Synthesis and enzymatic activation of N-[N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithiny]-L-phenylalanine, a candidate for antibody-directed enzyme prodrug therapy (ADEPT)

N-[N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithinyl]-L-phenylalanine (1), a carboxypeptidase A (CPA) cleavable prodrug was synthesized for use in an antibody directed strategy to improve the therapeutic selectivity of N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (2), an extremely potent nonpoly-glutamatable DHFR inhibitor which is also highly cytotoxic. Compound 1 was shown by HPLC analysis to give a >99% yield of 2 upon incubation with bovine CPA (bCPA) for 20 min at 25 degrees C. In a spectrophotometric kinetic assay with 50 microM dihydrofolate as the competing substrate in the presence of 65 microM NADPH, 1+bCPA stoichiometrically inhibited recombinant human DHFR (rhDHFR) with a K(i) of 0.35 pM. In contrast, 1 without bCPA was a poor inhibitor of rhDHFR (K(i)>10 microM). In a 72 h growth inhibition assay against cultured CCRF-CEM human leukemic lymphoblasts, the growth inhibitory activities of 1+bCPA, 2+bCPA, and 2 alone were the same (IC(50) 1.3-1.4 nM), whereas 1 in the absence of bCPA was >100-fold less potent (IC(50) 155 nM).     

Involvement of subcomplexes of 32 and 14 kDa subunits in RPA's DNA binding activity through redox change

The eukaryotic replication protein A (RPA) is a heterotrimeric protein complex. It consists of 70, 32, and 14 kDa subunits that are involved in DNA replication, repair, and genetic recombination. RPA is a 4-cysteine type zinc-finger protein. RPA's zinc-finger domain is not essential for DNA binding activity, but it is involved in the regulation of RPA's DNA binding activity through reduction-oxidation (redox). In this study, we show that yeast RPA's ssDNA binding activity is regulated by redox potential through its subcomplexes of 32 and 14 kDa subunits. In contrast, the subunits' complex, RPA70, formed a stable complex with ssDNA, even under non-reducing conditions. The addition of DTT and H202 had no effect on its DNA binding activity. In RPA70, since the addition of the subcomplexes of the 32 and 14 kDa subunits, it restored the modulating ssDNA binding activity to native RPA's DNA binding activity. These results suggest that the subcomplexes of the 32 and 14 kDa subunits may be involved in the modulating RPA's DNA binding activity through redox change. These studies, therefore, show the novel structure and function relationship of a multiprotein complex in that the role of a specific domain (or one subunit) is regulated by the other subunits.     

The cost of parental care: prey hunting in a digger wasp

Trivers's concept of parental investment is an integral partof modern evolutionary biology. "Parental investment" is definedas any parental expenditure that benefits a current progenyat the expense of a parent's ability to reproduce in the future.Because future costs are hard to quantify, other currencieswere used that were thought to be related to the actual costs.However, the validity of these alternative measures has rarely been established, at least in insects. Specifically, these measureswere not shown to represent costs at all. We investigated provisioningbehavior in a sphecid wasp, the European beewolf, Philanthustriangulum F., and tested whether prey hunting entails futurecosts to the female wasp and thus represents parental investment.We increased as well as decreased the females' hunting effortexperimentally and determined their hunting success on the following day. Furthermore, we analyzed the correlation betweenhunting rate of unrestricted females and their life span andassessed the effect of an experimentally decreased huntingeffort on life span. The future rate of bee hunting decreasedwhen hunting expenditure was increased (in the field) and viceversa (both in the field and in the laboratory). In contrast, there was no trade-off between hunting rate and life span, andlife span was not affected by an experimentally decreased huntingeffort (in the laboratory). Because prey hunting entails costsin terms of a reduced rate of prey hunting in the future, itmeets Trivers' definition of parental investment.     

Toxicity,persistence, and efficacy of spinosad,chlorfenapyr, and thiamethoxam on eggplant when applied against the eggplant flea beetle (Coleoptera: Chrysomelidae)

A laboratory bioassay was developed for determining the toxicity of spinosad, chlorfenapyr, and thiamethoxam against the eggplant flea beetle, Epitrix fuscula Crotch, on eggplant foliage. Four days after initial exposure, LC50 values were 1.99, 2.50, and 0.88 ppm for spinosad, chlorfenapyr, and thiamethoxam, respectively. By dividing the recommended field rate in ppm by the LC50 value, a field toxicity ratio was determined and ranged from 13.5 for spinosad to 73.9 for thiamethoxam. The high ratios suggest that field rates for all three insecticides could likely be reduced. This was supported by field studies in 2000 in which reduced rates of spinosad and thiamethoxam significantly reduced flea beetle numbers on eggplant. Mortality produced by thiamethoxam occurred more quickly than that for the other tested materials as shown with LT50 values of 1.8, 3.0, and 3.6 and days for thiamethoxam, chlorfenapyr, and spinosad, respectively. Persistence studies indicated that while all three of the tested compounds initially produced high levels of mortality, chlorfenapyr and thiamethoxam produced 50% or greater mortality after 6 d. Our data suggest that future management strategies for E. fuscula on eggplant can be successfully altered to meet the changing needs of the producer. Spinosad was recently registered, is effective against the E. fuscula, and offers a viable alternative to carbamate and pyrethroid insecticides. Thiamethoxam and chlorfenapyr offer high levels of toxicity to E. fuscula and upon registration will offer additional effective tools for management.     

Branchial chloride cells in sea bass (Dicentrarchus labrax) adapted to fresh water,seawater, and doubly concentrated seawater

Branchial chloride cells (CC) were studied in sea bass (Dicentrarchus labrax) maintained in seawater (SW: 35 per thousand) or gradually adapted to and subsequently maintained in fresh water (0.2 per thousand) or doubly concentrated seawater (DSW: 70 per thousand). Changes were observed in the location, number, and structure of CCs, that were discriminated by light, scanning, and transmission electron microscopy, as well as by immunofluorescence on the basis of their high Na(+)/K(+)-ATPase antigen content. The number of CCs increased in both fresh water and doubly concentrated seawater compared to control fish maintained in SW. In both experimental conditions, these cells were found on the gill filament (as in control fish) and even on the lamellae, especially in hypersaline conditions. Structural changes concerned the shapes and sizes of CCs and their apical outcrops and particularly the structures of their functional complexes (mitochondria, tubular system, and endoplasmic reticulum), which developed significantly in DSW adapted fish. The changes in the expression of the Na(+)/K(+)-ATPase were evaluated by assessing the enzyme's density at the ultrastructural level following immunogold labeling. This parameter was significantly higher in doubly concentrated seawater. The adaptative significance of the quantitative and morphofunctional changes in branchial chloride cells is discussed in relation to the original osmoregulatory strategy of this marine euryhaline teleost.     

Thiol antioxidants inhibit the adjuvant effects of aerosolized diesel exhaust particles in a murine model for ovalbumin sensitization

Although several epidemiological studies indicate a correlation between exposure to ambient particulate matter and adverse health effects in humans, there is still a fundamental lack of understanding of the mechanisms involved. We set out to test the hypothesis that reactive oxygen species are involved in the adjuvant effects of diesel exhaust particles (DEP) in a murine OVA sensitization model. First, we tested six different antioxidants, N-acetylcysteine (NAC), bucillamine (BUC), silibinin, luteolin, trolox (vitamin E), and ascorbic acid, for their ability to interfere in DEP-mediated oxidative stress in vitro. Of the six agents tested, only the thiol antioxidants, BUC and NAC, were effective at preventing a decrease in intracellular reduced glutathione:glutathione disulfide ratios, protecting cells from protein and lipid oxidation, and preventing heme oxygenase 1 expression. Therefore, we selected the thiol antioxidants for testing in the murine OVA inhalation sensitization model. Our data demonstrate that NAC and BUC effectively inhibited the adjuvant effects of DEP in the induction of OVA-specific IgE and IgG1 production. Furthermore, NAC and BUC prevented the generation of lipid peroxidation and protein oxidation in the lungs of OVA- plus DEP-exposed animals. These findings indicate that NAC and BUC are capable of preventing the adjuvant effects of inhaled DEP and suggest that oxidative stress is a key mechanistic component in the adjuvant effect of DEP. Antioxidant treatment strategies may therefore serve to alleviate allergic inflammation and may provide a rational basis for treating the contribution of particulate matter to asthmatic disease.     

Engineering of coordinated up- and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells

Production of O-linked oligosaccharides that interact with selectins to mediate cell-cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required.We report here the partial cloning of the CMP-sialic, acid:Galbeta1,3GalNAcalpha2,3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP-GlcNAc:Galbeta1,3GalNAc-R beta1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed "one-step" transient manipulation of multiple enzyme activities in the O-glycosylation pathway of a previously established CHO cell line already engineered to express alpha1,3-fucosyltransferase VI (alpha1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O-glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field.     

The genome of Methanosarcina mazei: evidence for lateral gene transfer between bacteria and archaea

The Archaeon Methanosarcina mazei and related species are of great ecological importance as they are the only organisms fermenting acetate, methylamines and methanol to methane, carbon dioxide and ammonia (in case of methylamines). Since acetate is the precursor of 60% of the methane produced on earth these organisms contribute significantly to the production of this greenhouse gas, e.g. in rice paddies. The 4,096,345 base pairs circular chromosome of M. mazei is more than twice as large as the genomes of the methanogenic Archaea currently completely sequenced (Bult et al., 1996; Smith et al., 1997). 3,371 open reading frames (ORFs) were identified. Based on currently available sequence data 376 of these ORFs are Methanosarcina-specific and 1,043 ORFs find their closest homologue in the bacterial domain. 544 of these ORFs reach significant similarity values only in the bacterial domain. They include 56 of the 102 transposases, and proteins involved in gluconeogenesis, proline biosynthesis, transport processes, DNA-repair, environmental sensing, gene regulation, and stress response. Striking examples are the occurrence of the bacterial GroEL/GroES chaperone system and the presence of tetrahydrofolate-dependent enzymes. These findings might indicate that lateral gene transfer has played an important evolutionary role in forging the physiology of this metabolically versatile methanogen.     

Seasonal variation of airborne fungal spore concentrations in a vineyard of North-West Spain

Conidial types collected daily in the air above a vineyard in northwest Spain were identified and counted. A total of 26 fungal spore types were recognised; ten of which (Cladosporium, Botrytis, Fusarium-Leptosphaeria type,Torula, Puccinia, Alternaria, Uncinula, Helminthosporium type,Agrocybe andStemphylium) gave a seasonal total concentration exceeding 1000 spores. Seasonal patterns are shown for 12 of the identified taxa.     

Mechanical advantage of the canine triangularis sterni

De Troyer, André, and Alexandre Legrand.Mechanical advantage of the canine triangularis sterni.J. Appl. Physiol. 84(2): 562-568, 1998.Recent studies on the canine parasternal intercostal,sternomastoid, and scalene muscles have shown that the maximal changesin airway opening pressure (Pao) obtained per unit muscle mass(Pao/m) during isolatedcontraction are closely related to the fractional changes in musclelength per unit volume increase of the relaxed chest wall. In thepresent study, we have examined the validity of this relationship for the triangularis sterni, an important expiratory muscle of the rib cagein dogs. Passive inflation above functional residual capacity (FRC)induced a virtually linear increase in muscle length, such that, with a1.0-liter inflation, the muscle lengthened by 17.9 ± 1.6 (SE) % of its FRC length. When the muscle in one interspace wasmaximally stimulated at FRC, Pao increased by 0.84 ± 0.11 cmH₂O. However, in agreement withthe length-tension characteristics of the muscle, when lung volume wasincreased by 1.0 liter before stimulation, the rise in Pao amounted to1.75 ± 0.12 cmH₂O. At thehigher volume, Pao/m thereforeaveraged + 0.53 ± 0.05 cmH₂O/g, such that the coefficientof proportionality between the change in triangularis sterni lengthduring passive inflation and Pao/m was the same as that previously obtained for the parasternalintercostal and neck inspiratory muscles. These observations,therefore, confirm that there is a unique relationship between thefractional changes in length of the respiratory muscles, bothinspiratory and expiratory, during passive inflation and theirPao/m. Consequently, the maximal effect of a particular muscle on the lung can be predicted on the basisof its change in length during passive inflation and its mass. Ageometric analysis of the rib cage also established that thelengthening of the canine triangularis sterni during passive inflationis much greater than the shortening of the parasternal intercostalsbecause, in dogs, the costal cartilages slope downward from thesternum.