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Introduction Differences in immune characteristics, including immune gene expression by peripheral blood mononuclear cells (PBMCs), correlating with herpes labialis and good or poor immune control of herpes simplex virus type 1 (HSV‐1), and how these characteristics change after dosing with squaric acid dibutyl ester (SADBE), were investigated. Methods PBMCs were collected from persons positive for IgG against HSV‐1 and having frequent, infrequent, or no herpes labialis outbreaks. The PBMCs were tested for proliferation against HSV‐1 and a fungal antigen (Candida) and immune gene expression in the presence of HSV‐1 and Candida. On day 1 after blood collection the subjects with frequent outbreaks were dosed topically on the arm once with SADBE, and their PBMCs were collected and tested 8 weeks later. Results Those with good immune control of their HSV‐1 infection (fewer outbreaks) differ from those with poorer immune control in these ways: (1) Greater PBMC proliferation in vitro to HSV‐1, HSV‐1‐infected cell extracts, and Candida considered together (P

Background/aim The insulin-like growth factor type 1 receptor (IGF-1R) is overexpressed in myelodysplastic syndrome (MDS) cells, and 765IGF-Methotrexate (IGF-MTX) is a conjugate of methotrexate and a variant of insulin-like growth factor-1 (IGF-1) designed to selectively target cancer cells through binding to IGF-1R. The aim of this study was to determine whether IGF-MTX would be effective to treat MDS. Patients and methods In this phase I clinical trial, two patients with high grade MDS or oligoblastic acute myeloid leukemia (O-AML) that had failed standard therapy were treated with IGF-MTX. Results No dose-limiting toxicity was observed. Both patients had stable or improved cell counts and CD34+ myelodysplastic cell counts and exceeded their life expectancy (both alive at 1.9 years despite a life expectancy of less than 6 months). Bone marrow blast counts decreased from 22% to 5% in one patient, and from 17% to 16% in the other. Conclusion In conclusion, IGF-MTX at 0.20 μM equivalents per kg was well tolerated, caused no cytopenia, and produced stable disease and extension of life.

This randomized placebo-controlled study examines the results of squaric acid dibutyl ester for the treatment of herpes labialis in adults.

The insulin-like growth factor receptor is overexpressed on many types of cancer cells and has been implicated in metastasis and resistance to apoptosis. We report here the development of a novel covalent conjugate that contains the antifolate drug methotrexate coupled to an engineered variant of insulin-like growth factor-1 (IGF-1), long-R3-IGF-1, which was designed to target methotrexate to tumor cells that overexpress the membrane IGF-1 receptor. The IGF-methotrexate conjugate was found to contain at least 4 methotrexate molecules per IGF-1 protein. The IGF-methotrexate conjugate bound to MCF7 breast cancer cells with greater than 3.3-fold higher affinity than unconjugated long-R3-IGF-1 in a competition binding assay against radiolabeled wild-type IGF-1. Compared with free methotrexate, the IGF-methotrexate conjugate required slightly higher concentrations to inhibit the in vitro growth of the human prostate cancer cell line LNCaP. In vivo, however, in a mouse xenograft model using LNCaP cells, the IGF-methotrexate conjugate was more effective than free methotrexate even at a 6.25-fold lower molar dosage. Similarly, MCF7 xenografts were inhibited more effectively by the IGF-methotrexate conjugate than free methotrexate, even at a 4-fold lower molar dosage. Our results suggest that the targeting of the IGF receptor on tumor cells and tumor-related tissues with IGF-chemotherapy conjugates may substantially increase the specific drug localization and therapeutic effect in the tumor.

We performed the first field-scale atrazine remediation study in the United States using chemically killed, recombinant organisms. This field study compared biostimulation methods for enhancing atrazine degradation with a novel bioaugmentation protocol using a killed and stabilized whole-cell suspension of recombinant Escherichia coli engineered to overproduce atrazine chlorohyrolase, AtzA. AtzA dechlorinates atrazine, producing non-toxic and non-phytotoxic hydroxyatrazine. Soil contaminated by an accidental spill of atrazine (up to 29,000 p.p.m.) supported significant populations of indigenous microorganisms capable of atrazine catabolism. Laboratory experiments indicated that supplementing soil with carbon inhibited atrazine biodegradation, but inorganic phosphate stimulated atrazine biodegradation. A subsequent field-scale study consisting of nine (0.75m3) treatment plots was designed to test four treatment protocols in triplicate. Control plots contained moistened soil; biostimulation plots received 300p.p.m. phosphate; bioaugmentation plots received 0.5% (w/w) killed, recombinant E. coli cells encapsulating AtzA; and combination plots received phosphate plus the enzyme-containing cells. After 8 weeks, atrazine levels declined 52% in plots containing killed recombinant E. coli cells, and 77% in combination plots. In contrast, atrazine levels in control and biostimulation plots did not decline significantly. These data indicate that genetically engineered bacteria overexpressing catabolic genes significantly increased degradation in this soil heavily contaminated with atrazine.

Two c-cytochromes extrinsically bound to the membranes of Nitrosomonas europaea have been identified. One is the tetraheme cytochrome c554, a protein previously described as soluble and periplasmic. Depending on the concentration of Fe and Cu in the growth medium, from 50 to 100% of the total cellular cytochrome c554 is membrane-associated. The cytochromes c554 found in the soluble or membrane fractions are identical in the spectroscopic, chromatographic, or primary structural properties examined. The interaction of cytochrome c554 with membranes is ionic in nature; it is disrupted by high concentrations of salt. Both membrane-derived and periplasmic forms of cytochrome c554 rebind tightly to membranes which have been washed free of the cytochrome. Cytochrome c554 binds to phospholipid vesicles, suggesting that phospholipids may play a role in the interaction of this cytochrome with the membrane. During the oxidation of NH2OH, the ability of the soluble hydroxylamine oxidoreductase (HAO) to transfer electrons to its natural electron acceptor, cytochrome c554, is substantially impaired when the latter is bound to phospholipid vesicles. The second c-cytochrome associated with membranes in N. europaea is identified as HAO based on its catalytic activity and the presence of a 464-nm ferrous absorption band. A small fraction of HAO is found to be membrane-bound and only in cells grown under low Fe/low Cu. This subpopulation of HAO can be released from the membranes without detergents.

The major enzymes associated with the oxidation of ammonia by chemolithoauthotrophic bacteria are studied, including some cytochromes c , are described and details of their properties are given.

Extracts from Clostridium pasteurianum were denatured in 7 M urea anaerobically to eliminate hydrogenase activity. Hydrogenase from the denatured extracts could be renatured by incubation anaerobically with sulfide, reduced thiol reagents, and Fe2+. Reconstituted extracts recovered 2% of the original hydrogen evolution activity. Reconstitution was also successful with purified hydrogenase I that was denatured anaerobically. This recovered 3.6% of its original hydrogen evolution activity. These results suggest that accessory proteins are not necessary for assembly of iron-only hydrogenases as they are for nickel-iron hydrogenases.

Atrazine chlorohydrolase (AtzA) from Pseudomonas sp. ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine. Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily. AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion. Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts. Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II). Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal. Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism.

The genome of Nitrosomonas europaea contains at least three copies each of the genes coding for hydroxylamine oxidoreductase (HAO) and cytochrome c554. A copy of an HAO gene is always located within 2.7 kb of a copy of a cytochrome c554 gene. Cytochrome P-460, a protein that shares very unusual spectral features with HAO, was found to be encoded by a gene separate from the HAO genes.

TPS2635 Background: The insulin-like growth factor 1 receptor (IGF-1R) is overexpressed in multiple malignancies, including breast, prostate, lung, colorectal, CLL, T-ALL, and mantle cell lymphoma....

H2 evolution by direct electron transfer from the dithionite-reduced photosystem I (PSI) complex to both hydrogenase I and hydrogenase II from Clostridium pasteurianum was observed. Evidence indicates that the electron carriers on PSI that transfer electrons to hydrogenase in this system are the FA/FB iron-sulfur clusters on the PsaC polypeptide, the terminal bound electron acceptors in PSI. Light-dependent H2 evolution was also observed, using high potential electron donors to PSI, from a combination of hydrogenase I and either solubilized purified PSI or thylakoids. Mediators capable of transferring electrons from the PSI complex to hydrogenase were not necessary for H2 evolution, indicating again that the mechanism of H2 evolution is direct electron transfer from PSI to hydrogenase, and that this can occur with light-reduced as well as chemically reduced PSI, and with PSI in thylakoids as well as the solubilized complex. Light-dependent H2 evolution was also observed from a mixture of thylakoids and the oxygen-resistant hydrogenase of Rhodococcus sp. MR11. These results suggest that direct electron transfer from PSI to hydrogenase could be engineered to occur in vivo in a photosynthetic organism to create an organism that would efficiently produce H2 from H2O.

Azotobacter vinelandii hydrogenase was purified aerobically with a 35% yield. The purified enzyme catalyzed H2 oxidation at much greater velocity than H2 evolution. There was a large difference in activation energy for the two reactions. EA was 10 kcal/mol for H2 oxidation and 22 kcal/mol for evolution. This difference in activation energies between the two reactions means that the ratio of oxidation velocity to evolution velocity drops from 70 at 33 degrees C to 8 at 48 degrees C. With D2 and H2O as substrates, both membranes and purified enzyme produced only H2 and no HD in the isotope exchange reaction. The velocity of isotope exchange was equal to the velocity of H2 evolution from reduced methyl viologen, indicating that the two reactions share the same rate-limiting step. D2 and H2 inhibited H2 evolution, but D2 did not inhibit isotope exchange. We conclude that H2 and D2 do not inhibit H2 evolution by competing with H+ for the active site of the reduced enzyme. The Km for D2 in isotope exchange is 40-times greater than its Km in D2 oxidation. The difference in Km cannot be accounted for by differences in kcat. We propose that redox environment regulates hydrogenase's affinity for D2 (and likely H2 as well).

Nitrosomonas europaea, a chemolithotrophic bacterium, was found to contain two copies of the gene coding for the presumed active site polypeptide of ammonia monooxygenase, the 32-kDa acetylene-binding polypeptide. One copy of this gene was cloned, and its complete nucleotide sequence is presented. Immediately downstream of this gene, in the same operon, is the gene for a 40-kDa polypeptide that copurifies with the ammonia monooxygenase acetylene-binding polypeptide. The sequence of the first 692 nucleotides of this structural gene, coding for about two-thirds of the protein, is presented. These sequences are the first sequences of protein-encoding genes from an ammonia-oxidizing autotrophic nitrifying bacterium. The two protein sequences are not homologous with the sequences of any other monooxygenase. From radioactive labelling of ammonia monooxygenase with [14C]acetylene it was determined that there are 23 nmol of ammonia monooxygenase per g of cells. The kcat of ammonia monooxygenase for NH3 in vivo was calculated to be 20 s-1.

Nitrosomonas is an obligately autotrophic and aerobic bacterium which produces energy for growth from the following reactions: NH3 + O2 + 2e- → NH2OH (ammonia monoxygenase, “AMO”) (8); NH2OH + H2O → 4e- + 4H+ + HNO2 (hydroxylamine oxidoreductase, “HAO”) (3) and 1/2 O2 + 2e- + 2H+ → H2O (terminal oxidase).

Photosynthetic state transitions occurring in nature are demonstrated. Chenopodium album leaves converted to state 1 and Ailanthus altissima leaves converted to an intermediate position between state 2 and state 1 at a time of day when these leaves were shaded by the canopy on a sunny day, while both plants' leaves were in state 2 at a time of day when they were not shaded. Filtering of white light by flasks of green algae also converted the light from causing state 2 in Chlorella vulgaris to causing state 1. Thus, light absorption by photosynthetic tissue can convert the natural light environment to one that causes state 1 in green plants.However, light absorption by water, by itself, up to a depth of 4.3m, does not change the light 2 character of sunlight.

The photosystem II reaction center as isolated (O Nanba, K Satoh [1987] Proc Natl Acad Sci USA 84: 109-112) is quite dilute and very unstable. Precipitating the complex with polyethylene glycol and resuspending it in buffer without detergent concentrates the reaction center and greatly improves its stability at 4°C in the dark as judged by light-induced electron transport activity. Furthermore, a procedure was developed to minimize photodestruction of polyethylene-glycol-concentrated material at room temperature in the light. The ability to stabilize the photosystem II reaction center should facilitate future photophysical, biochemical, and structural studies of the complex., Sponsored by the Division of Energy Biosciences, Office of Basic Energy Sciences, United States Department of Energy (contract No. 18-006-88) and by a grant from the Solar Energy Research Institute Director's Development Fund to M. S. H. M. is an Associated Western Universities Postgraduate Research Participant at SERI. A division of the Midwest Research Institute operated for the U.S. Department of Energy under contract No. DE-AC02-83CH-10093.