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     

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.     

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.     

Fine structural study of the statocysts in the veliger larva of the nudibranch,Rostanga pulchra

Summary The two statocysts of the veliger larva of Rostanga pulchra are positioned within the base of the foot. They are spherical, fluid-filled capsule that contain a large, calcareous statolith and several smaller concretions. The epithelium of the statocyst is composed of 10 ciliated sensory cells (hair cells) and 11 accessory cells. The latter group stains darkly and includes 2 microvillous cells, 7 supporting cells, and 2 glial cells. The hair cells stain lightly and each gives rise to an axon; two types can be distinguished. The first type, in which a minimum of 3 cilia are randomly positioned on the apical cell membrane, is restricted to the upper portion of the statocyst. The second type, in which 9 to 11 cilia are arranged in a slightly curved row, is found exclusively around the base of the statocyst. Each statocyst is connected dorso-laterally to the ipsilateral cerebral ganglion by a short static nerve, formed by axons arising from the hair cells. Ganglionic neurons synapse with these axons as the static nerve enters the cerebral ganglion. The lumen of the statocyst is continuous with a blind constricted canal located beneath the static nerve.A diagram showing the structure of the statocyst and its association with the nervous system is presented. Possible functions of the statocyst in relation to larval behavior are discussed.     

Development of a closed-loop artificial pancreas.

A closed-loop glucose controlled insulin infusion system was developed, consisting of elements for continuous blood glucose analysis, a computer control system, and infusion systems. Improvements include decreased size, cost reduction and better performance. The algorithm used was a piecewise linear representation of the sigmoidal curve commonly employed. The apparatus has been applied to simulation of the healthy beta cell and glucose clamp studies.     

Induction of stalk cell differentiation by cyclic-AMP in a susceptible variant of Dictyostelium discoideum.

The P-4 variant of Dictyostelium discoideum (DdH) was found to produce a great excess of stalk cells compared to the wild type DdH. If the vegetative cells of P-4 were repeatedly washed, the variant changed back to the wild type phenotype, and if cyclic-AMP was added to the washed P-4 cells, the variant character was restored. Furthermore, if the concentration of added cyclic-AMP was increased, it was possible to induce 100% stalk cells in P-4. Phosphodiesterase would cause the variant to change to the wild type, while 5-AMP and cyclic-nucleotides other than cyclic-AMP have no effect at all. Therefore it was concluded that cyclic-AMP plays a key role in stalk cell differentiation.A comparison between wild type DdH and the variant P-4 showed that DdH is ten times less sensitive to cyclic-AMP induction. They both produce the same amount of cyclic-AMP and extracellular phosphodiesterase, but the specific activity of P-4 cell-bound phosphodiesterase during development is significantly less than that in the DdH. One hypothesis that accounts for the P-4-DdH difference is that because of the lack of cell-bound phosphodiesterase, more cyclic-AMP enters the variant cells and hence more stalk cell differentiation.     

Carrier solutions for low-level intravenous insulin infusion.

In the use of low-level intravenous insulin infusion for treating diabetic hyperglycaemia and ketoacidosis adsorption of insulin to containers or plastic infusion apparatus results in significant losses of 60-80% of insulin in dilute physiological saline solution (40 U/l). It is therefore necessary to add protein to the carrier solution to minimize losses and maintain a constant delivery rate. Recovery studies showed that 3.5% w/v polygeline solution (polymer of degraded gelatin) was a suitable medium for this purpose, offering some advantages over human serum albumin. A minimum concentration of 0.5% polygeline was required to ensure adequate delivery of insulin to the patient.     

A gentle introduction to SNP analysis: resources and tools

Bioinformatics is the use of informatics tools and techniques in the study of molecular biology, genetic, or clinical data. The field of bioinformatics has expanded tremendously to cope with the large expansion of information generated by the mouse and human genome projects, as newer generations of computers that are much more powerful have emerged in the commercial market. It is now possible to employ the computing hardware and software at hand to generate novel methodologies in order to link data across the different databanks generated by these international projects and derive clinical and biological relevance from all of the information gathered. The ultimate goal would be to develop a computer program that can provide information correlating genes, their single nucleotide polymorphisms (SNPs), and the possible structural and functional effects on the encoded proteins with relation to known information on complex diseases with great ease and speed. Here, the recent developments of available software methods to analyze SNPs in relation to complex diseases are reviewed with emphasis on the type of predictions on protein structure and functions that can be made. The need for further development of comprehensive bioinformatics tools that can cope with information generated by the genomics communities is emphasized.     

A combinatorial system to examine the enzymatic repair of multiply damaged DNA substrates

DNA damage drives genetic mutations that underlie the development of cancer in humans. Multiple pathways have been described in mammalian cells which can repair this damage. However, most work to date has focused upon single lesions in DNA. We present here a combinatorial system which allows assembly of duplexes containing single or multiple types of damage by ligating together six oligonucleotides containing damaged or modified bases. The combinatorial system has dual fluorescent labels allowing examination of both strands simultaneously, in order to study interactions or competition between different DNA repair pathways. Using this system, we demonstrate how repair of oxidative damage in one DNA strand can convert a mispaired T:G deamination intermediate into a T:A mutation. We also demonstrate that slow repair of a T:G mispair, relative to a U:G mispair, by the human methyl-binding domain 4 DNA glycosylase provides a competitive advantage to competing repair pathways, and could explain why CpG dinucleotides are hotspots for C to T mutations in human tumors. Data is also presented that suggests repair of closely spaced lesions in opposing strands can be repaired by a combination of short and long-patch base excision repair and simultaneous repair of multiply damage sites can potentially lead to lethal double strand breaks.