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The aryl hydrocarbon receptor (AhR) mediates induction of CYP1A1 and CYP1B1 by 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (dioxin) via binding to xenobiotic-responsive elements (XREs) in their enhancer regions. CYP1A1 and CYPIB1 were both inducible by dioxin in human MCF-7 cells. However, only CYP1A1 was inducible in human HepG2 cells. Further experiments focused on providing an explanation for this last observation. Dioxin induced the recruitment of AHR and the transcriptional coactivators p300 and p300/cAMP response element-binding protein binding protein-associated factor (PCAF) to the CYP1B1 enhancer in HepG2 cells but failed to induce recruitment of RNA polymerase II (polII) or the TATA binding protein (TBP) and acetylations of histones 3 and 4 or methylation of histone 3 at the promoter. Because p300 was required for dioxin induction of the aforementioned histone modifications at the CYP1B1 promoter and for induction of CYP1B1 transcription (in MCF-7 cells), the recruitments of p300 and AhR, although necessary, are not sufficient for eliciting the above responses to dioxin. Cytosine residues within CpG dinucleotides at the enhancer, including those within the XREs, were partially methylated, whereas those at the promoter were fully methylated. Treatment of HepG2 cells with 5-aza-2′-deoxycytidine led to partial demethylation of the promoter, restored polII and TBP binding, and CYP1B1 inducibility. Thus, the deficiency of CYP1B1 induction in HepG2 cells is ascribable to cytosine methylation at the promoter, which prevents recruitment of TBP and polII. It is noteworthy that our data indicate that stable recruitment of p300 and PCAF to the CYP1B1 gene does not require their tethering to the promoter and to the enhancer.

The xenobiotic metabolizing enzymes Cyp1a1 and Cyp1b1 can be induced by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-rho-dioxin (dioxin) via the aryl hydrocarbon receptor (AhR). These genes are differentially induced by dioxin in different mouse cell lines. In the mouse hepatoma cell line Hepa1c1c7 (Hepa-1), the Cyp1a1 gene is induced to very high levels by dioxin, but the levels of Cyp1b1 mRNA are extremely low and are not inducible by dioxin. The reverse is the case for the mouse embryonic fibroblast cell line C3H10T1/2, in which Cyp1b1 is induced to very high levels by dioxin, but the levels of Cyp1a1 mRNA are extremely low and not inducible by dioxin. However, dioxin treatment leads to the recruitment of AhR to the enhancer regions of both genes in both cell lines. Somatic cell hybrid clones generated between the two cell lines display high levels of induction of both genes in response to dioxin. Strong reactivation of the Cyp1a1 gene was also observed in C3H10T1/2 cell line after treatment with the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine (5-AzadC) and the histone deacetylase inhibitor, trichostatin-A (TSA). However, only modest reactivation of Cyp1b1 was observed in Hepa-1 cells after 5-AzadC or TSA treatment. These data demonstrate that the presence or absence of binding of AhR to regulatory regions is not responsible for determining the differences in levels of induction of the two genes in these cell lines and indicate that DNA methylation plays a major role in silencing of Cyp1a1 gene expression in C3H10T1/2 cells, but appears to play only a minor role in silencing Cyp1b1 gene expression in Hepa-1 cells, which likely occurs principally because Hepa-1 cells lack a factor required for high levels of induction of this gene.

The Meningococcus Genome Informatics Platform (MGIP) is a suite of computational tools for the analysis of multilocus sequence typing (MLST) data, at http://mgip.biology.gatech.edu. MLST is used to generate allelic profiles to characterize strains of Neisseria meningitidis, a major cause of bacterial meningitis worldwide. Neisseria meningitidis strains are characterized with MLST as specific sequence types (ST) and clonal complexes (CC) based on the DNA sequences at defined loci. These data are vital to molecular epidemiology studies of N. meningitidis, including outbreak investigations and population biology. MGIP analyzes DNA sequence trace files, returns individual allele calls and characterizes the STs and CCs. MGIP represents a substantial advance over existing software in several respects: (i) ease of use--MGIP is user friendly, intuitive and thoroughly documented; (ii) flexibility--because MGIP is a website, it is compatible with any computer with an internet connection, can be used from any geographic location, and there is no installation; (iii) speed--MGIP takes just over one minute to process a set of 96 trace files; and (iv) expandability--MGIP has the potential to expand to more loci than those used in MLST and even to other bacterial species.

Although it is causative for many types of cancers, experimental and epidemiological evidence suggest that 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) may in fact protect against breast cancer. The mechanism(s) for this protection remain unclear. In an attempt to further elucidate this mechanism, we performed a microarray experiment to identify genes that were modulated upon dioxin treatment. We found that dioxin downregulated the messenger RNAs for the G-protein-coupled receptor, CXCR4, as well as its unique chemokine ligand, CXCL12, in MCF-7 breast cancer cells. We demonstrated that the corresponding proteins are also downregulated by dioxin. The interaction between CXCR4 and CXCL12 plays a central role in the metastasis of breast cancer, as disruption of the CXCL12/CXCR4 axis has been shown to limit the metastasis of breast cancer cells to the lung in mice. Utilizing an in vitro chemotaxis assay, we demonstrate that dioxin specifically inhibits the migration of MCF-7 cells toward CXCL12. We also show that dioxin reduces CXCR4 under hypoxia and CXCL12 under estradiol-induced conditions in MCF-7 cells. Finally, as the CXCR4/CXCL12 axis is implicated in the progression of numerous types of cancer, we identified several other cancer cell lines in which dioxin modulates CXCR4 and CXCL12 levels. We therefore propose that one mechanism whereby dioxin may protect against breast cancer is via downregulation of CXCR4 and CXCL12, thereby inhibiting progression of the disease. Further, other nontoxic ligands for the aryl hydrocarbon receptor (selective AHR modulators) may exert their protective effects by a similar mechanism.

Cytochrome P4502S1 (CYP2S1) is expressed at high levels in epithelial tissues and is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) via the aryl hydrocarbon receptor (AHR). Transcriptional initiation of mouse Cyp2s1 was found to occur at three regions, approximately 198, 102, and 22 nucleotides from the translational initiation codon. Approximately 400 nucleotides upstream of its translational initiation codon, mouse Cyp2s1 contains three overlapping xenobiotic-responsive element (XRE) sequences, which make a major contribution toward dioxin inducibility. Each XRE sequence in this trimeric XRE can bind the AHR/aryl hydrocarbon receptor nuclear translocator (ARNT) dimer in a dioxin-dependent fashion in vitro and can mediate dioxin-dependent transcription. Cyp2s1 is also markedly inducible by hypoxia. Induction is dependent on hypoxiainducible factor-1 (HIF-1) and is mediated in large part by three overlapping hypoxia response elements (HREs) embedded within the trimeric XRE segment. Although each HRE within this segment can bind HIF-1alpha/ARNT in vitro, the most 3' HRE contributes the most toward hypoxia inducibility. AHR/ARNT and HIF-1alpha/ARNT dimers bind to the region containing the trimeric XRE segment of the endogenous Cyp2s1 gene in vivo in a dioxin-dependent fashion and hypoxia-dependent fashion, respectively. These observations identify a novel regulatory cassette that mediates changes in Cyp2s1 expression.

The aryl hydrocarbon receptor nuclear translocator/hypoxia-inducible factor (ARNT/HIF-1 beta) mediates an organism's response to various environmental cues, including those to chemical carcinogens, such as 2,3,7,8-tetrachlorodibenzo-rho-dioxin (TCDD or dioxin), via its formation of a functional transcription factor with the ligand activated aryl hydrocarbon receptor (AHR). Similarly, tissue responses to hypoxia are largely mediated through the HIF-1 heterodimeric transcription factor, comprising hypoxia-inducible factor-1 alpha (HIF-1 alpha) and ARNT. The latter response is essential for a metabolic switch from oxidative phosphorylation to glycolytic anaerobic metabolism as well as for angiogenesis and has been implicated as necessary for growth in many solid tumors. In this report, we demonstrate that the thyroid hormone receptor/retinoblastoma-interacting protein 230 (TRIP230) interacts directly with ARNT and is essential for both hypoxic and TCDD-mediated transcriptional responses. We initially identified TRIP230 as an ARNT-interacting protein in a yeast two-hybrid assay screen. This interaction was confirmed in mammalian cell systems using co-immunoprecipitation and in mammalian two-hybrid assays. Furthermore, TRIP230 could be recorded at sites of activated transcription of either TCDD- or hypoxia-inducible genes in a stimulus-dependent fashion by chromatin immunoprecipitation analysis. Finally, using single-cell microinjection and RNA interference assays, we demonstrate that TRIP230 is indispensable for TCDD- and hypoxia-dependent gene transcription.

We are investigating a methanotrophic filter strategy for the in situ bioremediation of low levels of chlorinated aliphatic, volatile organic chemicals (VOCs). It is based on the use of pregrown, resting cells, instead of growth-nutrient stimulations. The economic feasibility of such a filter is dependent on its operational longevity at ground-water temperatures. The latter, in turn, is dependent on several key parameters, such as the bacterial attachment densities reached during the injection of the microbial suspension and the subsequent detachment-removal of cells from the filter over time. Scaled attachment/detachment experiments were carried out using a representative quartzitic sand in glass 1-cm x 10-cm columns to simulate a filter. A rosette-dominated form of Methylosinus trichosporium OB3b was isolated and used in these and the subsequent catalytic longevity experiments. Its initial attachment, employing Higgins' medium phosphate buffer, pH 7.0 (HPB), was 7.0 to 8.0 x 10(8) bacteria/g of dry sand. This was elevated to approximately 1.5 x 10(9) cells/g by including 1.0 mM MgCl(2), 100 muM FeSO(4), and 0.025% agar in the cell-suspension loading buffer. These loading additives also increased the time required to reach 50% cell detachment with HPB alone from 5 days to approximately 45 days. The functional longevity of a column biofilter, formed with resting-state rosette-enriched cells in the presence of the aforementioned additives, was determined at 21 degrees C by challenging it with weekly 12 h, approximately 250 ppb pulses of trichloroethylene (TCE). The column results indicate that for our attached-cell filter to biodegrade TCE levels of several hundred ppb sufficiently, to

XIAP is a key regulator of apoptosis, and its overexpression in cancer cells may contribute to their survival. The antiapoptotic function of XIAP derives from its BIR domains, which bind to and inhibit pro-apoptotic caspases. Most known IAP inhibitors are selective for the BIR3 domain and bind to cIAP1 and cIAP2 as well as XIAP. Pathways activated upon cIAP binding contribute to the function of these compounds. Inhibitors selective for XIAP should exert pro-apoptotic effects through competition with the terminal caspases. This paper details our synthetic explorations of a novel XIAP BIR2-selective benzazepinone screening hit with a focus on increasing BIR2 potency and overcoming high in vivo clearance. These efforts led to the discovery of benzoxazepinone 40, a potent BIR2-selective inhibitor with good in vivo pharmacokinetic properties which potentiates apoptotic signaling in a manner mechanistically distinct from that of known pan-IAP inhibitors.

A field test has demonstrated that an in situ biofilter using resting-state cells effectively remediated groundwater with about 425 ppb of trichloroethene (TCE) as the sole contaminant species. About 5.4 kg (dry weight equivalent) of a strain of methanotrophic bacteria (Methylosinus trichosporium OB3b) was suspended in 1800 L of groundwater (5.4 x 10{sup 9} cells/mL) and injected into an aquifer through a single well at a depth of 27 m, several meters below the water table. The injected groundwater was devoid of TCE and growth substrates but was amended with a phosphate solution (10 mM) to buffer the pH and phenol red (20 {mu}m) to act as a tracer. Approximately 50% of the injected bacteria attached to the sediments, forming an in situ, fixed-bed bioreactor of unknown geometry. Contaminated groundwater was subsequently withdrawn through the biofilter region by extracting at 3.8 L/min for 30 h and then at 2.0 L/min for the remaining 39 days of the field experiment. TCE concentrations in the extracted groundwater decreased from 425 to less than 10 ppb during the first 50h of withdrawal, which is equivalent to a 98% reduction. TCE concentration extracted through the biofilter gradually increased to background values at 40 daysmore » when the experiment was terminated. 31 refs., 7 figs., 2 tabs.« less

Methanotrophs have promising applications in the bioremediation of chlorinated hydrocarbons and in the production of a biopolymer, poly-beta-hydroxybutyrate (PHB). Batch bioreactor culture conditions were studied for the accumulation of PHB by methane-grown Methylosinus trichosporium OB3b, and to evaluate the effect of PHB on the bacterial capacity to degrade trichloroethylene (TCE), a common groundwater contaminant. The PHB content of the washed and lyophilized cells was measured by gas chromatography (GC), after hydrochloric acid (HCl) propanolysis. A differential GC-based assay was developed for the monomer and the polymer of beta-hydroxybutyrate utilizing 1% and 10% HCl (v/v) reaction mixtures, respectively. During bioreactor growth in a Cu-deficient modified Higgins' medium, the cells accumulated PHB upon depletion of nitrate. A biomass yield of 3.2 g dry wt/L and a PHB accumulation of approximately 10% (w/w) were reached after 140 to 160 h, without adversely affecting the propene or TCE epoxidation specific rate given by whole cells containing soluble methane monooxygenase (sMMO). The TCE biotransformation capacity ( approximately 0.25 mg TCE oxidized/mg dry cell wt) of resting cells containing approximately 10% PHB was consistently approximately 1.6-fold greater than that of cells containing only approximately 2% PHB. Higher levels (>10%) of accumulated PHB did not enhance this biotransformation capacity further. By replacing the bioreactor inlet air + CO(2) mixture with pure O(2) at approximately 85 h of batch operation, a PHB accumulation of approximately 45% was achieved after 160 h, but the whole-cell sMMO activity was markedly decreased. In contrast, cells grown in a 10 muM Cu-supplemented Higgins' nitrate minimal salts medium (particulate MMO formation) accumulated up to 50% PHB in only 120 h, coupled with a very high biomass yield of 18 g dry cell wt/L. High PHB accumulations above approximately 20% by both the -Cu and the +Cu grown cells resulted in a decreased ratio of the electronic cell count to the absorbance at 660 nm, which is commonly used to monitor bacterial growth. (c) 1996 John Wiley & Sons, Inc.

The synthesis and coordination chemistry of a new asymmetric multidentate ligand designed for modeling coordination number asymmetry at metal sites in binuclear metalloproteins are described. A binuclear copper complex of this ligand demonstrates proof-of-concept for inducing coordinative unsaturation at one metal of the binuclear pair, and subsequent reaction with azide illustrates site-directed reactivity.

A numerical model has been developed to simulate the biodegradation of trichloroethylene (TCE) by Methylosinus trichosporium OB3b bacteria as observed in a saturated, open system sand-bed experiment. The sand pack was inoculated with bacteria in a resting state (without nutrients) to form a 10 cm thick attached filter along the direction of flow. Degradation occurs through cometabolism of TCE to Cl− and primarily HCO3− as the major overall carbon product. A steady, 1·5 cm/h saturated flow was established in the sand pack, and was later doped (upstream of the attached filter) with a 13-day, 4 ppm pulse of TCE. Concentrations of TCE were monitored immediately upstream and downstream of the attached bacterial filter. Accounting for travel time through the test bed, observed downstream concentrations indicated degradation in the filter was complete for 0·5 days, substantially complete for another 1·5 days, and increasingly limited over the next 11 days, after which the outflow concentrations returned to zero, indicating the end of the pulse. Measurements of Cl− breakthrough were used to confirm TCE degradation quantitatively. The return of TCE in the downstream flow was anticipated from repetitive exposure, cyclic transfer experiments in sealed tubes which showed a limited capacity of the bacteria in the resting state to degrade TCE. Simulations based upon a previously-determined Michaelis-Menton relationship were used to successfully model the experimental results in the sand test-bed. Based upon the tube-transfer experiments, the rate law was adjusted to account for a fixed or limited capacity of the bacteria in the resting state to degrade TCE. The degradation capacity of sand-attached M. trichosporium OB3b has been estimated to be about 0·30 g TCE/g bacteria, and is not expected to be a leading limitation in implementing this approach for bioremediation in the field.

An in situ microbial filter technology is being tested and developed for remediating migrating subsurface plumes contaminated with low concentrations of trichloroethylene (TCE). The current focus is the establishment of a replenishable bioactive zone (catalytic filter) along expanding plume boundaries by the injection of a representative methanotrophic bacterium, Methylosinus trichosporium OB3b. This microbial filter strategy has been successfully demonstrated using emplaced, attached resting cells (no methane additions) in a 1.1 m flow-through test bed loaded with water-saturated sand. Two separate 24 h pulses of TCE (109 ppb and 85 ppb), one week apart, were pumped through the system at a flow velocity of 15 mm h−1; no TCE (< 0.5 ppb) was detected on the downstream side of the microbial filter. Subsequent excavation of the wet sand confirmed the existence of a TCE-bioactive zone 21 days after it had been created. An enhanced longevity of the cellular, soluble-form methane monooxygenase produced...

Continous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO2 addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 × 10−4 g Cu/g dry cell Wt) for the exclusive production of particulate MMo during continous culture growth. When the feed medium nitrate concentration was varied in the range of 5-50 mM, the whole-cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO2 gassing decreased pMMO activity by more than 30%. The steady-state cell density increased continuously over a 300-700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h−1 and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10 μM Cu, 80 μM Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h−1. Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2-15 μM, falling sharply at 20 μM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied. © 1992 John Wiley & Sons, Inc.

CXCR4 is a chemokine receptor frequently overexpressed on primary tumor cells. Organs to which these cancers metastasize secrete CXCL12, the unique ligand for CXCR4, which stimulates invasion and metastasis to these sites. Similar to our previous work with the chemoprotective phytochemical, 3,3′-diindolylmethane (DIM), we show here that genistein also downregulates CXCR4 and CXCL12 and subsequently lowers the migratory and invasive potentials of breast and ovarian cancer cells. Moreover, genistein and DIM elicit a significantly greater cumulative effect in lowering CXCR4 and CXCL12 levels than either compound alone. Our data suggest a novel mechanism for the protective effects of phytochemicals against cancer progression and indicate that in combination, these compounds may prove even more efficacious.

Cytochrome P450 (CYP) 1A1 and CYP1B1 are inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) in the human breast cancer cell line, MCF-7. Since CYP1A1 was inducible to a much greater degree than CYP1B1, we hypothesized that there may be differences in coactivator recruitment to the promoter and/or enhancer regions of these genes. Dioxin treatment leads to recruitment of the aryl hydrocarbon receptor to the enhancer regions but not to the proximal promoter regions of both the CYP1A1 and CYP1B1 genes. On the other hand, dioxin treatment facilitated recruitment of RNA polymerase II to the promoters but not the enhancer regions. Dioxin treatment also elicited recruitment of the transcriptional coactivators, steroid receptor coactivator 1 (SRC-1) and steroid receptor coactivator 2 (SRC-2) and p300, which possess intrinsic histone acetyltranferase activities, to both genes, whereas Brahma (BRM)/Switch 2-related gene 1 (BRG-1), a subunit of nucleosomal remodeling factors, was recruited more robustly to CYP1A1 relative to CYP1B1. Small inhibitory RNA-mediated knockdown of p300 and SRC-2 adversely affected dioxin induction of both genes, whereas knockdown of BRM/BRG-1 reduced CYP1A1 induction but had little, if any, effect on CYP1B1 induction. These results suggest that nucleosomal remodeling is less significant for dioxin-mediated induction of CYP1B1 than that of CYP1A1 and may be related to the more modest inducibility of the former. Interestingly, simultaneous knockdown of SRC-2 and BRM/BRG-1 had no greater effect on CYP1A1 induction than knockdown of each coactivator individually, while simultaneous knockdown of p300 and BRM/BRG-1 had a much greater effect than knockdown of each individual gene, suggesting that the recruitment of SRC-2 to CYP1A1 depends upon BRM/BRG-1, while the recruitments of p300 and BRM/BRG-1 are independent of each other. These observations provide novel insights into the functional roles of the endogenous coactivators in dioxin induction of the human CYP1A1 and CYP1B1 genes in their natural chromosomal configurations.

Cruciferous vegetables are thought to protect against numerous types of cancer. 3,3'-Diindolylmethane (DIM) is an acid-catalyzed product generated during the consumption of cruciferous vegetables and appears to be chemoprotective for breast cancer. The interaction between the chemokine receptor, CXCR4, and its unique ligand, CXCL12, is known to mediate the progression and metastasis of breast and other cancers. Organs to which these cancers metastasize secrete CXCL12, which binds to CXCR4 expressed on the surface of primary cancer cells. This process subsequently stimulates the invasive properties of the cancer cells and attracts them to the preferred organ sites of metastases. We have found that DIM down-regulates both CXCR4 and CXCL12 in MCF-7 and MDA-MB-231 breast cancer cells as well as in BG-1 ovarian cancer cells at the transcriptional level and in an estrogen-independent manner. We demonstrate that the potential of MDA-MB-231 and BG-1 cells for chemotaxis and invasion towards CXCL12, but not towards IL-6 or fetal bovine serum, respectively, is inhibited by DIM. Furthermore, we show that DIM down-regulates CXCR4 under hypoxia and CXCL12 under estradiol-inducing conditions. Our data suggest that one mechanism whereby DIM protects against breast, ovarian, and possibly other cancers is through the repression of CXCR4 and/or CXCL12, thereby lowering the invasive and metastatic potential of these cells.

This article presents data gathered from young people in a poor urban community in New York City, the South Bronx. It seeks to help public health professionals better understand young people’s perceptions of violence in the context of their daily lives. Sources of data include a street survey, five focus groups, interviews with incarcerated young males, and observations of several youth programs. These data suggest that violence is pervasive in the lives of both young men andwomen, although gender plays an important role in shaping the experience of violence. Other factors that influence the experience of violence include patterns of substance use, availability and use of weapons, and a perception that the police do not respect young people. Despite numerous challenges, many young people do take actions to reduce violence. The article suggests actions public health professionals can take to strengthen the ability of families, schools, youth organizations, and young people themselves to reduce violence in low-income urban communities.

Two thermophilic bacteria, Thermus aquaticus ATCC 25104 and Thermus species ATCC 27978, were investigated for their abilities to degrade BTEX (benzene, toluene, ethylbenzene, and xylenes). Thermus aquaticus and the Thermus sp. were grown in a nominal medium at 70 degrees C and 60 degrees C, respectively, and resting cell suspensions were used to study BTEX biodegradation at the same corresponding temperatures. The degradation of BTEX by these cell suspensions was measured in sealed serum bottles against controls that also displayed significant abiotic removals of BTEX under such high-temperature conditions. For T. aquaticus at a suspension density of only 1.3 x 10(7) cells/mL and an aqueous total BTEX concentration of 2.04 mg/L (0.022 mM), benzene, toluene, ethylbenzene, m-xylene, and an unresolved mixture of o-and p-xylenes were biodegraded by 10, 12, 18, 20, and 20%, respectively, after 45 days of incubation at 70 degrees C. For the Thermus sp. at a suspension density of 1.1 x 10(7) cells/mL and an aqueous total BTEX concentration of 6.98 mg/L (0.079 mM), benzene, toluene, ethylbenzene, m-xylene, and the unresolved mixture of o-and p-xylenes were biodegraded by 40, 35, 32, 33, and 33%, respectively, after 45 days of incubation at 60 degrees C. Raising the BTEX concentrations lowered the extents of biodegradation. The biodegradations of both benzene and toluene were enhanced when T. aquaticus and the Thermus sp. were pregrown on catechol and o-cresol, respectively, as carbon sources. Use of [U-(14)C]benzene and [ring-(14)C]toluene verified that a small fraction of these two compounds was metabolized within 7 days to water-soluble products and CO(2) by these nongrowing cell suspensions. Our investigation also revealed that the nominal medium can be simplified by eliminating the yeast extract and using a higher tryptone concentration (0.2%) without affecting the growth and BTEX degrading activities of these cells. (c) 1995 John WileySons, Inc.

Batch culture conditions were established for the formation of H(2)-driven whole-cell soluble or particulate methane monooxygenase (sMMO or pMMO) activity in the obligate methanotroph, Methylosinus trichosporum Ob3b, to expand its potential uses in groundwater bioremediation and the production of specific chemicals. Addition of either Ni and H(2) to a nitrate-containing minimal salts growth medium or Ni and Mo to a nitrate-lacking growth medium (induces a nitrogenase that generates intracellular H(2)) markedly enhanced both the hydrogenase and the accompanying washed-cell H(2)-driven MMO activities of shake-flask cultured cells. For sMMO containing cells, H(2) provided in vitro reducing power for the oxidation of chlorinated solvents such as chloroform and trichloroethylene. Cell cultivations under N(2)-fixing conditions in a 5-L bioreactor, however, required an initial nitrate concentration of at least 1 to 2 mM to achieve high biomass yields (5 to 7 g of dry cell wt/L) for cells producing H(2)-driven sMMO or pMMO activity. Elevation of the initial medium nitrate concentration to 20 mM shortened the culture time for pMMO producing cells by 40%, yet still generated an equivalent growth yield. High nitrate also shortened the culture time for sMMO containing cells by approximately 25%, but it lowered their biomass yield by 26%. Upon storage for 5 weeks at room temperature, washed resting-state cells retained 90% and 70% of their H(2)-driven sMMO and pMMO activity, respectively. This makes their practical use quite feasible. (c) 1995 John WileySons, Inc.

A study of the random motility and chemotaxis of Methylosinus trichosporium OB3b was conducted by using Palleroni-chamber microcapillary assay procedures. Under the growth conditions employed, this methanotroph was observed qualitatively with a microscope to be either slightly motile or essentially nonmotile. However, the cells did not not respond in the microcapillary assays in the manner expected for nonmotile Brownian particles. As a consequence, several hydrodynamic effects on these Palleroni microcapillary assays were uncovered. In the random-motility microcapillary assay, nondiffusive cell accumulations occurred that were strongly dependent upon cell concentration. An apparent minimal random-motility coefficient (mu) for this bacterial cell of 1.0 x 10(-7) cm2/s was estimated from microcapillary assays. A simple alternative spectrophotometric assay, based upon gravitational settling, was developed and shown to be an improvement over the Palleroni microcapillary motility assay for M. trichosporium OB3b in that it yielded a more-accurate threefold-lower random-motility coefficient. In addition, it provided a calculation of the gravitational-settling velocity. In the chemotaxis microcapillary assay, the apparent chemotactic responses were strongest for the highest test-chemical concentrations in the microcapillaries, were correlated with microcapillary fluid density, and were strongly dependent upon the microcapillary volume. A simple method to establish the maximal concentration of a chemical that can be tested and to quantify any contributions of abiotic convection is described. Investigators should be aware of the potential problems due to density-driven convection when using these commonly employed microcapillary assays for studying cells which have low motilities.

The obligatory methanotroph, Methylosinus trichosporium OB3b, was studied to optimize the batch culture conditions for the formation of particulate methane monooxygenase (pMMO) in a nitrate minimal salts medium. The important medium components investigated were copper, carbon dioxide, and nitrate. The whole-cell specific pMMO activity decreased sharply with increasing copper concentrations in the range of 10-40 microM and remained constant upon further increases of the copper concentration to 120 microM. The cell growth rate (micro), on the other hand, decreased over the entire range (10-120 microM) of copper concentrations tested. When pMMO was produced in a bioreactor with an optimal initial copper concentration of 10 microM, M. trichosporium OB3b exhibited a much faster overall growth rate and a higher whole-cell propene epoxidation activity compared to our earlier study, in which soluble methane monooxygenase (sMMO) was produced with copper-deficient medium. The addition of external carbon dioxide to the bioreactor culture eliminated an initial lag period in the cell growth. When the standard culture medium nitrate concentration (10 mM) was depleted, the pMMO activity, but not the growth rate, decreased rapidly. The whole-cell specific pMMO activity could be maintained by subsequent supplementation of nitrate. A 4-fold higher initial culture medium nitrate concentration of 40 mM, however, resulted in slower cell growth and lower pMMO activity. These observations demonstrate that, in addition to affecting the exclusive production of pMMO, copper also has an important previously unrecognized role in enhancing the growth rate of M. trichosporium OB3b. They also indicate that for the optimal batch production of pMMO with the minimal medium under study, nitrate should be supplied intermittently during the course of cultivation until other culture medium components become growth-limiting.

To characterize the reaction conditions that generate frameshift mutagens in cooked meats, we have concentrated on a supernatant fraction (S2) prepared from (1:1) aqueous homogenates of lean round steak. Soluble compounds

The cooking of foods derived from muscle generates heterocyclic amines that are very potent bacterial mutagens and carcinogens in mice, rats, and monkeys. Presently 12 mutagenic compounds have been found in cooked foods derived from the Western Diet. Only 6 of these compounds have been definitely identified. Specifically designed monoclonal antibodies can detect nanogram amounts each of 2-amino-1-methyu1-6-phenylimidazo[4,5-f]pyridine(PhIP), 2-amino-3-methylimidazo[4,5f] quinoline(IQ), and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQx) in the meat matrix, but specific quantitation requires a prior HPLC separation before use of antibodies for identification. Modeling of PhIP (20 ppb in beef) formation under dry heating conditions using heavy isotope incorporation coupled with MS and NMR shows that all the C and N atoms in the PhIP are contributed by creatin(ine) and phenylalanine. PhIP is both a potent frameshift mutagen in Salmonella bacteria and a potent inducer of mutations, sister chromatid exchange and chromosomal aberrations in Chinese Hamster Cells in culture, and of chromosome damage in mouse bone marrow. Using accelerator mass spectroscopy, we can measure in a linear fashion one MeIQz DNA adduct per 1011nucleotides. N-OH-PhIP which is generated by both IA1 and IA2 forms of cytochrome P-450 appears to be a proximal mutagen.

Fried beef and commercial beef extracts contain Ames/Salmonella frameshift mutagens that form at relatively low temperatures (100–200°C). To investigate the types of natural components in beef muscle that give rise to frameshift mutagenic activity, we previously devised a model boiling system and demonstrated that all of the Salmonella TA1538 activity is formed from H2O‐soluble, < 500 molecular wt compounds that are present in round steak supernatant fractions (S1 and S2). S2 is derived from S1, the soluble fraction of homogenized beef, by a brief 30 min boil and centrifugation. We now report that proteolysis of beef S1 with papain, trypsin, or chymotrypsin (± carboxypeptidase A) increases the mutagenic activity of boiled S2 by 1.8–4.5 fold over the baseline range of 90–100 TA1538 revertants/108 bacteria/g dry beef/14 h at pH 4.0. Sequential treatment of beef S1 with chymotrypsin followed by carboxypeptidase A is the most efficient mutagen enhancing digestion (415 revertants/g dry beef or 7.7 rev...

Mutagenic 1,2-dicarbonyls have been reported to occur in coffee and other beverages and in various foods. We have measured the induction of sister-chromatid exchanges (SCEs) and endoreduplicated cells (ERCs) to determine the genotoxicity of various 1,2-dicarbonyl compounds in Chinese hamster ovary (CHO) AUXB1 cells and human peripheral lymphocytes. The 1,2-dicarbonyls glyoxal, methylglyoxal and kethoxal each induced highly significant increases in both SCEs and ERCs in AUXB1 cells. Glyoxal and kethoxal induced SCEs but not ERCs in human peripheral lymphocytes. In addition, hydrogen peroxide induced highly significant levels of SCEs and ERCs in AUXB1 cells. Bisulfite, which reacts with carbonyl groups to form addition products, significantly reduced the frequency of SCEs and the proportion of ERCs when glyoxal, methylglyoxal, kethoxal and diacetyl were administered to AUXB1 cells. In addition, bisulfite blocked the formation of ERCs, but not SCEs, induced by hydrogen peroxide. These in vitro results suggest that 1,2-dicarbonyls may play an important role in the genotoxicity of some foods and beverages.

An extensively boiled supernatant2 (S2) fraction from beef round steak contains two major Salmonella frameshift mutagens, designated as HPLC peaks A and B. These same mutagens arise, but in different proportions, when S2 is boiled with creatine phosphate (CP), and they are produced in much greater quantities from a boiled mixture of S2 + L‐tryptophan (Trp) + CP + FeSO4 (S2 ∗). A third mutagen, peak C, is also generated in the S2 ∗ mixture. Mutagen peaks A, B, and C were purified to homogeneity and shown by their absorption spectra, mass fragmentation patterns, and other data to be 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), 3‐amino‐l‐methyl‐5H‐pyrido[4,3‐b]‐indole (Trp‐P‐2), and 3‐amino‐l,4‐dimethyl‐5H‐pyrido[4,3‐b]indole (Trp‐P‐1), respectively. This is the first demonstration that IQ, Trp‐P‐2, and Trp‐P‐1 can form under aqueous conditions at 100°C from reactions between low molecular wt precursors that are present in meat juice. Further simplification of and studies with the S2 fraction of be...

It was reported earlier that methylcobalamin (MeB‐12) plus K2PtCl6 or Na2PtCl6 under acidic conditions yields a single square planar Pt2+ species, MePtCl2‐ 3. A reaction between MeB‐12 and PtCl4 in...

Cellular resistances to the toxic effects of two environmentally and industrially important platinum (Pt) complexes (K2PtCl6 and Pt(SO4)2) were induced separately in Chinese hamster ovary cells (CHO‐S) by continuous exposure to the compounds for 5 and 4 months, respectively. The two resulting cell lines, 220R (resistant to 220 μM K2PtCl6) and 550R (resistant to 550 μM Pt(SO4)2) have resistant phenotypes which are stable for at least 55 population doublings in the absence of a selective agent (i.e., Pt). Each resistance is quite specific with respect to the Pt atom as well as the attached ligands, but both cell lines show some cross resistance to other Pt complexes and to one other Noble metal complex (K3 RhCl6). X‐Ray fluorescence determinations of Pt in parental CHO‐S and 220R cells cultured in the presence and absence of K2PtCl6 demonstrated that very little of this compound is taken up by either cell line. However, comparable analyses revealed that the amount of Pt accumulated by 550 cells gro...

6 platinum (Pt) compounds were compared in suspension cultured Chinese hamster ovary (CHO-S) cells with respect to their inhibition of growth, their reduction of cloning efficiency, and their induction of mutants resistant to 200 microM (30 micrograms/ml) 8-azaguanine (8-AG) and 3 mM ouabain (OUA), respectively. The toxicity of these compounds can be ranked by the medium concentrations which decrease suspension growth/or cloning efficiency by 50%: cis-Pt(NH3)2-Cl2 (0.9/1.5 microM) greater than Pt(SO4)2 + methylcobalamin (MeB-12) methylation product (20/10 microM) greater than K2PtCl4 (32/50 microM) = K2PtCl6 (34/50 microM) = MePtCl2-3 (60/50 microM) greater than Pt(SO4)2 (66/105 microM). Following 20 h exposures to concentrations which resulted in relative survivals of 80-2%, none of the foregoing compounds increased consistently the frequency of OUA(R) mutants above the spontaneous frequency (6.0 x 10(-6)). Parallel treatments with 800 microM (100 micrograms/ml) ethyl methanesulfonate (EMS) increased the OUA(R) mutant frequency 10--12-fold. Using 8-AG for mutant selection, dose-dependent increases of 5--7-fold above the spontaneous frequency (3--8 x 10(-5) were obtained with cis-Pt(NH3)2Cl2, Pt(S04)2, and the product from Pt(SO4)2 + MeB-12. Identical 20 h exposures to varying amounts of K2PtCl4, K2PtCl6, and MePtCl2-3 did not induce 8-AG(R) mutants. Optimal detection of Pt-induced 8-AG(R) mutants required 7 post-treatments, expression doublings in suspension culture. Under our selection conditions 8/8 spontaneous and 24/24 Pt-induced 8-AG(R) variants contained reduced hypoxanthine-guanine phosphoribosyl transferase (HGPRT) specific activities (means ranging from 3 to 11% of the parental CHO-S cells). When compared from linear plots of the 8-Ag(r) frequency against the initial medium concentration, cis-Pt(NH3)2Cl2 is 134 times and Pt(SO4)2 si 3.5 times more mutagenic than EMS. However, on a cell-survival basis EMS is 8--10-fold more mutagenic than these two Pt-compounds. 6-Thioguanine (10 microM) can be substituted for 8-AG to assay mutant induction by cis-Pt(NH3)2Cl2 and Pt(SO4)2 in CHO-S cells. The sensitivity of the CHO-S HGPRT locus for detecting mutagenesis by Pt complexes can be increased several fold by continuous subculture in the presence of these agents for 10--25 population doublings. By this procedure K2PtCl6 is seen to be weakly mutagenic and 20 microM Pt(SO4)2 produces 8-AG(R) mutants at frequencies requiring 7--8-fold higher concentrations when a fixed 20 h exposure is used.

5-Methyltetrahydrohomofolate (5-MeH 4 -homofolate) is a substrate for the cobalamin (B-12) methyltransferases in Escherichia coli B, rabbit liver, HeLa S 3 cells, and Chinese hamster ovary cells. Each of these B-12 enzymes catalyzes 5-MeH 4 -homofolate-homocysteine transmethylation at one-tenth the rate of methionine synthesis from 5-MeH 4 -folate. Only one stereoisomer in dl -5-MeH 4 -homofolate is active enzymically. Reduced higher 5-alkyl homofolates and folates are weak competitive inhibitors of 5-MeH 4 -folate, but are inactive as substrates. The K m of l -5-MeH 4 -homofolate for the E. coli B enzyme (80 μ m ) is greater than that of 5-MeH 4 -folate (35 μ m ), but its K m for the Chinese hamster ovary cell transmethylase (20 μ m ) is less than that of 5-MeH 4 -folate (35 μ m ). l -H 4 -Homofolate is a potent competitive inhibitor ( K i = 56 nM) for the E. coli B thymidylate synthetase compared to 5-MeH 4 -homofolate ( K i = 56 μM); it is also inactive as a cofactor. However, l -H 4 -homofolate is a cofactor for both the HeLa S 3 and Chinese hamster ovary cell thymidylate synthetases, giving 22% of the activity observable with l -H 4 -folate. Moreover, its K m as a cofactor is only 2.1 μ m compared to 17 μ m for l -H 4 -folate. dl -5-MeH 4 -Homofolate inhibits the growth of Chinese hamster ovary cells in a reversible manner, but the inhibition is not related to the amount of B-12 holomethyltransferase in the cells.

Fried beef and commercial beef extract contain Ames/ Salmonella frameshift mutagens that require microsomal (S‐9) activation. To ascertain which fraction(s) of beef muscle contain(s) the essential precursors, aqueous (1:1) homogenates of lean round steak were centrifuged to give an insoluble residue1 (R1) and a soluble supernatant1 (S1). S1 was then boiled for 30 min and again centrifuged, yielding a residue2 (R2) and a soluble supernatant2 (S2). S2 represents only 5% of the meat dry wt and it contains only 10∗ of the H2O‐soluble protein, but it contributes all of the S‐9 dependent Salmonella TA1538 mutagenic activity in boiled homogenates. Mutagen formation from S2 boiled for 0–30 h at a constant volume increases exponentially with time and displays sharp pH optima at 4.0 and 9.0. By molecular ultrafiltration the pH 4.0 mutagen precursors in S2 have molecular wts < 500. They are also stable to lyophilization. These observations and the disappearance of certain amino acids upon boiling at pH 4.0 ...

Two glycine-requiring Chinese hamster ovary (CHO) auxotrophs (GLYB and AUXB2) representative of the Gly − mutant classes B and C are shown to have defects in folate metabolism. These defects result in 10-fold lower rates of whole cell l -[U- 14 C]serine-to-[ 14 C]glycine conversion relative to the parental CHO lines (2 vs 20 nmol/h/10 6 cells). This restriction in serine hydroxymethyltransferase (SHMT) activity is localized in the mitochondria. Intact mitochondria from GLYB and AUXB2 convert labeled serine to glycine at 1–4% the rate and with only 1–3% of the total capacity of parental CHO mitochondria. Yet, GLYB and AUXB2 contain parental cell amounts of cytosolic and mitochondrial SHMT, the latter displaying normal substrate K m values. The whole cell and mitochondrial impairments in glycine formation are corrected in GLYB (but not AUXB2) by a prior growth with 100 μ m dl -folinate. They are also partially restored in spontaneous or chemically induced Gly + revertants of GLYB and AUXB2. Subcellular fractionation experiments suggest that a low content (one-fifth parental) of mitochondrial folylpolyglutamates contributes to the auxotrophy of GLYB. Solubilized mitochondrial SHMT from both Gly − auxotrophs cleaves the alternate substrates allothreonine and β-phenylserine at parental CHO rates, without tetrahydrofolate. Consequently, both GLYB and AUXB2 exhibit normal growth rates in the presence of folate plus 1 m m dl -allothreonine (without glycine). These studies demonstrate that mitochondrial SHMT is potentially functional in the Gly − mutant classes B (GLYB) and C (AUXB2). The impaired SHMT activity in vivo and in isolated mitochondria may result from a deficiency in mitochondrial recycling of 5,10-methylenetetrahydrofolate back to tetrahydrofolate. The multiple glycine + adenosine + thymidine auxotroph CHO AUXB1 closely mimics GLYB and AUXB2 with respect to whole cell and mitochondrial glycine synthesis, but the primary cause is different. It is due to a lack of folylpolyglutamate synthetase activity.

Aerobic incubation of μM levels of K2PtCl6 and methylcobalamin (MeB‐12) at pH 2.0 (0.01 M HC1) results in the complete conversion of MeB‐12 to aquoB‐12 without the accumulation of any corrinoid intermediates. Platinic sulfate (Pt(SO4)2) is also reactive; whereas, K2PtCl4 alone is unreactive. Soluble lead salts containing Pb2+ were likewise inactive; however, partial to complete demethylation was observed by prolonged incubation with fine suspensions of Pb4+ compounds. Demethylation of [Me‐14C]MeB‐12 with Pb4+ oxides was accompanied by a proportionate volatilization of the label and only unreacted radioactive MeB‐12 was detected upon paper electrophoresis. In contrast, demethylation with K2PtCl6 occurs with a high recovery of nonvolatile 14C which migrates with a major zone of Pt upon electrophoresis. There is no selective loss of 3H when [Me‐3H]MeB‐12 reacts with K2PtCl6. The Me‐Pt product has been isolated and characterized with respect to its light absorption spectrum in aqueous solutions.

A methyl‐Pt compound formed by incubating K2PtCl6 with methylcobalamin (MeB‐12) is further characterized with respect to its chloride content, its Pt oxidation state, and its electro‐phoretic mobility. The complexed chloride has been determined from nuclear magnetic resonance (NMR) spectra of the Cl displaced by competing thiocyanate (SCN ) anions. Release of 3 chlorides per Pt combined with a Me/Pt ratio of 1.0 indicates a metal coordination number of 4. This is in agreement with X‐ray photoelectron spectra (ESCA) which show that the majority of the Me‐Pt product contains Pt in the +2 valence state. Based on this data and the anionic nature of the product at pHs from 2.5–7.5 it 2‐is presently represented as MePtCl3. In comparison to the closely related complexes K2PtCl4 and K2PtCl6, MePtCl3 ‐2 is slightly less cytotoxic to an auxotrdphic Chinese hamster ovary cell line (CHO AUXB1). All three of these chloro‐Pt compounds at concentrations of 15–70 μ? induce a 6–10 fold increase in the frequency o...

Upon the addition of AMe to a 10 μM solution of B 12 enzyme in a FH 4 + DTT reducing system containing homocysteine, the absorption spectrum of the cobalamin chromophore changed from a B 12r type of spectrum (470–475 mμ peak) to a B 12s spectrum (maxima at 385 mμ and 460 mμ). These spectral changes began immediately after the injection of AMe at 37° and became maximal 6–12 minutes later. The original B 12r spectrum was restored after approximately 24 minutes but was converted back to a B 12s spectrum upon a second addition of exogenous AMe. In a parallel experiment the transient appearance of the B 12s -like absorption spectrum was correlated with the metabolism of methyl- 14 C-AMe, i. e., the transfer of methyl- 14 C groups to homocysteine. Steady-state catalysis of N 5 -methyl- 14 C-FH 4 -homocysteine transmethylation in the FH 4 + DTT reducing system was dependent on exogenous AMe and a non-enzymatic reduction of free hydroxy-B 12 to B 12s was not observed in this reducing system.

Extensively purified E. coli B cobalamin methyltransferase was resolved into an apoenzyme plus an unbound, 1-electron-reduced, cobalamin (B 12r ) upon incubation for 15 min at 37 ° with 6.0 m urea containing dithiothreitol (DTT). Apoenzyme which had been prepared by urea resolution was reconstituted 75–80% into a radioactive holoenzyme complex upon a 5-min incubation at 37 ° with 10 nmoles of either methyl- 14 CB 12 or propyl- 14 CB 12 per mg of protein. Reconstituted methyl- 14 CB 12 enzyme was light-stable (100 W tungsten lamp, 0 °) unless acidified and was almost completely demethylated by homocysteine. Incubation of resolved apoenzyme with deoxyadenosyl-B 12 , hydroxy-B 12 , cyano-B 12 , B 12r , diaquocobinamide, and methylcobinamide resulted in negligible holoenzyme formation. However, under the same conditions sulfito-B 12 yielded 48–52% holoenzyme. Urea-prepared apoenzyme differed from the purified B 12 holoenzyme with respect to its sedimentation coefficient. Based on a sedimentation coefficient of 7.0 S for the purified B 12 holoenzyme the apoenzyme sedimented at a slightly slower rate with a relative coefficient equal to 6.2 S . Binding methyl-B 12 ( 3 H) to the apoenzyme yielded a reconstituted methyl-B 12 ( 3 H) holoenzyme with a sedimentation coefficient of 7.0 S . Unpurified holoenzyme and unpurified apoenzyme in cell-free, sonic extracts also centrifuged differently, like purified holoenzyme and urea-resolved apoenzyme, respectively.

The methylcobalamin (MeB 12 ) homocysteine transmethylase activity and the B 12 -dependent 5-methyltetrahydrofolate (5-MeH 4 -folate) homocysteine transmethylase activity in cell-free extracts of E. coli B are catalytic functions of separate sites on a single enzyme-protein. Whether these two transmethylases exactly co-purify from extracts, and are protected against p -chloromercuribenzoate (pCMB), however, depends on whether or not the cells were previously cultured in the presence of approximately 1 × 10 −8 m cyanocobalamin (CNB 12 ). E. coli B (met H − ) contains a defective 5-MeH 4 -folate apoenzyme which does not tightly bind B 12 as a prosthetic group. While the folate-inactive apoenzyme from the mutant strain still catalyzes MeB 12 homocysteine transmethylation, this second site on the defective protein is not protected by media CNB 12 against pCMB inactivation. Both transmethylase activities are repressed 50% by growth in the presence of 10 m l-methionine.

Through the combined use of cellulose-polyacetate and disc-gel electrophoresis, a 200-fold purified preparation of Escherichia coli B 5-methyltetrahydrofolate-homocysteine methyltransferase was further fractionated into a single, protein-staining band. Associated with this band, as in the initial cobalamin (B 12 ) holoenzyme preparation, was a methyl-B 12 -homocysteine methyltransferase activity. Several types of experiments support the view that 5-methyltetrahydrofolate-homocysteine transmethylation (Reaction 1) and methyl-B 12 -homocysteine transmethylation (Reaction 2) occur at separate sites on the B 12 -protein. Both sites are specific for a cobalt-methyl group, however. An inhibited (Reaction 1) propyl-B 12 enzyme catalyzed Reaction 2 with no exchange or decomposition of the propyl-B 12 group. Reconstituted holoenzyme containing a tritiated B 12 group catalyzed Reaction 2 with little loss of its tightly bound chromophore. Aquo-B 12 , but not propyl-B 12 , markedly inhibited Reaction 2. Distinctly different pH-activity curves were observed for the two activities. The apparent K m of urea-resolved apoenzyme for methyl-B 12 as a prosthetic group (Reaction 1) was 2.0 μ m ; whereas, the K m of holoenzyme for methyl-B 12 as a substrate (Reaction 2) was >2.5 m m . At a methyl-B 12 concentration of 2.5 m m Reaction 2 was catalyzed at approximately the rate of Reaction 1.

A simple method is described whereby millimicromole amounts of extensively purified N5-methyltetrahydrofolate-homocysteine transmethylase (vitamin B12 transmethylase) can be chemically propylated to yield an inhibited vitamin B12 enzyme which is reactivated with light. The procedure consists of a short incubation of the enzyme with propyl iodide in the presence of reduced flavin mononucleotide and dithiothreitol under hydrogen gas. Formation of a propylated (inhibited) enzyme was correlated with alterations in the visible absorption spectrum of the enzyme. When propyl-1-14C bromide was the alkylating agent a radioactive enzyme was formed which lost radioactivity upon exposure to light. The effects of several reaction parameters on the extent of propylation were examined as was the effect of propylation on four separate transmethylation reactions catalyzed by the vitamin B12 enzyme preparations. Transmethylation reactions which require catalytic amounts of S-adenosylmethionine (AMe) as a cofactor or in which AMe itself is the substrate methyl donor were inhibited by propylation; however, methyl group transfer from methyl-B12 (5,6-dimethylbenzimidazolylcobamide methyl) to homocysteine was not affected by propylation. Micromolar concentrations of both AMe and N5-methyltetrahydrofolate markedly inhibited propylation as did higher levels of methyl iodide. For the inhibition of propylation by low concentrations of N5-methyltetrahydrofolate a noninhibitory amount of AMe was essential.

Further experimental observations support previous circumstantial evidence that binding activity released by osmotic shock functions in the initial step of cyanocobalamin (cyano-B12) transport. A similar specificity for cobalamins (as opposed to cobinamides) was found for the inhibition of cyano-B12[3H] uptake at 37 °, the repression of uptake, the inhibition of cyano-B12[3H] adsorption to whole cells at 0 °, and the inhibition of cyano-B12[3H]-binding to osmotic shock protein at 4 °. In confirmation of these findings, the rate of dicyanocobinamide [3H] uptake at 37 ° was only about 5% of that for cyano-B12[3H]. Osmotic shock, which was shown earlier to reduce 37 ° uptake, also decreased by 3–4-fold the amount of cyano-B12[3H] which whole cells could adsorb at 0 °. Moreover, when cyano-B12[3H] was adsorbed at 0 °, about 80% of it was lost when the washed cells were subsequently shocked. Only 7% was lost, however, if the cells were incubated several minutes at 37 ° to permit transport of the preadsorbed cyano-B12[3H]. Additionally, 0 °-adsorbed cyano-B12[3H] could be removed partially by subjecting whole cells to gel-filtration; whereas, 37 °-transported isotope could not. This and other data strongly indicate that the 0 °-adsorption to whole cells represents the attachment of cyano-B12[3H] to the binders released upon osmotic shock treatment. A cell wall location for the binders and a facilitated-diffusion uptake mechanism are also suggested by the combined data presented here and earlier.

Tritiated cyanocobalamin ([3H]cyano-B12) uptake was maximal at pH 6.0–8.0, required phosphate buffer, was proportional to the cell concentration, was inhibited reversibly by Tris+, and was markedly temperature dependent (optimal, 37–47 °). Starved cells showed a 3–4-fold dependency on glucose and their glucose-dependent uptake was decreased by certain inhibitors of energy metabolism. However, the primary role of glucose in uptake may be to maintain cell wall structure rather than to supply chemical energy for transport per se. The Km for [3H]cyano-B12 uptake was 1.0 × 10−8 m and the Vmax was 2.3 pmoles/min/mg of dry cell wt. Unchanged [3H]-cyano-B12 is the major cobalamin after 5 min of uptake. Treatment with Tris-EDTA reduced the initial uptake rate by 40–50%, but osmotic shock decreased it an additional 5–8-fold. No detectable 5-methyltetrahydrofolate-homocysteine B12 methyltransferase was released by osmotic shock. However, treatment with Tris-EDTA alone, as well as osmotic shock, did release from the cells a heat-labile, ammonium sulfate-precipitable, binding protein. By gel filtration, its molecular weight and Stokes radius were 22,000 and 15.6 A, respectively. A very large binder (mol wt >200,000) was also found in Tris-EDTA and shock fluids.The 4 ° dissociation constant for the reversible complexes between [3H]cyano-B12 and both binders was 0.6 × 10−8 m . At 31 ° it was 0.5 × 10−8 m .

Leucine transaminase was purified extensively from pig heart muscle. It appeared to be nearly homogeneous by the criteria of both starch gel and free boundary electrophoresis and ultracentrifugation. The molecular weight was found to be 75,000. Purified leucine transaminase is yellow, and our best preparations contained 1 mole of bound pyridoxal phosphate per 75,000 g of protein. The spectrum of the enzyme has absorption maxima at 414 mµ and 326 mµ in addition to the characteristic 280-mµ peak. Addition of an amino acid substrate shifted the 414-mµ peak to 326 mµ, whereupon addition of the corresponding α-keto acid partially reversed this shift. Incubation of the enzyme with an amino acid substrate plus a high concentration of phosphate yielded an inactive apoenzyme which could be reconstituted with pyridoxal phosphate. Loss of activity was correlated with a loss of absorption in the visible region. The enzyme activity was inhibited by carbonyl reagents. Reduction with sodium borohydride completely shifted the 414-mµ peak to 320 mµ and destroyed the transaminase activity. Acid hydrolysis of the reduced enzyme yielded a fluorescent compound which was tentatively identified as e-pyridoxal lysine. Leucine transaminase displayed a bell-shaped pH-activity curve with an apparent optimum at pH 8.3 to 8.5; however, it was most stable at pH 6 to 7. At pH 8.5 and in the presence of 0.1 m β-mercaptoethanol, this single enzyme catalyzed transamination between l-glutamate, l-leucine, l-valine, or l-isoleucine and their α-keto forms. Under arbitrary conditions, the relative rates of transamination were of the same order of magnitude with a maximum difference of 5-fold.

The kinetics of transamination reactions catalyzed by pig heart "branched chain amino acid" aminotransferase was investigated. The activation of aged preparations by 2-mercaptoethanol, previously noted, was associated with increases in both the maximum velocity and enzyme-substrate affinities. This implies a protein conformational change. The sharp pH optimum observed with standard assay conditions reflects a change in the rate-limiting half-reaction from that of leucine with the phosphopyridoxal form of the enzyme to that of α-ketoglutarate with the phosphopyridoxamine form as the pH is raised. Whereas nonpolar monocarboxylic acids and dicarboxylic acids which are substrate analogues both inhibited strongly at the optimum pH of 8.3, only the monocarboxylic acids were effective at higher pH values. The kinetics of the reaction of l-leucine with α-ketoglutarate could be adequately described by a binary rate equation as would be expected for a transamination reaction. Exchange transamination reactions between homologous amino acid keto acid substrate pairs were also shown and found to proceed at rates comparable to that observed with leucine and α-ketoglutarate.

A simple colorimetric assay for measuring leucine-α-ketoglutarate transamination is described. It is reproducible, sensitive, and specific for α-ketoisocaproate. α-Ketoglutarate interferes only in that high concentrations decrease the color yield. This assay is suitable for detailed kinetic studies, even with crude tissue extracts, provided one corrects for this effect of α-ketoglutarate. A separate method was also described whereby transamination between isoleucine (or valine) and α-ketoglutarate can be measured, although this assay is not specific for the leucine transaminase in crude tissue extracts. The α-ketoisocaproate assay was used to examine subcellular fractions of pig heart muscle. Most of the activity was found in the soluble fraction.

The control mechanisms of N5,10-methylene-H4-folate metabolism and de novo methyl group synthesis were studied in a mutant Escherichia coli K12, which is auxotrophic for either l -methionine or vitamin-B12. N5,10-Methylene-H4-folate reductase was found to be repressed at high media concentrations of l -methionine. Growth at high media concentrations of purines led to a partial repression of N5,10-methylene-H4-folate dehydrogenase. This enzyme was also found to be inhibited in vitro by purine ribonucleotide triphosphates, and the kinetics of the inhibition indicated that they were competitive with triphosphopyridine nucleotide. Tracer studies of the incorporation of 3-14C- l -serine into nucleic acid, protein, and phospholipid correlated with the preceding in vivo and in vitro effects.