Application of the empirical force field to macrocyclic ion carriers,siderophores, and biomimetic analogs

The empirical force field (EFF), developed by Prof. Lifson, was applied to the study of macrocyclic alkali ion carriers and to di- and tripodal and open chain siderophores and synthetic biomimetic molecules binding transition metals. The highly symmetric nature of these structures facilitated a favorable coordination geometry of the ligating groups about the metal, which helped organize the entire molecule into a fairly rigid structure. In our combined experimental-theoretical approach, EFF calculations were used to help predict likely candidates to synthesize, and provided a wealth of structural data to complement what we learned from the spectroscopic measurements, while feedback from these measurements allowed us to continue improving the EFF itself. The simple, highly modular design of the biomimetic analogs allowed rapid synthesis and systematic examination of a large number of related structures, as well as facilitating an efficient, piecewise conformational scanning for the theoretical calculations. In the early years, we focused on macrocyclic polylactones and lactams binding monovalent alkali ions, particularly the natural products enniatin and valinomycin, including inside a crystal lattice. Later we switched to bi- and tridentate siderophores, natural microbial iron carriers, and synthetic biomimetic analogs-in particular, of enterobactin, ferrichrome, and ferrioxamine B. Over the years a large number of biomimetic siderophores have been prepared, some active in a broad range of microorganisms while others are highly species specific. The results of this work have broad applications in many areas, including the design of novel drugs and antimicrobial agents, helical polymeric structures, and polynuclear metal complexes.     

Flyingfish Spawning (Parexocoetus brachypterus) in the Northeastern Gulf of Mexico

A spawning aggregation of Parexocoetus brachypterus was observed in the northeastern Gulf of Mexico over the outer continental shelf, 100km south of Mobile, Alabama. The event was considered to be a spawning aggregation given the remarkably high abundance of flyingfish observed, together with unusually vigorous flying, jumping, and swimming activity near the surface, and observations of both males and females exuding ripe sex products when landed. The spawn occurred during May 2001 just after moonrise 2 days post full moon. More than 1000000 flyingfish were estimated to have participated in the spawn. Male flyingfish were three times more abundant than females, consistent with observations of 3–4 flyingfish grouped together before and after the spawning aggregation, if several males were simultaneously pursuing a single female. Although egg attachment to flotsam is the only reproductive mode previously described for flyingfishes with demersal eggs, the P. brachypterus spawn occurred in the absence of flotsam. Other possible egg development modes include egg suspension upon current, mid-water suspension above pycnocline or upon topographically induced turbulence, or benthic.     

Coupled biogeochemical and hydrological responses of streams and rivers to drought

• 1 Severe or extreme droughts occurred about 10% of the time over a 105‐year record from central New Mexico, U.S.A., based on the Palmer Drought Severity Index.
• 2 Drought lowers water tables, creating extensive areas of groundwater recharge and fragmenting reaches of streams and rivers. Deeper groundwater inputs predominate as sources of surface flows during drought. Nutrient inputs to streams and rivers reflect the biogeochemistry of regional ground waters with longer subsurface residence times.
• 3 Inputs of bioavailable dissolved organic carbon to surface waters decrease during drought, with labile carbon limitation of microbial metabolism a byproduct of drought conditions.
• 4 Decreased inputs of organic forms of carbon, nitrogen and phosphorus and a decrease in the organic : inorganic ratio of nutrient inputs favours autotrophs over heterotrophs during drought.
• 5 The fate of autotrophic production during drought will be strongly influenced by the structure of the aquatic food web within impacted sites.

     

Expression and tyrosine phosphorylation of Cbl regulates macrophage chemokinetic and chemotactic movement

Primary macrophages isolated from hck(-/-)fgr(-/-) mice display altered morphology and F-actin cytoskeletal structures and reduced migration. The ability of phorbol myristyl acetate (PMA), a protein kinase C activator that has been reported to increase macrophage spreading and carcinoma cell motility, to rescue these hck(-/-)fgr(-/-) defects was tested. Although PMA-treated wild-type and hck(-/-)fgr(-/-) macrophages exhibited a similar flattened, spread phenotype, PMA did not rescue the hck(-/-)fgr(-/-) macrophage migration defect. Instead, both PMA-treated wild type and hck(-/-)fgr(-/-) macrophages were defective in spontaneous and chemotactic migration and tyrosine phosphorylation of the Cbl protooncoprotein was decreased in both. Moreover, c-cbl(-/-) macrophages displayed the same impairment of motility as hck(-/-)fgr(-/-) macrophages and a similar morphology with less polarization and more dorsal ruffling than wild-type macrophages. As Hck and Fgr expression and activity were not decreased in c-cbl(-/-) macrophages, these results suggest that Cbl is likely to be an important downstream mediator of the Src family kinase-regulated macrophage motility pathway.     

The mechanism underlying T cell help for induction of an antigen-specific in vivo humoral immune response to intact Streptococcus pneumoniae is dependent on the type of antigen

Little is known concerning the role of T cells in regulating an anti-polysaccharide Ig response to an intact pathogen. We previously reported that the in vivo Ig responses to Streptococcus pneumoniae (strain R36A), specific for pneumococcal surface protein A (PspA) and for the phosphorylcholine (PC) determinant of C-polysaccharide, were both dependent on TCR-alphabeta(+) T cells and B7-dependent costimulation, although only PspA-specific memory was generated. In this report, we show that the T cell help underlying these two Ag-specific Ig responses is distinct. Using H-Y-specific T cell transgenic mice made "nonleaky" by crossing with mice genetically deficient for TCR-alpha, we demonstrate that the T cell help for the anti-PC, in contrast to the anti-PspA, response is TCR-nonspecific and occurs normally in the absence of germinal center formation, although it is still dependent on B7-dependent costimulation. Consistent with these data, we demonstrate, using cathepsin S(-/-) mice, that although the anti-PC response is largely dependent on CD4(+) T cells, there is a reduced (or lack of) dependence, relative to the anti-PspA response, on the generation of new peptide-MHC class II complexes. In this regard, the T cell help for an optimal anti-PC response is delivered more rapidly than that required for an optimal anti-PspA response. Collectively, these data demonstrate a novel accelerated TCR-nonspecific B7-dependent form of T cell help for augmenting a polysaccharide-specific Ig response to an intact bacterium without the generation of memory.     

Evaluation of the antigenotoxic potential of monomeric and dimeric flavanols, and black tea polyphenols against heterocyclic amine-induced DNA damage in human lymphocytes using the Comet assay

The polyphenolic dimers, epicatechin-4beta-8-catechin (B1), epicatechin-4beta-8-epicatechin (B2), catechin-4beta-8-catechin (B3), catechin-4beta-8-epicatechin (B4), and the gallate ester epicatechin-4beta-8-epicatechin gallate (B'2G) were isolated from grape seeds, and theaflavins and theafulvins from black tea brews. The ability of these naturally-occurring polyphenols to afford protection against the genotoxicity of the heterocyclic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was compared with that of the monomeric tea flavanols, (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG). Genotoxic activity was evaluated in human peripheral lymphocytes using the Comet assay. At the concentration range of 1-100 microM, neither the monomeric nor the dimeric flavanols prevented the lymphocyte DNA damage induced by Trp-P-2. In contrast, both of the black tea polyphenols, theafulvins and theaflavins, at a dose range of 0.1-0.5 mg/ml, prevented, in a concentration-dependent manner, the DNA damage elicited by Trp-P-2. Finally, neither the monomeric and dimeric polyphenols (100 microM) nor the theafulvins and theaflavins (0.5mg/ml) caused any DNA damage in the human lymphocytes. These studies illustrate that black tea theafulvins and theaflavins, if absorbed intact, may contribute to the anticarcinogenic potential associated with black tea intake.     

Spatial constraint of peatland butterfly occurrences within a heterogeneous landscape

The occurrence of ten butterfly taxa (Clossiana eunomia dawsonii, Clossiana freija, Clossiana frigga, Clossiana titania, Coenonympha inornata, Erebia discoidalis, Incisalia augustinus, Lycaena dorcas, Lycaena epixanthe, Oeneis jutta) was analyzed within three acid peatland habitat types from the Lake Superior drainage basin of northwestern Wisconsin. Both first- (nearest-neighbor spatial analysis) and second-order (Ripley's K) spatial point process statistics were used to identify the extents over which each distribution pattern significantly deviated from random expectations. Versions of these tests were used that identified significant spatial pattern uncorrelated to habitat location and habitat preference. These analyses documented non-random occurrence patterns in seven species. Deviations from random were largely confined to two extents: <50 km and 70–100+ km. The majority of non-random patterns at <50 km extents were examples of aggregation, while the majority of non-random patterns noted at the 70–100+ km scale were examples of segregation. These results demonstrate that even for winged animals inside a limited landscape, spatially constrained processes can be important determinants of distribution. It is likely that metapopulation dynamics and dispersal limitation help explain why aggregation is dominant at small scales. The mechanisms underlying the predominance of segregation at large scales are less clear, but may be related to migration history and/or weak environmental gradients.     

Effectiveness of an antagonist to gonadotrophin releasing hormone on the FSH and LH response to GnRH in perifused equine pituitary cells,and in seasonally acyclic mares

We wish to use a gonadotrophin-releasing hormone (GnRH) antagonist in the mare as a tool for investigating the control of the oestrous cycle. The aim of this study was to test the effectiveness of the antagonist cetrorelix by testing both in vitro, using perifused equine anterior pituitary cells, and in vivo in seasonally acyclic mares. Pituitary cells were prepared and after 3-4 days incubation, loaded onto columns and given four pulses of GnRH (at 0, 30, 60 and 90 min; dose-response study). After the second GnRH pulse, infusion of cetrorelix began (0, 100, 1000 and 2000 pmol/l) and continued until the end of the experiment. To mimic luteal phase conditions, cells were pre-incubated and perifused with progesterone (25 nmol/l) and GnRH pulses given at 0, 90, 180 and 270 min. Cetrorelix (0 or 1000 pmol/l) began after the second GnRH pulse. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) concentrations were measured in 5 min fractions. Both FSH and LH response areas (above baseline) after GnRH were inhibited by 1000 pmol/l cetrorelix (P < 0.01, P < 0.01, respectively) but not by 100 pmol/l cetrorelix. Similarly, in the presence of progesterone, cetrorelix inhibited the FSH (P < 0.001) and LH (P = 0.0002) response area. Seasonally acyclic mares, pre-treated for 3 days with progesterone (150 mg i.m. per day) were given cetrorelix as (i) a loading dose of 1 microg/kg then infusion at 2.2 ng/(kg min) for 90 min, (ii) a s.c. injection at 20 microg/kg, (iii) infusion at 2.2 ng/(kg min) for 48 h, and (iv) no cetrorelix (control mares). At 90 min, 6, 24 and 48 h after cetrorelix was first administered, mares were given a bolus injection of GnRH (22.2 ng/kg i.v.) and the FSH and LH responses measured. All doses of cetrorelix inhibited the FSH response at 90 min. The response was no longer suppressed at 6 h in the 90 min infusion group, showing a rapid recovery from inhibition. At 24 h, the FSH responses in the injected and 48 h infusion group were suppressed. The LH concentrations were low and showed no significant changes. This study has defined the time course and dose of cetrorelix with respect to its effect on FSH in the horse. It is concluded that cetrorelix could be used to elucidate the role of FSH in follicular development in cyclic mares.