Lymphocyte activation and serine-esterase induction following recombinant interleukin-2 infusion for lymphomas and acute leukaemias

Summary C57BL mice inoculated with radiation leukemia virus (RadLV) develop preleukemic cells long before the onset of leukemia. These cells are potentially immunogenic but fail to elicit an immune response in the host because of the appearance of virus-specific suppressor T cells. We have studied the effect of polysaccharide K (PSK) on the generation of RadLV-specific cell-mediated immune responses in vitro. Long-term exposure to PSK in culture potentiated the ability of immunized T cells to respond to a RadLV-induced lymphoma. It also abrogated the suppressive activity of suppressor T cells and simultaneously boosted the ability of reactive T cells to respond. The dual immunostimulating activity of PSK resulted in the generation of T cytotoxic lymphocytes that could lyse lymphoma cells in vitro. The results suggest that PSK could be used as a prophylactic immune response modifier in preleukemia.     

A population‐based temporal logic gate for timing and recording chemical events

Engineered bacterial sensors have potential applications in human health monitoring, environmental chemical detection, and materials biosynthesis. While such bacterial devices have long been engineered to differentiate between combinations of inputs, their potential to process signal timing and duration has been overlooked. In this work, we present a two‐input temporal logic gate that can sense and record the order of the inputs, the timing between inputs, and the duration of input pulses. Our temporal logic gate design relies on unidirectional DNA recombination mediated by bacteriophage integrases to detect and encode sequences of input events. For an E. coli strain engineered to contain our temporal logic gate, we compare predictions of Markov model simulations with laboratory measurements of final population distributions for both step and pulse inputs. Although single cells were engineered to have digital outputs, stochastic noise created heterogeneous single‐cell responses that translated into analog population responses. Furthermore, when single‐cell genetic states were aggregated into population‐level distributions, these distributions contained unique information not encoded in individual cells. Thus, final differentiated sub‐populations could be used to deduce order, timing, and duration of transient chemical events.     

Open reading frame 1 of the Norwalk-like virus Camberwell: completion of sequence and expression in mammalian cells

The ORF1 sequence was determined for Camberwell virus, a genogroup 2 Norwalk-like virus, completing the full genome of 7,555 nucleotides. ORF1 cDNA was cloned into a simian virus 40-based expression vector, and the viral proteins synthesized following transfection into COS cells were analyzed. By using antisera directed against the helicase, protease, or polymerase regions, eight polypeptides ranging in size from 19 to 117 kDa were detected by radioimmunoprecipitation. The cleavage sites determining the amino and carboxy termini of the 3C-like protease were identified at E(1008)/A and E(1189)/G, respectively.     

Single amino acid substitution in Bacillus sphaericus phenylalanine dehydrogenase dramatically increases its discrimination between phenylalanine and tyrosine substrates

Homology-based modeling of phenylalanine dehydrogenases (PheDHs) from various sources, using the structures of homologous enzymes Clostridium symbiosum glutamate dehydrogenase and Bacillus sphaericus leucine dehydrogenase as a guide, revealed that an asparagine residue at position 145 of B. sphaericus PheDH was replaced by valine or alanine in PheDHs from other sources. This difference was proposed to be the basis for the poor discrimination by the B. sphaericus enzyme between the substrates L-phenylalanine and L-tyrosine. Residue 145 of this enzyme was altered, by site-specific mutagenesis, to hydrophobic residues alanine, valine, leucine, and isoleucine, respectively. The resultant mutants showed a high discrimination, above 50-fold, between L-phenylalanine and L-tyrosine. This higher specificity toward L-phenylalanine was due to K(m) values for L-phenylalanine lowered more than 20-fold compared to the values for L-tyrosine. The greater specificity for L-phenylalanine in the wild-type Bacillus badius enzyme, which has a valine residue in the corresponding position, was also found to be largely due to a lower K(m) for this substrate. Activities were also measured with a range of six amino acids with aliphatic, nonpolar side chains, and with the corresponding oxoacids, and in all cases the specificity constants for these substrates were increased in the mutant enzymes. As with phenylalanine, these increases are mainly attributable to large decreases in K(m) values.     

Identification of H-specific determinants in flagellin of four Escherichia coli strains

The H serogroup of Escherichia coli is determined by the flagellar antigen, flagellin. Sequence analysis of the flagellin gene, fliC, reveals a central variable region and the highly conserved N- and C-termini. This variable region has been shown to encode both H-specific and cross-reactive epitopes. Using polyclonal antibodies, we mapped the linear H-specific determinants in flagellin from four E. coli serotypes O157:H10, 0138:H14, O157:H42 and O157:H43. The specificity of all potential fragments was verified with 52 ECRC (Escherichia coli Reference Center) H-specific antisera. Our results indicated that: (a) a specific determinant of H10 flagellin (1263 bp long) maps to the region covering amino acid residues 305-331; (b) a specific determinant of H14 flagellin (1653 bp long) maps to the region covering amino acid residues 430-461; (c) a specific determinant of H42 flagellin (1281 bp long) maps to a region covering amino acid residues 171-201; and (d) a specific determinant of H43 flagellin (1506 bp long) maps to a region covering amino acid residues 200-260.     

Characterization of a C-C bond hydrolase from Sphingomonas wittichii RW1 with novel specificities towards polychlorinated biphenyl metabolites

Sphingomonas wittichii RW1 degrades chlorinated dibenzofurans and dibenzo-p-dioxins via meta cleavage. We used inverse PCR to amplify dxnB2, a gene encoding one of three meta-cleavage product (MCP) hydrolases identified in the organism that are homologues of BphD involved in biphenyl catabolism. Purified DxnB2 catalyzed the hydrolysis of 8-OH 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) approximately six times faster than for HOPDA at saturating substrate concentrations. Moreover, the specificity of DxnB2 for HOPDA (k(cat)/K(m) = 1.2 x 10(7) M(-1) s(-1)) was about half that of the BphDs of Burkholderia xenovorans LB400 and Rhodococcus globerulus P6, two potent polychlorinated biphenyl (PCB)-degrading strains. Interestingly, DxnB2 transformed 3-Cl and 4-OH HOPDAs, compounds that inhibit the BphDs and limit PCB degradation. DxnB2 had a higher specificity for 9-Cl HOPDA than for HOPDA but a lower specificity for 8-Cl HOPDA (k(cat)/K(m) = 1.7 x 10(6) M(-1) s(-1)), the chlorinated analog of 8-OH HOPDA produced during dibenzofuran catabolism. Phylogenetic analyses based on structure-guided sequence alignment revealed that DxnB2 belongs to a previously unrecognized class of MCP hydrolases, evolutionarily divergent from the BphDs although the physiological substrates of both enzyme types are HOPDAs. However, both classes of enzymes have mainly small hydrophobic residues lining the subsite that binds the C-6 phenyl of HOPDA, in contrast to the bulky hydrophobic residues (Phe106, Phe135, Trp150, and Phe197) found in the class II enzymes that prefer substrates possessing a C-6 alkyl. Thr196 and/or Asn203 appears to be an important determinant of specificity for DxnB2, potentially forming hydrogen bonds with the 8-OH substituent. This study demonstrates that the substrate specificities of evolutionarily divergent hydrolases may be useful for degrading mixtures of pollutants, such as PCBs.     

A novel property of spider silk: chemical defence against ants

Spider webs are made of silk, the properties of which ensure remarkable efficiency at capturing prey. However, remaining on, or near, the web exposes the resident spiders to many potential predators, such as ants. Surprisingly, ants are rarely reported foraging on the webs of orb-weaving spiders, despite the formidable capacity of ants to subdue prey and repel enemies, the diversity and abundance of orb-web spiders, and the nutritional value of the web and resident spider. We explain this paradox by reporting a novel property of the silk produced by the orb-web spider Nephila antipodiana (Walckenaer). These spiders deposit on the silk a pyrrolidine alkaloid (2-pyrrolidinone) that provides protection from ant invasion. Furthermore, the ontogenetic change in the production of 2-pyrrolidinone suggests that this compound represents an adaptive response to the threat of natural enemies, rather than a simple by-product of silk synthesis: while 2-pyrrolidinone occurs on the silk threads produced by adult and large juvenile spiders, it is absent on threads produced by small juvenile spiders, whose threads are sufficiently thin to be inaccessible to ants.