Engineering lysine‐rich caleosins as carrier proteins to render biotin as a hapten on artificial oil bodies for antibody production

It has been demonstrated that caleosin alone is sufficient to stabilize artificial oil bodies. A series of recombinant caleosins, mutated with 3, 5, 8, 11, 13, 15, and 17 extra Lys residues and over‐expressed in Escherichia coli, were used as carrier proteins to render biotin as a hapten on the surface of artificial oil bodies for antibody production. Biotinylation levels of the recombinant caleosins were step‐wisely elevated as the number of extra Lys residues increased, and the biotinylated Lys residues were identified by mass spectrometric analysis. Polyclonal antibodies against biotin were successfully generated in rats injected with artificial oil bodies constituted with each of the biotinylated caleosins. Moreover, those generated via the biotinylated caleosins with eight or more extra Lys residues no longer recognized caleosin. It appears that engineered Lys‐rich caleosins are suitable carrier proteins for the production of antibodies against small molecules. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Secretion of Recombinant Pediocin PA-1 by Bifidobacterium longum, Using the Signal Sequence for Bifidobacterial α-Amylase

A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial α-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1.     

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

Continuous culture of a recombinantEscherichia coli and plasmid maintenance for tryptophan production

Summary Production of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 trpLDtrpR ^ts tna ^– (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.     

A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure

We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine. Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer. However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-biphosphate (Fru-1,6-P2). The mutation has not significantly changed the catalytic properties of the dimer (Kd NADH, Km pyruvate, Ki oxamate and kcat), but has weakened the binding of Fru-1,6-P2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect. We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-1,6-P2, is important for allosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism.     

Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity

Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.     

Effect of biological toxins on gap-junctionai intercellular communication in Chinese hamster V79 cells

Since chemical modulation of gap junctional intercellular communication has been implicated in several toxicological endpoints, a study to examine the ability of several biological toxins to inhibit this process was undertaken. Eight biological toxins were tested for their ability to inhibit metabolic cooperation, a measure of gap-junctional intercellular communication, in the Chinese V79 cell system. Aplysiatoxin, anhydrodebromoaplysiatoxin and debromoaplysiatoxin showed the strongest ability to inhibit metabolic cooperation while T2-toxin and vomitoxin inhibited metabolic cooperation to a lesser degree. Afatoxin B₁, afatoxin B₂ and palytoxin were inactive in the Chinese V79 system. Palytoxin, which was extremely cytotoxic, might act as a tumor promoter if it induces compensatory hyperplasia in vivo.Abbreviations 6-TG 6-thioguanine - TPA 12-0-tetradecanoylphorbol-13-acetate     

The fine structure of the newly discovered propodial ganglia of the veliger larva of the nudibranch Onchidoris bilamellata

Summary Two pairs of ganglia are found in the propodial region of the veliger of Onchidoris bilamellata: the anterolateral pair is located at the foremost corners of the propodium, and the frontal pair is located beside the propodial midline. Both sets of ganglia are positioned below the epidermis, and they are joined to the cerebral ganglia by large, common connectives. Each ganglion possesses sensory cells, nerve cells and sheath cells, and the frontal pair contains a complement of secretory cells. Externally, the propodial ganglia are manifested as sensory fields. The fields of the anterolateral pair are elliptical in shape, and each appears as a band of cilia bordering an unciliated zone. The region devoid of cilia is composed of ordinary epidermal cells, whereas the ciliated portion is comprised of dendritic endings originating from cells in the ganglion. Dendrites arise from one type of sensory cell and pass through the epidermis in bundles. Each dendrite terminates as a single cilium at the epidermal surface. Sensory fields of the frontal ganglia are key-shaped and oppose one another on the anterior end of the foot. Each field appears as a flat, circular, unciliated region which extends into a ciliated groove that runs dorsally toward the mouth. The groove contains the terminals of secretory cells, ciliated sensory cells, and the cell bodies of nonciliated sensory cells. The nonciliated sensory cells, characterized by a microvillous apex, are the dominant cells in the flattened circular zone. The space between the frontal ganglia and the epidermis is bridged by bundles of processes which are similar to those of the anterolateral ganglia. However, these tracts contain collections of the apical processes of secretory cells, the dendrites of ciliated sensory cells, and the axons of nonciliated sensory cells. Morphological and behavioral evidence indicates that the propodial ganglia serve a chemosensory function during settlement and metamorphosis.     

Genotype of Yupik Eskimos with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Summary An A-to-G transition in the second intron was the sole mutation detected in four Yupik Eskimo patients with salt-wasting congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. Allele-specific hybridization should be an efficient means of performing prenatal diagnosis of the disease in this highly inbred population.     

The molecular analyses of an antimorphic mutation of Drosophila melanogaster, Scutoid

A dominant mutation of Drosophila melanogaster, Scutoid (Sco), acts as an antimorphic allele of the no-ocelli (noc) gene. In Sco the noc region has been transposed from 35B to 35D on chromosome arm 2L and the noc gene is now adjacent to snail (sna). Induced revertants of Sco are frequently mutant for sna or are aberrations broken very close to sna. A molecular analysis of the Sco chromosome has confirmed that noc is transposed and fused to the sna region. However, only part of the noc region is included within the transposition. The breakpoints of 19 chromosomally aberrant Sco revertants have been mapped at the molecular level. Fourteen of these breakpoints map to the noc region, spread over about 80 kb of DNA. The breakpoints of the remaining five are not within the DNA of the noc region and appear to map within sequences from the sna region. This has been shown directly for three of these, those associated with T(2;3)ScoR+13, In(2L)ScoR+24 and In(2L)ScoR+26. Thus mutation of either noc or sna, genes which are apparently unrelated in their wild-type functions, can revert the antimorphic phenotype of Sco.