The design of potent hydrazones and disulfides as cathepsin S inhibitors

The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis. The nature and mechanism of these adducts was explored. Kinetic analysis of the benzoyl hydrazones indicate that these inhibitors are acting as irreversible inhibitors of Cathepsin S. Additionally, the benzoylhydrazones were shown to be potent inhibitors of Cathepsin S processing of Class II associated invariant peptide both in vitro and in vivo.     

Doxorubicin immunoconjugates containing bivalent,lysosomally-cleavable dipeptide linkages

Bivalent doxorubicin (DOX)-dipeptides (16a-c) were prepared and conjugated to the monoclonal antibody BR96. The dipeptides are cleaved by lysosomal proteases following internalization of the resulting immunoconjugates. Conjugate 18b demonstrated antigen-specific in vitro tumor cell killing activity (IC(50)=0.2 microM) that was equipotent to DOX with a near doubling of drug molecules/MAb. Size exclusion chromatography showed 18b to be a noncovalent dimer that was formed immediately upon conjugation.     

Quantifying the effects of switchgrass (Panicum virgatum) on deep organic C stocks using natural abundance 14C in three marginal soils

Perennial bioenergy crops have been shown to increase soil organic carbon (SOC) stocks, potentially offsetting anthropogenic C emissions. The effects of perennial bioenergy crops on SOC are typically assessed at shallow depths (<30 cm), but the deep root systems of these crops may also have substantial effects on SOC stocks at greater depths. We hypothesized that deep (>30 cm) SOC stocks would be greater under bioenergy crops relative to stocks under shallow‐rooted conventional crop cover. To test this, we sampled soils to between 1‐ and 3‐m depth at three sites in Oklahoma with 10‐ to 20‐year‐old switchgrass (Panicum virgatum) stands, and collected paired samples from nearby fields cultivated with shallow rooted annual crops. We measured root biomass, total organic C, ¹⁴C, ¹³C, and other soil properties in three replicate soil cores in each field and used a mixing model to estimate the proportion of recently fixed C under switchgrass based on ¹⁴C. The subsoil C stock under switchgrass (defined over 500–1500 kg/m² equivalent soil mass, approximately 30–100 cm depth) exceeded the subsoil stock in neighboring fields by 1.5 kg C/m² at a sandy loam site, 0.6 kg C/m² at a site with loam soils, and showed no significant difference at a third site with clay soils. Using the mixing model, we estimated that additional SOC introduced after switchgrass cultivation comprised 31% of the subsoil C stock at the sandy loam site, 22% at the loam site, and 0% at the clay site. These results suggest that switchgrass can contribute significantly to subsoil organic C—but also indicated that this effect varies across sites. Our analysis shows that agricultural strategies that emphasize deep‐rooted grass cultivars can increase soil C relative to conventional crops while expanding energy biomass production on marginal lands.     

Plant invasion alters nitrogen cycling by modifying the soil nitrifying community

Plant invasions have dramatic aboveground effects on plant community composition, but their belowground effects remain largely uncharacterized. Soil microorganisms directly interact with plants and mediate many nutrient transformations in soil. We hypothesized that belowground changes to the soil microbial community provide a mechanistic link between exotic plant invasion and changes to ecosystem nutrient cycling. To examine this possible link, monocultures and mixtures of exotic and native species were maintained for 4 years in a California grassland. Gross rates of nitrogen (N) mineralization and nitrification were quantified with ¹⁵N pool dilution and soil microbial communities were characterized with DNA‐based methods. Exotic grasses doubled gross nitrification rates, in part by increasing the abundance and changing the composition of ammonia‐oxidizing bacteria in soil. These changes may translate into altered ecosystem N budgets after invasion. Altered soil microbial communities and their resulting effects on ecosystem processes may be an invisible legacy of exotic plant invasions.     

Mechanisms for Soil Moisture Effects on Activity of Nitrifying Bacteria

Moisture may limit microbial activity in a wide range of environments including salt water, food, wood, biofilms, and soils. Low water availability can inhibit microbial activity by lowering intracellular water potential and thus reducing hydration and activity of enzymes. In solid matrices, low water content may also reduce microbial activity by restricting substrate supply. As pores within solid matrices drain and water films coating surfaces become thinner, diffusion path lengths become more tortuous, and the rate of substrate diffusion to microbial cells declines. We used two independent techniques to evaluate the relative importance of cytoplasmic dehydration versus diffusional limitations in controlling rates of nitrification in soil. Nitrification rates in shaken soil slurries, in which NH(inf4)(sup+) was maintained at high concentrations and osmotic potential was controlled by the addition of K(inf2)SO(inf4), were compared with rates in moist soil incubations, in which substrate supply was controlled by the addition of NH(inf3) gas. Comparison of results from these techniques demonstrated that diffusional limitation of substrate supply and adverse physiologic effects associated with cell dehydration can explain all of the decline in activity of nitrifying bacteria at low soil water content. However, the relative importance of substrate limitation and dehydration changes at different water potentials. For the soil-microbial system we worked with, substrate limitation was the major inhibiting factor when soil water potentials were greater than -0.6 MPa, whereas adverse physiological effects associated with cell dehydration were more inhibiting at water potentials of less than -0.6 MPa.     

Water stress effects on toluene biodegradation by Pseudomonas putida

We quantified the effects of matric and solute waterpotential on toluene biodegradation by Pseudomonasputida mt-2, a bacterial strain originally isolated fromsoil. Across the matric potential range of 0 to – 1.5 MPa,growth rates were maximal for P. putida at – 0.25MPa and further reductions in the matric potentialresulted in concomitant reductions in growth rates.Growth rates were constant over the solute potential range0 to – 1.0 MPa and lower at – 1.5 MPa. First ordertoluene depletion rate coefficients were highest at0.0 MPa as compared to other matric water potentialsdown to – 1.5 MPa. Solute potentials down to – 1.5 MPadid not affect first order toluene depletion ratecoefficients. Total yield (protein) and carbon utilizationefficiency were not affected by water potential, indicatingthat water potentials common to temperate soils were notsufficiently stressful to change cellular energyrequirements. We conclude that for P. putida: (1)slightly negative matric potentials facilitate faster growthrates on toluene but more negative water potentials resultin slower growth, (2) toluene utilization rate per cell massis highest without matric water stress and is unaffected bysolute potential, (3) growth efficiency did not differ acrossthe range of matric water potentials 0.0 to – 1.5 MPa.     

Cell killing by lysosomotropic detergents

We have studied the mechanism by which lysosomotropic detergents kill baby hamster kidney cells. Lysosomotropic detergents are lysosomotropic amines (compounds with pK between 5 and 9, such as imidazole or morpholine) containing straight-chain hydrocarbon "tails" of 9-14 carbon atoms (Firestone, R. A., J. M. Pisano, and R. J. Bonney. 1979, J. Med. Chem., 22:1130-1133). Using lucifer yellow CH as a specific fluorescent label for lysosomes, it was shown by light microscopy that N-dodecyl (C12)-imidazole acted rapidly to damage lysosomes, causing leakage of dye into the cytoplasm. This was followed at later times by vacuolization, blebbing of the plasma membrane, cell rounding, and cell death. 3H-labeled C12-imidazole rapidly diffused into cells where much of it was trapped in lysosomes as shown by its co-migration with lysosomes in Percoll gradients. Cells preincubated with C12-imidazole released it slowly into C12-imidazole-free media, permitting the cells to be killed by the preincubation dose. Cell killing by the lysosomotropic detergents exhibited strongly sigmoidal dose-response curves. The sensitivity of baby hamster kidney cells to killing by C12-imidazole was density dependent, the cells being most sensitive at lowest cell densities, and relatively resistant at confluence. The amount of 3H-C12-imidazole taken up by the cells was also density dependent, with highest specific uptake occurring at the lowest cell density. A rise in lysosomal pH, measured in fluoresceinated dextran-labeled cells, commenced immediately upon addition of C12-imidazole to cells, and continued for over an hour. This was followed after a lag of 1-2 h by inhibition of protein and RNA synthesis and by lactate dehydrogenase release. Ionophores or lysosomotropic amines, such as methylamine, that raise intralysosomal pH provided substantial protection of the cells from killing by lysosomotropic detergents. These findings provide strong support for the idea that lysosomotropic detergents kill cells by disrupting lysosomes from within.     

Respiratory syncytial virus (RSV) F, G, M2 (22K), and N proteins each induce resistance to RSV challenge, but resistance induced by M2 and N proteins is relatively short-lived.

The ability of recombinant vaccinia viruses that separately encoded 9 of the 10 known respiratory syncytial virus (RSV) proteins to induce resistance to RSV challenge was studied in BALB/c mice. Resistance was examined at two intervals following vaccination to examine early (day 9) as well as late (day 28) immunity. BALB/c mice were inoculated simultaneously by the intranasal and intraperitoneal routes with a recombinant vaccinia virus encoding one of the following RSV proteins: F, G, N, P, SH, M, 1B, 1C, or M2 (22K). A parainfluenza virus type 3 HN protein recombinant (Vac-HN) served as a negative control. One half of the mice were challenged with RSV intranasally on day 9, and the remaining animals were challenged on day 28 postvaccination. Mice previously immunized by infection with RSV, Vac-F, or Vac-G were completely or almost completely resistant to RSV challenge on both days. In contrast, immunization with Vac-HN, -P, -SH, -M, -1B, or -1C did not induce detectable resistance to RSV challenge. Mice previously infected with Vac-M2 or Vac-N exhibited significant but not complete resistance on day 9. However, in both cases resistance had largely waned by day 28 and was detectable only in mice immunized with Vac-M2. These results demonstrate that F and G proteins expressed by recombinant vaccinia viruses are the most effective RSV protective antigens. This study also suggests that RSV vaccines need only contain the F and G glycoproteins, because the immunity conferred by the other proteins is less effective and appears to wane rapidly with time.     

Partial characterization of a glucocorticoid suppressible mitogenic activity secreted from a rat hepatoma cell line hypersensitive to the antiproliferative effects of glucocorticoids

We have previously shown that glucocorticoids suppress the proliferation of Fu5 hepatoma cells and have selected subclones which are either hypersensitive (BDS1) or resistant (EDR3) to the antiproliferative effects of dexamethasone, a synthetic glucocorticoid. BDS1 cells externalize a glucocorticoid suppressible mitogenic activity (denoted GSM) which stimulated [3H]thymidine incorporation in quiescent, serum-starved Balb/c 3T3 cells. Glucocorticoid treatment of BDS1 cells reduced the secreted levels of GSM activity by approximately 20-fold in comparison to untreated cells. The GSM activity was constitutively secreted from a glucocorticoid receptor minus variant (EDR3) demonstrating that the suppression of this mitogenic activity is a new glucocorticoid hormone response which required a functional receptor. GSM activity was sensitive to sulfhydryl reducing agents or trypsin, stable to heat and acid treatments and fractionated in gel filtration columns with a native molecular weight of approximately Mr 30,000. The persistence of this size for mitogenic activity after electrophoretic fractionation in nonreducing sodium dodecyl sulfate-poly-acrylamide gels suggested that the GSM activity is comprised of a single protein. Total secreted protein isolated from untreated BDS1, but not dexamethasone-treated BDS1, stimulated 3T3 cells to grow in transformed-appearing large colonies in soft agar and to display multiple layering and elongated spindle-like morphology on solid substratum. The addition of both insulin and EGF to conditioned medium protein isolated from glucocorticoid-treated BDS1 cells restored full induction of 3T3 cell anchorage-independent growth while insulin restored full and EGF partial mitogenic stimulation of these fibroblasts. These results suggest that the GSM activity acts in a pathway common to that of insulin or EGF in fibroblasts.