Contributions of CDR3 to V H H domain stability and the design of monobody scaffolds for naive antibody libraries

Camelids produce functional antibodies devoid of light chains. Autonomous heavy chain variable (V(H)H) domains in these molecules have adapted to the absence of the light chain in the following ways: bulky hydrophobic residues replace small aliphatic residues in the former light chain interface, and residues from the third complementarity-determining region (CDR3) pack against the framework and stabilize the global V(H)H domain fold. To determine the specific roles of CDR3 residues in framework stabilization, we used nai;ve phage-displayed libraries, combinatorial alanine-scanning mutagenesis and biophysical characterization of purified proteins. Our results indicate that in the most stable scaffolds, the structural residues in CDR3 reside near the boundaries of the loop and pack against the framework to form a small hydrophobic core. These results allow us to differentiate between structural CDR3 residues that should remain fixed, and CDR3 residues that are tolerant to substitution and can therefore be varied to generate functional diversity within phage-displayed libraries. These methods and insights can be applied to the rapid design of heavy chain scaffolds for the identification of novel ligands using synthetic, antibody-phage libraries. In addition, they shed light on the relationships between CDR3 sequence diversity and the structural stability of the V(H)H domain fold.     

Comprehensive mutagenesis of the C-terminal domain of the M13 gene-3 minor coat protein: the requirements for assembly into the bacteriophage particle

Filamentous bacteriophage assemble at the host membrane in a non-lytic process; the gene-3 minor coat protein (P3) is required for release from the membrane and subsequently, for recognition and infection of a new host. P3 contains at least three distinct domains: two N-terminal domains that mediate host recognition and infection, and a C-terminal domain (P3-C) that is required for release from the host cell following phage assembly and contributes to the structural stability of the phage particle. A comprehensive mutational analysis of the 150 residue P3-C revealed that only 24 side-chains, located within the last 70 residues of sequence, were necessary for efficient incorporation into a wild-type coat. The results reveal that the requirements for the assembly of P3 into the phage particle are quite lax and involve only a few key side-chains. These findings shed light on the functional and structural requirements for filamentous phage assembly, and they may provide guidelines for the engineering of improved coat proteins as scaffolds for phage display technology.     

Protective role of zinc in nickel induced hepatotoxicity in rats

This study was planned to determine the protective role of zinc, if any, in attenuating the toxicity induced by nickel sulfate in rat liver. Female Sprague Dawley (SD) rats received either nickel alone in the dose of 800 mg/l in drinking water, zinc alone in the dose of 227 mg/l in drinking water, and nickel plus zinc or drinking water alone for a total duration of eight weeks. The effects of different treatments were studied on various parameters in rat liver which include antioxidant enzymes, levels of nickel and zinc and histoarchitecture at the light microscopic level. Further, the activities of hepatic marker enzymes AST and ALT were also studied in rat serum. Nickel treatment to the normal control animals, resulted in a significant increase in lipid peroxidation and enzyme activities of catalase and glutathione-S-transferase. On the contrary, nickel treatment to normal rats caused a significant inhibition in the levels of reduced glutathione. Superoxide dismutase activity was found to be decreased which however was not significant. Interestingly, when Zn was supplemented to nickel treated rats, the activities of catalase, and glutathione-S-transferase and the levels of GSH and lipid peroxidation came back to within normal limits. Activities of serum AST and ALT were increased significantly following nickel treatment to normal rats. Simultaneous zinc administration to nickel treated rats tended to restore the altered levels of AST and ALT. Normal control and zinc treated animals revealed normal histology of liver. On the other hand, nickel treated animals showed alterations in normal hepatic histoarchitecture which comprise of vacuolization of the hepatocytes and dilatation of sinusoids as well as increase in the number of bi-nucleated cells. Administration of zinc to nickel treated rats resulted in marked improvement in the structure of hepatocytes, thus emphasizing the protective potential of zinc in restoring the altered hepatic histoarchitecture. The nickel administration to normal rats indicated increased concentrations of nickel and decreased concentrations of zinc. However, zinc effectively brought the altered levels of nickel and zinc to within normal range. The study concludes that zinc has the potential in alleviating the toxic effects of nickel in rat liver because of its property to induce metallothionein (S-rich protein) as a free radical scavenger, or its indirect action in reducing the levels of oxygen reactive species.     

Phylogenetic analysis of the vertebrate galectin family

Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biological processes such as morphogenesis, control of cell death, immunological response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.     

Acid-base effects on intestinal Cl- absorption and vesicular trafficking

In rat ileum and colon, apical membrane exchange and net Cl^- absorption are stimulated by increases in PCO₂ or . Because changes in PCO₂ stimulate colonic Na⁺ absorption, in part, by modulating vesicular trafficking of the Na⁺/H⁺ exchanger type 3 isoform to and from the apical membrane, we examined whether changes in PCO₂ affect net Cl^- absorption by modulating vesicular trafficking of the exchanger anion exchanger (AE)1. Cl^- transport across rat distal ileum and colon was measured in the Ussing chamber, and apical membrane protein biotinylation of these segments and Western blots of recovered proteins were performed. In colonic epithelial apical membranes, AE1 protein content was greater at PCO₂ 70 mmHg than at PCO₂ 21 mmHg but was not affected by pH changes in the absence of CO₂. AE1 was internalized when PCO₂ was reduced and exocytosed when PCO₂ was increased, and both mucosal wortmannin and methazolamide inhibited exocytosis. Wortmannin also inhibited the increase in colonic Cl^- absorption caused by an increase in PCO₂. Increases in PCO₂ stimulated ileal Cl^- absorption, but wortmannin was without effect. Ileal epithelial apical membrane AE1 content was not affected by PCO₂. We conclude that CO₂ modulation of colonic, but not ileal, Cl^- absorption involves effects on vesicular trafficking of AE1. PCO₂; ileum; colon; anion exchanger 1; Na⁺/H⁺ exchanger type 3     

Ionic calcium levels in oviduct explant-conditioned media from an Australian marsupial, the brushtail possum (Trichosurus vulpecula) and its relevance to in vitro fertilization

Gametes from the brushtail possum (Trichosurus vulpecula), an Australian marsupial, require exposure to oviductal cells and/or their secretions before sperm binding and penetration of the zona pellucida can occur. Sperm-egg fusion, the next critical step in fertilization has not previously been reported in vitro. Here we describe the refinement of an oviduct epithelial cell (OEC) explant culture system using two different media to obtain in vitro sperm-egg fusion in the brushtail possum for the first time. Conditioned media from OEC explant cultures were supplemented with either 1% fetal calf serum (FCS) or 1 mg/ml polyvinyl alcohol and used for co-culture of epididymal sperm and superovulated eggs. Under these conditions zona penetration rates varied from 0 to 46% and sperm-egg fusion from 0 to 20%. Analysis of explant conditioned media indicated that qualitative and quantitative differences between batches could account, at least partially, for the large variability in zona penetration rates. Conditioned media that contained approximately 1 mM of ionic calcium were most effective for achieving sperm capacitation, zona binding, and penetration and sperm-egg fusion. The reorientation of the sperm head to T-shape, an indicator of capacitation in the brushtail possum, was closely linked with the concentration of calcium present in vitro.     

Bioregulation of carbohydrate metabolism in relation to source-sink operation during grain-filling phase of growth in wheat

Mobilization of free sugars from vegetative tissues to grain and their transformation to starch in relation to activities of some relevant enzymes during growth and development were investigated in wheat (Triticum aestivum L.). Vegetative tissues, viz. flag-leaf, flag-leaf sheath, nodes and internodes contained high concentration of free sugars from 70 DAS to 18 DPA and that was in the order of accumulation--flag-leaf sheath> flag-leaf and internodes > nodes. In these tissues, major portion of 14C appeared in endogenous sucrose, irrespective of the nature of (U-14C]-sugars supplied. In photosynthetic structures above flag-leaf node, namely peduncle, rachis and bracts, the free sugar make-up was maximum at anthesis (90 DAS). Activity of soluble acid invertase (EC 3.2.1.26) was high in these tissues during early stages of grain growth but reverse was true for soluble neutral invertase (EC 3.2.1.27) activity. In apical and basal portions of grain, free sugars were more or less similarly distributed in concentration. Linear and rapid accumulation of starch in endosperm paralleled with a decline in accumulation of this polymer in pericarp-aleurone. In the latter tissue, the activities of starch hydrolyzing enzymes, i.e alpha- and beta-amylases (3.2.1.1 and 3.2.1.2) were high during initial stages of grain growth. During active grain-filling, alkaline inorganic pyrophosphatase (EC 3.6.1.1) seemed to play a vital role during starch accumulation in endosperm, whereas the involvement of 3-PGA phosphatase (EC 3.1.3.38) was almost confined to pericarp-aleurone. Impairement of ear head photosynthesis by shading depressed starch synthesis (approximately 50%) indicating, thereby, the significant role of current photosynthates during grain-filling. The results suggested that grain growth in wheat was influenced by an efficient operation of source as well as regulatory factors, including enzymes, constituting intrinsic potential of grain sink.     

Protection by SIV VLP DNA prime/protein boost following mucosal SIV challenge is markedly enhanced by IL-12/GM-CSF co-administration

The ever increasing number of people infected by human immunodeficiency virus (HIV) throughout the world renders the development of effective vaccines an urgent priority. Herein, we report on an attempt to induce and enhance antiviral responses using a deoxyribonucleic acid (DNA) prime/virus-like particles (VLP) protein boost strategy adjuvanted with interleukin (IL)-12/GM-CSF in rhesus macaques challenged with simian immunodeficiency virus (SIV). Thus, groups of monkeys were administered three consecutive doses of pVecB7 a plasmid expressing VLP with or without plasmids expressing IL-12 and GM-CSF at weeks 0, 13 and 26. The VLP boost was administered at week 39 with or without IL-12. All monkeys were challenged intrarectally with SIVsmE660 2 months following the protein boost. All except one immunized monkey became infected. While all immunized monkeys showed a marked reduction of acute viral peaks, reduction of viral load set points was only achieved in groups whose prime-boost immunizations were supplemented with IL-12/GM-CSF (prime) and/or with IL-12 (boost). Control of viremia correlated with lack of disease progression and survival. Detection of virus in rectal washes at 1 year post-challenge was only successful in monkeys whose immunizations did not include cytokine adjuvant, but these loads did not correlate with plasma viral loads. In summary, use of IL-12 and/or GM-CSF was shown to provide significant differences in the outcome of SIV challenge of prime/boost immunized monkeys.     

A Single Amino Acid Change in the Hemagglutinin Protein of Measles Virus Determines Its Ability To Bind CD46 and Reveals Another Receptor on Marmoset B Cells

This paper provides evidence for a measles virus receptor other than CD46 on transformed marmoset and human B cells. We first showed that most tissues of marmosets are missing the SCR1 domain of CD46, which is essential for the binding of Edmonston measles virus, a laboratory strain that has been propagated in Vero monkey kidney cells. In spite of this deletion, the common marmoset was shown to be susceptible to infections by wild-type isolates of measles virus, although they did not support Edmonston measles virus production. As one would expect from these results, measles virus could not be propagated in owl monkey or marmoset kidney cell lines, but surprisingly, both a wild-type isolate (Montefiore 89) and the Edmonston laboratory strain of measles virus grew efficiently in B95-8 marmoset B cells. In addition, antibodies directed against CD46 had no effect on wild-type infections of marmoset B cells and only partially inhibited the replication of the Edmonston laboratory strain in the same cells. A direct binding assay with insect cells expressing the hemagglutinin (H) proteins of either the Edmonston or Montefiore 89 measles virus strains was used to probe the receptors on these B cells. Insect cells expressing Edmonston H but not the wild-type H bound to rodent cells with CD46 on their surface. On the other hand, both the Montefiore 89 H and Edmonston H proteins adhered to marmoset and human B cells. Most wild-type H proteins have asparagine residues at position 481 and can be converted to a CD46-binding phenotype by replacement of the residue with tyrosine. Similarly, the Edmonston H protein did not bind CD46 when its Tyr481 was converted to asparagine. However, this mutation did not affect the ability of Edmonston H to bind marmoset and human B cells. The preceding results provide evidence, through the use of a direct binding assay, that a second receptor for measles virus is present on primate B cells.     

Absence of enantioselectivity in the pharmacodynamics of P450 2B induction by 5-ethyl-5-phenylhydantoin in the male rat liver or in cultured rat hepatocytes

To explore the enantioselectivity of ligand interaction with the putative phenobarbital receptor, the pharmacodynamics of cytochrome P450 2B (CYP2B) induction by racemic 5-ethyl-5-phen-ylhydantoin and its two enantiomers were investigated in the male F344/NCr rat and in cultured adult male rat hepatocytes. Steady-state serum drug concentrations, measured following 14 days of administration of the compounds in the diet (0-1320 ppm, n = 3 rats per group), were used as an approximation of intrahepatocellular drug concentration. The serum xenobiotic concentrations associated with half-maximal hepatic CYP2B induction were 5-10 μM, based on measurement of pentoxy- or benzyloxyresorufin O-dealkylation activities, or immunoreactive CYP2B1 protein. The corresponding potency values in the hepatocyte culture experiments were 8-12 μM, based on measurement of total cellular RNA coding for CYP2B1. In both the in vivo and the hepatocyte culture experiments, the potencies for CYP2B induction were essentially equivalent for the racemate and the individual enantiomers of 5-ethyl-5-phenylhydantion. In the case of this compound, there would appear to be no enantioselectivity for CYP2B induction. This finding may be interpreted as evidence against receptor mediation in the induction of CYP2B activity, although it is also possible that a receptor is involved that does not exhibit enantioselectivity.