) stresses the need for sustainable agricultural practices to reduce changes in flow regimes and water quality.In this research editorial, we make four points relative to solving water resource issues: (1) they are complex problems and difficult to solve; (2) some progress has been made on solving these issues; (3) external nonstationary drivers such as land use changes, climate change and variability, and shifts in markets, policies, and regulations warrant constant vigilance to assure that presumed improvements are being attained; and (4) we are poised to make substantial progress on these challenges over the next 10 to 20 years if critical steps are…

XK469 (1) is among the most highly and broadly active antitumor agents to have been evaluated in our laboratories. Subsequent developmental studies led to the entry of (R)-(+) 1 (NSC 698215) into phase 1 clinical trials (NIH UO1-CA62487). The antitumor mechanism of action of 1 remains to be elucidated, which has prompted a sustained effort to elaborate a pharmacophoric pattern of 1. The present study focused on a strategy of synthesis and biological evaluation of topologically based, bioisosteric replacements of the quinoxaline moiety in the lead compound (1) by quinazoline (4a−d), 1,2,4-benzotriazine (12a−18b), and quinoline (21a−g) ring systems. The synthetic approach to each of the bioisosteres of 1 utilized the methodology developed in previous work (see Hazeldine, S. T.; Polin, L.; Kushner, J.; Paluch, J.; White, K.; Edelstein, M.; Palomino, E.; Corbett, T. H.; Horwitz, J. P. Design, Synthesis, and Biological Evaluation of Analogues of the Antitumor Agent 2-{4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469). J. Med. Chem. 2001, 44, 1758−1776.), which is extended to the procurement of the benzoxazole (23a,b), benzthiazole (23c,d), pyridine (25a,b), and pyrazine (27) congeners of 1. Only quinoline analogues, bearing a 7-halo (21a,b,d,e) or a 7-methoxy substituent (21g), showed antitumor activities (Br > Cl > CH3O > F ≈ I), at levels comparable to or greater than the range of activities manifested by 1 and corresponding analogues. At high individual dosages, the (S)-(−) enantiomers of 1 and 21b,d all produce a reversible slowing of nerve−conduction velocity in the mice, the onset of which is characterized by a distinctive dysfunction of the hind legs, causing uncoordinated movements. The condition resolves within 5−10 min. However, at higher dosages, which approach a lethal level, the behavior extended to the front legs, lasting from 20 min to 1 h. By contrast, the (R)-(+) forms of these same agents did not induce the phenomenon of slowing of nerve−conduction velocity.

2-{4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469) is among the most highly and broadly active antitumor agents to have been evaluated in our laboratories and is currently scheduled to enter clinical trials in 2001. The mechanism or mechanisms of action of XK469 remain to be elaborated. Accordingly, an effort was initiated to establish a pharmacophore hypothesis to delineate the requirements of the active site, via a comprehensive program of synthesis of analogues of XK469 and evaluation of the effects of structural modification(s) on solid tumor activity. The strategy formulated chose to dissect the two-dimensional parent structure into three regions&sbd;I, ring A of quinoxaline; II, the hydroquinone connector linkage; and III, the lactic acid moiety&sbd;to determine the resultant in vitro and in vivo effects of chemical alterations in each region. Neither the A-ring unsubstituted nor the B-ring 3-chloro-regioisomer of XK469 showed antitumor activity. The modulating antitumor effect(s) of substituents of differing electronegativities, located at the several sites comprising the A-ring of region I, were next ascertained. Thus, a halogen substituent, located at the 7-position of a 2-{4-[(2-quinoxalinyl)oxy]phenoxy}propionic acid, generated the most highly and broadly active antitumor agents. A methyl, methoxy, or an azido substituent at this site generated a much less active structure, whereas 5-, 6-, 8-chloro-, 6-, 7-nitro, and 7-amino derivatives all proved to be essentially inactive. When the connector linkage (region II) of 1 was changed from that of a hydroquinone to either a resorcinol or a catechol derivative, all antitumor activity was lost. Of the carboxylic acid derivatives of XK469 (region III), i.e., CONH2, CONHCH3, CON(CH3)2, CONHOH, CONHNH2, CN, or CN4H (tetrazole), only the monomethyl- and N,N-dimethylamides proved to be active.

Normal human skin fibroblasts cultured in vitro exhibit specific binding sites for 125I-labelled transferrin. Kinetic studies revealed a rate constant for association (Kon) at 37 degrees C of 1.03×10(7) M-1 X min-1. The rate constant for dissociation (Koff) at 37 degrees C was 7.9×10(-2) X min-1. The dissociation constant (KD) was 5.1×10(-9) M as determined by Scatchard analysis of binding and analysis of rate constants. Fibroblasts were capable of binding 3.9×10(5) molecules of transferrin per cell. Binding of 125I-labelled diferric transferrin to cells was inhibited equally by either apo-transferrin or diferric transferrin, but no inhibition was evident with apo-lactoferrin, iron-saturated lactoferrin, or albumin. Preincubation of cells with saturating levels of diferric transferrin or apo-transferrin produced no significant change in receptor number or affinity. Preincubation of cells with ferric ammonium citrate caused a time- and dose-dependent decrease in transferrin binding. After preincubation with ferric ammonium citrate for 72 h, diferric transferrin binding was 37.7% of control, but no change in receptor affinity was apparent by Scatchard analysis. These results suggest that fibroblast transferrin receptor number is modulated by intracellular iron content and not by ligand-receptor binding.

The number of diferic transferrin receptors on HeLa cells decreases when cells are grown in iron-supplemented media. The experiments reported here suggest that heme is the iron-containing compound which serves as the signal for receptor number regulation. When HeLa cells were grown in the presence of hemin, transferrin receptor number decreased to a greater degree than when cells were grown in equivalent amounts of iron supplied as ferric ammonium citrate. Incubation of cells in conditions which increased cellular heme content resulted in a decrease in cellular transferrin receptors. Incubating cells with 5-aminolevulinic acid (thus bypassing the rate-limiting step in heme biosynthesis, 5-aminolevulinic acid synthase) led to a decrease in transferrin receptor number. Incubation of cells with an inhibitor of heme oxygenase, Sn-protoporphyrin IX, also led to a decrease in transferrin receptor number. When cellular heme content was decreased by inhibiting heme synthesis with succinylacetone (an inhibitor of 5-aminolevulinic acid dehydratase), or by depriving cells of iron with deferoxamine, an increase in HeLa cell transferrin receptor number was seen. When HeLa cells were incubated with inducers of heme oxygenase (CoCl2, SnCl2, Co-protoporphyrin IX), transferrin receptor number also increased. The effects of all compounds which alter transferrin receptor number were dependent on the concentration of the supplement, as well as the duration of the supplementation. These experiments suggest that intracellular heme content may be an important signal controlling transferrin receptor number.

We have cloned and sequenced a full-length cDNA coding for human uroporphyrinogen decarboxylase. The deduced 367-amino acid sequence is consistent with the molecular weight, the partial amino acid sequence of cyanogen bromide peptides, and the total amino acid composition of the purified enzyme. Southern analysis of human genomic DNA shows that its gene is present as a single copy in the human genome, and Northern analysis demonstrates the presence of a single size species of mRNA in erythroid and non-erythroid tissues and in several cultured cell lines. We have also demonstrated that the level of uroporphyrinogen decarboxylase mRNA is markedly increased in tissues or cell lines of erythroid origin and that this is due to a tissue-specific transcriptional activation of the uroporphyrinogen decarboxylase gene.

HeLa cells were found to have a single class of non-interacting receptors specific for transferrin. Both apotransferrin and diferric transferrin competed equally with 125I-diferric transferrin for receptor binding. Transferrin binding was temperature-dependent and reversible. Binding of transferrin to cells exhibited a KD of 27 nM with a maximum binding capacity of 1.8-3.7 x 10(6) molecules/cell. Cells grown in the presence of diferric transferrin or in the presence of ferric ammonium citrate exhibited a concentration- and time-dependent decrease in 125I-diferric transferrin binding. The decrease in binding activity reflected a reduction in receptor number rather than an alteration in ligand receptor affinity. Growth of cells in saturating concentrations of apotransferrin did not cause a decrease in receptor number. When iron-treated cells were removed to media free of ferric ammonium citrate, the receptor number returned to control values by 40 h. When receptors were removed with trypsin, cells grown and maintained in ferric ammonium citrate-supplemented media demonstrated a rate of receptor reappearance 47% that of control cells grown in ferric ammonium citrate-free media. Cells grown in media supplemented with diferric transferrin or ferric ammonium citrate exhibited an increase in cytosolic iron content. The transferrin receptor number returned to normal after cells were removed to unsupplemented media, despite persistent elevation of cytosolic iron content. Increased iron content did not appear to be the sole factor determining receptor number.

The subcellular location of radiolabeled transferrin (125I-Tf), internalized during cellular iron uptake, and the cellular distribution of transferrin (Tf) receptors were studied in cultured HeLa cells. Cells were incubated at 37 degrees C with 125I-Tf(Fe)2. Forty per cent of the labeled ligand was associated with cell surface receptors. The remaining 60% was internalized as shown by the inability to dissociate 125I-Tf from cells by competition with excess Tf(Fe)2 or treatment of cells with 0.2 M acetic acid containing 0.5 M NaCl. Subcellular fractionation studies using sucrose density gradients indicated that internalized Tf was localized in a membranous vesicle distinct from lysosomes, Golgi apparatus, endoplasmic reticulum, or plasma membranes. The subcellular distribution of Tf receptors was studied using an assay for detergent solubilized receptors. Even without preincubation with ligand, the majority of cellular Tf receptors were localized intracellularly in a vesicle with the same buoyant density as the vesicle containing internalized 125I-Tf. Using an assay for occupied receptors, we demonstrated that the same vesicle contained both internal receptors and internalized ligand. A portion (20%) of the intracellular receptor pool was insensitive to trypsin treatment of whole cells at 37 degrees C suggesting that during the experimental time period (20-30 min) this portion did not recycle to the cell surface. We propose that during cellular iron uptake, Tf receptor-ligand complexes are internalized and directed to a nonlysosomal compartment where iron is released, followed by recycling to the cell surface of an intact Tf receptor-apo-Tf complex.

The molecular basis of the uroporphyrinogen decarboxylase defect in eleven yeast ‘uroporphyric’ mutants was investigated. Uroporphyrinogen decarboxylase, an enzyme of the haem-biosynthetic pathway, catalyses the decarboxylation of uroporphyrinogen to coproporphyrinogen and is encoded by the HEM12 gene in the yeast Saccharomyces cerevisiae. The mutations were identified by sequencing the mutant hem12 alleles amplified in vitro from genomic DNA extracted from the mutant strains. Four mutations leading to the absence of enzyme protein were found: one mutation caused the substitution of the translation initiator Met to Ile, a two-base deletion created a frameshift at codon 247 and two nonsense mutations were found at codons 50 and 263. Four different point mutations were identified in seven ‘leaky’ mutants with residual modified uroporphyrinogen decarboxylase activity; each of three mutations was found in two independently isolated mutants. The nucleotide transitions resulted in the amino acid substitutions Ser-59 to Phe, Thr-62 to Ile, Leu-107 to Ser, or Ser-215 to Asn, all located in or near highly conserved regions. The results suggest that there is a single active centre in uroporphyrinogen decarboxylase, the geometry of which is affected in the mutant enzymes.

The purpose of this study was to assess the ability of hepatocytes to synthesize porphyrins and heme from delta-aminolevulinic acid derived from gamma, delta-dioxovalerate and alanine. An alternate pathway for delta-aminolevulinic acid synthesis, in contrast to the condensation of succinate and glycine by delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] has been suggested. This has been supported by the isolation of gamma, delta-dioxovalerate transaminase from liver mitochondria (Varticovski, L., Kushner, J. P. & Burnham, B. F. (1980) J. Biol. Chem. 255, 3742-3747). Gamma, delta-dioxovalerate transaminase catalyzes the formation of delta-aminolevulinic acid by a transamination reaction involving gamma, delta-dioxovalerate and alanine. To assess the significance of this alternate route, we prepared suspensions of respiring rat hepatocytes, which were incubated with optimal concentrations of various additives and then analyzed spectrophotometrically for synthesized porphyrins. No porphyrin synthesis was detected in cell suspensions incubated with succinate(1 mM) and glycine (20 mM). Cell suspensions incubated with gamma, delta-dioxovalerate (0.5-1.0 mM) and alanine (20 mM) synthesized 0.19 nmol of porphyrin per 10(7) cells per 2 hr (SD, 0.057). Cell suspensions incubated with delta-aminolevulinic acid (0.1 mM) synthesized 1.26 nmol of porphyrin per 10(7) cells per 2 hr (range, 1.18-1.32). Incubations with chemically synthesized gamma, delta-dioxo[5-14C]valerate were followed by extraction and purification of porphyrin esters and heme. Liquid scintillation counting revealed radiolabel incorporation into both porphyrins and heme. These studies demonstrate significant tetrapyrrole synthesis by the gamma, delta-dioxovalerate transaminase reaction. That gamma, delta-dioxovalerate is an important precursor of heme in vivo must be considered.

The figures quoted above and the tables outlined, indicate a maternal mortality per 1000 of 2.7, a stillbirth rate of 22, and a neonatal death rate of 13. The number of maternal and fetal deaths has reached a low figure at the Bronx Hospital. This has been brought about, first, by concentration of care upon the mother in the last three months of pregnancy; second, by a general attitude of conservatism during labor, with interference only when delay and procrastination might possibly result in morbidity or death.

The cesarean operation is not to be regarded as a panacea for all types of labor and while the operation itself may be simple, it has a real mortality both for the baby and the mother. These must be considered before subjecting any woman to such form of interference.

Out of 22,705 deliveries at the Bronx Hospital, 19 were delivered by craniotomy, a percentage of 0.084. In these 19 cases there were two deaths, a percentage of 10.5.