Mechanical model for theoretical determination of maximum running speed in mammals

A mechanical model for the determination of maximum speed in terrestrial tetrapods, designed for application to extinct species, is proposed. Only external bone measures and average body mass estimations are used as input data, and the hypothesis is made that leg bones are strong enough to endure the stress of running at maximum speed at a certain universal safety factor. The model is applied to a broad sample of living mammalian species to test its predictive power, and it is found to provide very good estimates of maximum running speed.     

High-affinity inhibition of a family of Plasmodium falciparum proteases by a designed adaptive inhibitor

Drug development against viral or microbial targets is often compounded by the existence of naturally occurring polymorphisms or drug resistant mutations. In the case of Plasmodium falciparum, the etiological agent of malaria, four related and essential proteases, plasmepsin I, II, and IV and the histo-aspartyl protease (HAP), have been identified in the food vacuole of the parasite. Since all of these enzymes are involved in the hemoglobin degradation of infected victims, the simultaneous inhibition of the four enzymes can be expected to lead to a faster starvation of the parasite and to delay the onset of drug resistance, since four enzymes will need to mutate in a concerted fashion. This study describes the design of an adaptive inhibitor intended to inhibit the entire plasmepsin family. Adaptive inhibitors bind with extremely high affinity to a primary target within the family and maintain significant affinity against the remaining members. This objective is accomplished by engineering the strongest and most specific interactions of the inhibitor against conserved regions of the binding site and by accommodating target variations by means of flexible asymmetric functional groups. Using this approach, we have designed an inhibitor with subnanomolar affinity (0.5 nM) against the primary target, plasmepsin II, and with no loss or a very small loss of affinity against plasmepsin IV, I, and HAP (K(i) ratios of 0.4, 7.1, and 17.7, respectively). The core of the inhibitor is defined by an allophenylnorstatine scaffold. Adaptability is provided by an asymmetric amino indanol functional group facing one of the key variable regions in the binding site. Adaptive inhibitors, which display high affinity against several variations of a primary target, are expected to play an important role in the chemotherapy of infectious diseases.     

A major role for a set of non-active site mutations in the development of HIV-1 protease drug resistance

A major problem in the chemotherapy of HIV-1 infection is the appearance of drug resistance. In the case of HIV-1 protease inhibitors, resistance originates from mutations in the protease molecule that lower the affinity of inhibitors while still maintaining a viable enzymatic profile. Drug resistance mutations can be classified as active site or non-active site mutations depending on their location within the protease molecule. Active site mutations directly affect drug/target interactions, and their action can be readily understood in structural terms. Non-active site mutations influence binding from distal locations, and their mechanism of action is not immediately apparent. In this paper, we have characterized a mutant form of the HIV-1 protease, ANAM-11, identified in clinical isolates from HIV-1 infected patients treated with protease inhibitors. This mutant protease contains 11 mutations, 10 of which are located outside the active site (L10I/M36I/S37D/M46I/R57K/L63P/A71V/G73S/L90M/I93L) and 1 within the active site (I84V). ANAM-11 lowers the binding affinity of indinavir, nelfinavir, saquinavir, and ritonavir by factors of 4000, 3300, 5800, and 80000, respectively. Surprisingly, most of the loss in inhibitor affinity is due to the non-active site mutations as demonstrated by additional experiments performed with a protease containing only the 10 non-active site mutations (NAM-10) and another containing only the active site mutation (A-1). Kinetic analysis with two different substrates yielded comparable catalytic efficiencies for A-1, ANAM-11, NAM-10, and the wild-type protease. These studies demonstrate that non-active site mutations can be the primary source of resistance and that their role is not necessarily limited to compensate deleterious effects of active site mutations. Analysis of the structural stability of the proteases by differential scanning calorimetry reveals that ANAM-11 and NAM-10 are structurally more stable than the wild-type protease while A-1 is less stable. Together, the binding and structural thermodynamic results suggest that the non-active site mutants affect inhibitor binding by altering the geometry of the binding site cavity through the accumulation of mutations within the core of the protease molecule.     

The CD43 coreceptor molecule recruits the zeta-chain as part of its signaling pathway

CD43 is an abundant cell surface sialoglycoprotein implicated in hemopoietic cell adhesion and activation. Cell stimulation through CD43 results in recruitment of different signaling proteins, including members of the Src family kinases, Syk, phospholipase Cgamma2, the adapter protein Shc, the guanine nucleotide exchange factor Vav, and activation of protein kinase C. In this study, we report that in human T lymphocytes, the zeta-chain is part of the CD43 signaling pathway. Upon CD43 engagement, the zeta-chain was tyrosine-phosphorylated, generating docking sites for tyrosine-phosphorylated zeta-associated protein of 70 kDa and Vav. In vitro kinase assays suggested that zeta-associated protein of 70 kDa could account for the kinase activity associated with the zeta-chain following CD43 engagement. Cross-linking CD43 on the surface of the Lck-deficient JCaM.1 cells failed to phosphorylate the zeta-chain and associated proteins, suggesting that Lck is a key element in the CD43 signaling pathway leading to zeta phosphorylation. CD43 engagement with beads coated with anti-CD43 mAb resulted in concentration of the zeta-chain toward the bead attachment site, but interestingly, the distribution of the T cell Ag receptor complex remained unaffected. Recruitment of the zeta-chain through CD43-mediated signals was not restricted to T lymphocytes because phosphorylation and redistribution of the zeta-chain was also observed in NK cells. Our results provide evidence that the zeta-chain functions as a scaffold molecule in the CD43 signaling pathway, favoring the recruitment and formation of downstream signaling complexes involved in the CD43-mediated cell activation of T lymphocytes and other leukocytes such as NK cells.     

HIV-1 p55Gag encoded in the lysosome-associated membrane protein-1 as a DNA plasmid vaccine chimera is highly expressed,traffics to the major histocompatibility class II compartment,and elicits enhanced immune responses

Several genetic vaccines encoding antigen chimeras containing the lysosome-associated membrane protein (LAMP) translocon, transmembrane, and cytoplasmic domain sequences have elicited strong mouse antigen-specific immune responses. The increased immune response is attributed to trafficking of the antigen chimera to the major histocompatibility class II (MHC II) compartment where LAMP is colocalized with MHC II. In this report, we describe a new form of an HIV-1 p55gag DNA vaccine, with the gag sequence incorporated into the complete LAMP cDNA sequence. Gag encoded with the translocon, transmembrane and cytoplasmic lysosomal membrane targeting sequences of LAMP, without the luminal domain, was poorly expressed, did not traffic to lysosomes or MHC II compartments of transfected cells, and elicited a limited immune response from DNA immunized mice. In contrast, addition of the LAMP luminal domain sequence to the construct resulted in a high level of expression of the LAMP/Gag protein chimera in transfected cells that was further increased by including the inverted terminal repeat sequences of the adeno-associated virus to the plasmid vector. This LAMP/Gag chimera with the complete LAMP protein colocalized with endogenous MHC II of transfected cells and elicited strong cellular and humoral immune responses of immunized mice as compared with the response to DNA-encoding native Gag, with a 10-fold increase in CD4+ responses, a 4- to 5-fold increase in CD8+ T-cell responses, and antibody titers of >100,000. These results reveal novel roles of the LAMP luminal domain as a determinant of Gag protein expression, lysosomal trafficking, and possibly of the immune response to Gag.     

Effect of the polycation nature on the structure of layer-by-layer electrostatically self-assembled multilayers of polyphenol oxidase

Self-assembled multilayers comprised of alternate layers of polyphenol oxidase (PPO) and poly(allylamine) (PAH) or PPO and poly(diallyldimethylamine) (PDDA), deposited on a 3-mercaptopropanesulfonic acid (MPS)-modified gold surface, were studied "in-situ" (under water) by means of ellipsometry and quartz crystal microbalance (QCM), and "ex-situ" (in open air) by ellipsometry and fourier transform infrared reflection-absorption spectroscopy (FT-IRRAS). Optical ellipsometric properties of (PAH)(n)(PPO)(n) and (PDDA)(n)(PPO)(n) multilayers were obtained at two wavelengths, employing an isotropic single-layer model with the substrate parameters measured after thiol adsorption. Film thickness as well as ellipsometric mass values based on the de Feijter equation were also evaluated. The quartz crystal impedance analysis showed that self-assembled multilayers behaved as acoustically thin films, and therefore, the shifts observed in the film inductive impedance parameter were interpreted in terms of gravimetric mass. The enzyme mass up-take in each adsorption step was determined on PAH or on PDDA topmost layer. A comparative study between the ellipsometric thickness and acoustic mass values allowed us to obtain average values of "apparent" densities of (2.1 +/- 0.1) and (2.4 +/- 0.1) g cm(-3) for (PAH)(n)(PPO)(n) and (PDDA)(n)(PPO)(n) multilayers, respectively. The content of water included in the open polymer-enzyme structure was evaluated by comparison of QCM and ellipsometric mass values. FT-IRRAS spectra of each layer in (PAH)(n)(PPO)(n) and (PDDA)(n)(PPO)(n) films were recorded, and the intensity ratio of the amide bands was evaluated to obtain information about layer-by-layer enzyme conformation. An enzyme/polycation distribution model for (PAH)(n)(PPO)(n)and (PDDA)(n)(PPO)(n) multilayer structures is presented on the basis of combined ellipsometric, QCM, and FT-IRRAS results.     

Altered intestinal transport of amino acids in cirrhotic rats: the effect of insulin-like growth factor-I

The intestine is an important target organ for insulin-like growth factor-I (IGF-I), an anabolic hormone synthesized in the liver upon growth hormone (GH) stimulation. Levels of IGF-I are reduced in cirrhosis, and altered GH/IGF-I axis may contribute to malnutrition in cirrhotic patients. Our aim was to study Na(+)-dependent jejunal transport of amino acids (L-leucine, L-proline, L-glutamic acid, and L-cysteine) in cirrhotic rats and to analyze the effect of IGF-I on this function. IGF-I or saline was administered for 2 wk to rats with CCl(4)-induced cirrhosis and saline was administered to healthy control rats. Transport of amino acids was assessed in brush-border membrane vesicles (BBMV) using (14)C- or (35)S-labeled amino acids, and the kinetic constants V(max) and K(t) were determined. Na(+)-independent uptake of L-leucine, L-proline, L-glutamic acid, and L-cysteine by BBMV was similar in all groups. Na(+)-dependent uptake of all four amino acids was significantly diminished in cirrhotic rats compared with both controls and IGF-I-treated cirrhotic rats. The latter two groups exhibited similar V(max) and K(t), whereas untreated cirrhotic rats had reduced V(max) and increased K(t) compared with normal controls and IGF-I-treated cirrhotic animals. In conclusion, the transport of all four tested amino acids by BBMV is impaired in cirrhotic rats, and low doses of IGF-I can correct this defect.     

Effects of Resource-Island Soils, Competition, and Inoculation with Azospirillum on Survival and Growth of Pachycereus pringlei, the Giant Cactus of the Sonoran Desert

Resource‐island soils formed by some plants in arid lands are capable of supporting certain plants that do not normally establish in surrounding areas free of vegetation. We determined growth responses of Pachycereus pringlei (cardon), the giant columnar cactus, whose widespread, finely branched, subsurface root systems stabilize desert soils, to four soils collected from within or outside of resource islands. Traits of cardon grown in soils from mature (MM) or young (YM) Prosopis articulata (mesquite), mature Olnea testosa (ironwood, MI), or bare areas (BA) were compared, to determine differences between the effects of soils due to the identity or the stage of development of nurse plants. The levels of soil N, P, and C contents were in the order MM> > YM > MI BA. The BA soil had the coarsest and MM soil the finest texture. Cardon was also grown in pot cultures inoculated with the plant‐growth‐promoting bacterium Azospirillum brasilense, or in association with a competing grass, Sorghum bicolor (sorghum). Competition did not affect survival rates of cardon in any of the soils after six months of growth, but decreased biomass accumulation by up to 90% in the best (MM) soil. Inoculation of cardon seeds with A. brasilense did not affect survival but resulted in significantly better root and shoot growth, and this effect increased linearly as soil nutrients declined. In the best soil (MM), A. brasilense had no effect on cardon growth, but in the poorest soil (BA) shoot dry mass was almost 60% and root length over 100% greater as a result of inoculation, with responses in the other two soils intermediate. This effect did not appear to be owing to N₂ fixation, as nitrogenase activity (acetylene reduction) was not detected in any of the treatments. Soil formation by selected nurse trees in arid areas is an important factor in plant establishment and growth, and the present results indicate that these processes can be impeded or facilitated by the introduction of competing or beneficial organisms. The use of beneficial microorganisms associated with roots may accelerate the restoration of disturbed areas. Key words: Azospirillum, desert soils, nitrogen fixation, nurse plant, Pachycereus pringlei, plant survival, resource‐island.