Isoprene emissions in Africa inferred from OMI observations of formaldehyde columns.

We use 2005-2009 satellite observations of formaldehyde (HCHO) columns from the OMI instrument to infer biogenic isoprene emissions at monthly 1 × 1° resolution over the African continent. Our work includes new approaches to remove biomass burning influences using OMI absorbing aerosol optical depth data (to account for transport of fire plumes) and anthropogenic influences using AATSR satellite data for persistent small-flame fires (gas flaring). The resulting biogenic HCHO columns (ΩHCHO) from OMI follow closely the distribution of vegetation patterns in Africa. We infer isoprene emission (E ISOP) from the local sensitivity S = ΔΩHCHO / ΔE ISOP derived with the GEOS-Chem chemical transport model using two alternate isoprene oxidation mechanisms, and verify the validity of this approach using AMMA aircraft observations over West Africa and a longitudinal transect across central Africa. Displacement error (smearing) is diagnosed by anomalously high values of S and the corresponding data are removed. We find significant sensitivity of S to NOx under low-NOx conditions that we fit to a linear function of tropospheric column NO2. We estimate a 40% error in our inferred isoprene emissions under high-NOx conditions and 40-90% under low-NOx conditions. Our results suggest that isoprene emission from the central African rainforest is much lower than estimated by the state-of-the-science MEGAN inventory.

Nutrition and wound healing.

Continued research and development in the field of wound healing holds the potential to affect both quality of life and incidence of mortality. For the health care provider to promote successful wound healing, an understanding of the function of nutrients in inflammation and tissue growth is helpful. The intent of this paper is to discuss the metabolic and cellular pathways crucial to wound healing and identify appropriate nutritional interventions and clinical applications.

Molecular mechanism of sequence-specific termination of lentiviral replication.

The central termination sequence (CTS) terminates (+) strand DNA synthesis in certain lentiviruses. The molecular mechanism underlying this event, catalyzed by equine infectious anemia virus reverse transcriptase (EIAV RT), was evaluated by pre-steady-state kinetic techniques. Time courses in nucleotide incorporation using several DNA substrates were biphasic, consistent with release of enzyme from extended DNA being the rate-limiting step for turnover. While the burst amplitude reflecting the amount of functional RT-DNA complex was sequence-dependent, rate constants for initial product formation were not. Filter binding assays indicate the K(d) for CTS-containing substrate is only 2-fold higher than a random DNA and cannot account entirely for the large diminution in burst amplitudes. Measurements of processive DNA replication on a millisecond time scale indicate that the rate of polymerization is unaffected by the T(6)-tract within the CTS. However, termination products accumulate due to a substantial increase in the rate of nonproductive enzyme-nucleic acid complex formation after incorporation of four to five adenosines of a T(6)-tract within the CTS. During strand displacement synthesis through the CTS, products accumulate after incorporation of three to four adenosines. The rate of polymerization during strand displacement synthesis decreases 2-fold while the rate of nonproductive enzyme-nucleic acid complex formation is identical in the absence or presence of the displacement strand. These results have allowed us to develop a model for CTS-induced termination of (+) strand synthesis.

Genetic variation in physiological sensitivity to estrogen in mice.

Genetic variation in susceptibility to endocrine disruption by estrogenic agents was examined in juvenile male mice. Mice were implanted with increasing doses of estradiol (E2) at 3 weeks of age and reproductive responses were determined 3 weeks later. Greater than 16-fold differences in susceptibility to the disruption of reproductive development by E2 were detected between strains of mice. CD-1 was much more resistant to the inhibition of testes weight, vesicular gland weight and spermatogenesis by increasing doses of E2. Spermatid maturation was eliminated by low doses of E2 in unselected strains such as C17/Jls and C57BL/6J. In contrast, widely used, large litter size selected CD-1 mice showed little or no inhibition in spermatogenesis even in response to 16-fold higher doses of E2. Testicular sulfotransferase activity (EST) per gram body weight was 3.5-fold higher in untreated CD-1 than in B6 strain males. This suggests that genetic differences in testicular EST activity may play a critical role in the detoxification of estrogens. These and other findings emphasize the need to identify and study genetic variation in sensitivity to estrogen in laboratory animal models used to assess the risk of xenobiotic estrogen exposure.

Reassessment of models used to test xenobiotics for oestrogenic potency is overdue.

Product safety bioassays need to include data from animals with susceptible genotypes or the potential for environmental compounds to disrupt reproductive development in hormonally sensitive populations may be greatly underestimated. The continued use of resistant animal models is likely to result in allowable releases of toxic levels of oestrogenic agents that could differentially disrupt reproductive development and function of sensitive genotypes, leading to reproductive failure and loss or extinction of susceptible individuals, populations and species. Rather than ignoring the role of genetic differences in susceptibility to oestrogenic agent-induced carcinogenicity and endocrine disruption, government agencies should support efforts to identify the genetic mechanisms involved in these responses, and to screen for and develop strains of mice and rats which are sensitive to the induction of genotoxicity/carcinogenicity as well as the inhibition of reproductive development and function by oestrogenic agents. Such sensitive strains would be even more optimal for testing chemicals for endocrine disruptor activity.

Solution conformation of EcoRI restriction endonuclease changes upon binding of cognate DNA and Mg2+ cofactor.

EcoRI endonuclease has two tryptophans at positions 104 and 246 on the protein surface. A single tryptophan mutant containing Trp246 and a single cysteine labeling site at the N-terminus was used to determine the position of the N-terminus in the protein structure. The N-termini of EcoRI endonuclease are essential for tight binding and catalysis yet are not resolved in any of the crystal structures. Resonance energy transfer was used to measure the distance from Trp246 donor to IAEDANS or MIANS acceptors at Cys3. The distance is 36 A in apoenzyme, decreasing to 26 A in the DNA complex. Molecular modeling suggests that the N-termini are located at the dimer interface formed by the loops comprising residues 221-232. Protein conformational changes upon binding of cognate DNA and cofactor Mg(2+) were monitored by tryptophan fluorescence of the single tryptophan mutant and wild-type endonuclease. The fluorescence decay of Trp246 is a triple exponential with lifetimes of 7, 3.5, and 0.7 ns. The decay-associated spectra of the 7- and 3.5-ns components have emission maxima at approximately 345 and approximately 338 nm in apoenzyme, which shift to approximately 340 and approximately 348 nm in the DNA complex. The fluorescence quantum yield of the single tryptophan mutant drops 30% in the DNA complex, as compared to 10% for wild-type endonuclease. Fluorescence changes of Trp104 upon binding of DNA were inferred by comparison of the decay-associated spectra of wild type and single tryptophan mutant. Fluorescence changes are related to changes in proximity and orientation of quenching functional groups in the tryptophan microenvironments, as seen in the crystal structures.

Genetic control of hormone-induced ovulation rate in mice.

The nature of genetic differences in ovarian responsiveness to gonadotropins was examined in mouse strains and subspecies. Hormone-induced ovulation rate (HIOR) differed 5-fold between Mus musculus strains A/J (10.3 +/- 1.6 eggs in cumulus) and C57BL/6J (B6) (47.3 +/- 2.5 eggs in cumulus), and 6-fold among Mus spretus lines and crosses. Subspecies differed up to 10-fold in HIOR (Mus spretus/Ros: 4.8 +/- 1.0 eggs in cumulus versus B6). An additional experiment examined the genetics of HIOR in crosses. The number of eggs ovulated in response to equine chorionic gonadotropin (CG)/human CG averaged 8.4 +/- 0.9 in A/J, 40.7 +/- 1.7 in B6, 33.9 +/- 1.6 in B6AF1, and 20.2 +/- 0.3 in (B6xA)xA backcrosses. The 5-fold genetic differences in hormone-induced ovulation rate between Mus musculus strains A/J and B6 segregated in backcrosses as though they were controlled by the action of approximately 3 loci with major effects. This study demonstrates genetic variation in HIOR both within and between mouse subspecies, and provides confirmation that genetic differences are a major source of variation in the regulation of ovarian responsiveness to gonadotropins.

Mapping genes that control hormone-induced ovulation rate in mice.

The present study mapped quantitative trait loci (QTL) that control 6-fold genetic differences in hormone-induced ovulation rate (HIOR) between C57BL/6J (B6) (HIOR = 54) and A/J strain mice (HIOR = 9). (The gene name is Ovulation Rate Induced [ORI] QTL and the gene symbol is Oriq.) QTL linkage analysis was conducted on 167 (B6xA)xA backcross mice at 165 loci. Suggestive B6 ORI QTL that control the number of eggs in cumulus mapped, as follows, near: Cyp19 and D9Mit4 on chromosome (Chr) 9 (Oriq1); D2Mit433 on Chr2 (Oriq2); D6Mit316 on Chr6 (Oriq3); DXMit22 on ChrX (Oriq4) and were associated with a 2.7, 2.7, 2.6, and 4.2 egg increases in HIOR, respectively. Oriq3 was significant (LOD = 3.45) based on composite interval mapping. QTL linkage analysis of the number of eggs matured by endogenous gonadotropins and ovulated by eCG mapped a significant Oriq5 to Chr 10 and suggestive Oriq to Chr 6, 7, and X. These data provide the first molecular genetic markers for reproductive QTL that control major differences in ovarian responsiveness to gonadotropins. These and closely linked syntenic molecular markers will enable a more accurate prediction of ovarian responsiveness to gonadotropins and provide selection criteria for improving reproductive performance in diverse mammalian species.

Genetic variation in susceptibility to endocrine disruption by estrogen in mice.

Large (more than 16-fold) differences in susceptibility to disruption of juvenile male reproductive development by 17beta-estradiol (E2) were detected between strains of mice. Effects of strain, E2 dose, and the interaction of strain and E2 dose on testes weight and spermatogenesis were all highly significant (P < 0.0001). Spermatid maturation was eliminated by low doses of E2 in strains such as C57BL/6J and C17/Jls. In contrast, mice of the widely used CD-1 line, which has been selected for large litter size, showed little or no inhibition of spermatid maturation even in response to 16 times as much E2. Product safety bioassays conducted with animals selected for fecundity may greatly underestimate disruption of male reproductive development by estradiol and environmental estrogenic compounds.

N-termini of EcoRI restriction endonuclease dimer are in close proximity on the protein surface.

The N-terminal region of EcoRI endonuclease is essential for cleavage yet is invisible in the 2.5 A crystal structure of endonuclease-DNA complex [Kim, Y., Grable, J. C., Love, R., Greene, P. J., Rosenberg, J. M. (1990) Science 249, 1307-1309]. We used site-directed fluorescence spectroscopy and chemical cross-linking to locate the N-terminal region and assess its flexibility in the absence and presence of DNA substrate. The second amino acid in each subunit of the homodimer was replaced with cysteine and labeled with pyrene or reacted with bifunctional cross-linkers. The broad absorption spectra and characteristic excimer emission bands of pyrene-labeled muteins indicated stacking of the two pyrene rings in the homodimer. Proximity of N-terminal cysteines was confirmed by disulfide bond formation and chemical cross-linking. The dynamics of the N-terminal region were determined from time-resolved emission anisotropy measurements. The anisotropy decay had two components: a fast component with rotational correlation time of 0.3-3 ns representing probe internal motions and a slow component with 50-100 ns correlation time representing overall tumbling of the protein conjugate. We conclude that the N-termini are close together at the dimer interface with limited flexibility. Binding of Mg2+ cofactor or DNA substrate did not affect the location or flexibility of the N-terminal region as sensed by pyrene fluorescence and cross-linking, indicating that substrate binding is not accompanied by folding or unfolding of the N-terminus.

Toward understanding tryptophan fluorescence in proteins.

A general approach to dissecting the complex photophysics of tryptophan is presented and used to elucidate the effects of amino acid functional groups on tryptophan fluorescence. We have definitively identified the amino acid side chains that quench tryptophan fluorescence and delineated the respective quenching mechanisms in a simple model system. Steady-state and time-resolved fluorescence techniques, photochemical H-D exchange experiments, and transient absorption techniques were used to measure individual contributions to the total nonradiative rate for deactivation of the excited state, including intersystem crossing, solvent quenching, and excited-state proton and electron transfer rates. Eight amino acid side chains representing six functional groups quench 3-methylindole fluorescence with a 100-fold range in quenching rate constant. Lysine and tyrosine side chains quench by excited-state proton transfer; glutamine, asparagine, glutamic and aspartic acid, cysteine, and histidine side chains quench by excited-state electron transfer. These studies provide a framework for deriving detailed structural and dynamical information from tryptophan fluorescence intensity and lifetime data in peptides and proteins.

Self-assembly of designed antimicrobial peptides in solution and micelles.

Hydrophobic interactions are responsible for stabilizing leucine zippers in peptides containing heptad repeats. The effects of substituting leucine by phenylalanine and alanine by glycine on the self-assembly of coiled-coils were examined in minimalist antimicrobial peptides designed to form amphipathic alpha-helices. The secondary structure of these peptides was monitored in solution and in diphosphocholine (DPC) micelles using circular dichroism spectroscopy. The leucine peptides (KLAKLAK)3 and (KLAKKLA)n (n = 3, 4) become alpha-helical with increasing concentrations of salt, peptide, and DPC. The aggregation state and equilibrium constant for self-association of the peptides were measured by sedimentation equilibrium. The glycine peptide (KLGKKLG)3 does not self-associate. The leucine peptides and phenylalanine peptides (KFAKFAK)3 and (KFAKKFA)n (n = 3, 4) are in a monomer-tetramer equilibrium in solution, with the phenylalanine zippers being 2-4 kcal/mol less stable than the equivalent leucine zippers. Thermodynamic parameters for the association reaction were calculated from the temperature dependence of the association constants. Leucine zipper formation has DeltaCp = 0, whereas phenylalanine zipper formation has a small negative DeltaCp, presumably due to the removal of the larger surface area of phenylalanine from water. Self-association of the peptides is coupled to formation of a hydrophobic core as detected using 1-anilino-naphthalene-8-sulfonate fluorescence. Carboxyfluorescein-labeled peptides were used to determine the aggregation state of (KLAKKLA)3 and (KLGKKLG)3 in DPC micelles. (KLAKKLA)3 forms dimers, and (KLGKKLG)3 is a monomer. Aggregation appears to correlate with the cytotoxicity of these peptides.

Readiness: observations and comments from a medical team deployment.

The evolving strategy of the United States in dealing with the changing world order calls for a force structure capable of fighting and winning two nearly simultaneous major regional conflicts and conducting a range of other military operations. Readiness is a key factor in this new strategy. Consequently, major paradigm shifts are occurring within the Air Force Medical Service. Maintaining current and accurate medical records on personnel to meet deployment requirements is a significant challenge. Historically, time and resources are consumed determining the deployability of troops prior to a deployment. This adds to the cost of doing business and increases the time required to clear the deploying team, even though there is an established process to avoid these very problems. The experience of a recent medical team deployment to Bosnia is discussed. Future directions given the implementation of TRI-CARE, the Preventive Health Assessment Program, and the Strategic Health Resourcing Plan are also considered.

Time-resolved fluorescence studies of tomaymycin bonding to synthetic DNAs.

Tomaymycin reacts covalently with guanine in the DNA minor groove, exhibiting considerable specificity for the flanking bases. The sequence dependence of tomaymycin bonding to DNA was investigated in synthetic DNA oligomers and polymers. The maximum extent of bonding to DNA is greater for homopurine and natural DNA sequences than for alternating purine-pyrimidine sequences. Saturation of DNA with tomaymycin has little effect on the melting temperature in the absence of unbound drug. Fluorescence lifetimes were measured for DNA adducts at seven of the ten unique trinucleotide bonding sites. Most of the adducts had two fluorescence lifetimes, representing two of the four possible binding modes. The lifetimes cluster around 2-3 ns and 5-7 ns; the longer lifetime is the major component for most bonding sites. The two lifetime classes were assigned to R and S diastereomeric adducts by comparison with previous NMR results for oligomer adducts. The lifetime difference between binding modes is interpreted in terms of an anomeric effect on the excited-state proton transfer reaction that quenches tomaymycin fluorescence. Bonding kinetics of polymer adducts were monitored by fluorescence lifetime measurements. Rates of adduct formation vary by two orders of magnitude with poly(dA-dG).poly(dC-dT), reacting the fastest at 4 x 10(-2) M-1 s-1. The sequence specificity of tomaymycin is discussed in light of these findings and other reports in the literature.

Genetic variation in plasma androgens and ovarian aromatase activity during mouse pregnancy.

Genetic variation in fetal survival, maternal plasma androgen levels, and ovarian aromatase activity was examined mid (Day 9) and late gestation (Day 16) in strains of mice that differ in reproductive performance (A/J, C57BL/6J, C8/JIs, C17/JIs, and S15/JIs). At both gestational stages, females selected for large litter size (S15/JIs) carried more fetuses than any of the other strains examined. Particularly at midpregnancy, S15/JIs females also maintained higher plasma levels of androstenedione and testosterone relative to both control strains, C8/JIs and C17/JIs. Consistent with previously reported changes in peripheral estrogen levels during mouse pregnancy, aromatase activity was higher on Day 16 than on Day 9. This study demonstrates genetic variation in fetal survival that is correlated with increased maternal androgen levels. A stage-specific gestational increase in aromatase activity occurs in several strains of mice and is associated with elevated plasma estrogen during the second half of pregnancy.

Time-resolved fluorescence studies of tomaymycin bonding to DNA.

Tomaymycin is an antibiotic that reacts at guanine N2 in the minor groove of the DNA helix. The number and type of tomaymycin-DNA adducts present on natural sequence DNA were identified using time-resolved fluorescence spectroscopy. At low bonding density, only two discrete species were observed with lifetimes of 4.3 and 7.1 ns and relative amplitudes of 40% and 60%. These two lifetime species are proposed to represent either R5' or S5' and S3' binding modes at the preferred bonding sequence 5'-AGA. R and S denote the configuration at C11 of tomaymycin, and 5' and 3' describe the orientation of the aromatic ring on the covalently modified strand. These two species were present over a range of solution conditions, including pH, nucleotide to drug ratio, DNA concentration, and DNA size. They have the same emission spectra, but slightly shifted absorption spectra. The weak temperature dependence of the fluorescence lifetimes presumably is due to the excited-state proton-transfer reaction that quenches tomaymycin fluorescence. The rate of formation of the longer lifetime species of DNA adduct is about twice as fast as that of the shorter lifetime species. Under saturating conditions, the fluorescence decay shows a bimodal lifetime distribution whether analyzed by least-squares assuming a Gaussian distribution model or by the maximum entropy method. The two groups of lifetimes are centered around 2-3 and 6-6.6 ns, reflecting multiple species on different bonding sequences.