Analysis of a glucose-containing tetrasaccharide by high-performance liquid affinity chromatography

In the present work we have explored conditions for using a pulsed amperometric detector for on-line analysis of oligosaccharides eluted from a high-performance liquid affinity chromatography column. A monoclonal antibody that specifically binds a glucose-containing oligosaccharide is coupled to a SelectiSphere-10-activated tresyl column. The system is eluted isocratically and easily detects 10 ng of the oligosaccharide with a linear response up to 250 ng. Analysis of both serum and urine samples from normal individuals and patients with acute pancreatitis gives a single retarded peak with a retention time identical to that of authentic (Glc)4. Retarded material pooled from several analyses of urine was positively identified as (Glc)4 by GC-MS analysis. As this method requires little cleanup and no chemical derivitization of the sample and is performed rapidly (less than 20 min) at sensitivities of at least 10 micrograms/liter in biological fluids, it represents a substantial improvement over previous GC-MS, radioimmunoassay, and enzyme-linked immunoadsorbent assay methods used to determine (Glc)4.     

Detection and isolation of oligosaccharides with Lea and Leb blood group activities by affinity chromatography using monoclonal antibodies

Affinity columns prepared by immobilizing monoclonal antibodies that specifically recognize the Lea or the Leb blood group antigens can be used for analytical or preparative isolation of oligosaccharides with the corresponding reactivities. The number of immobilized functional antibody combining sites on a column and the dissociation constants for standard oligosaccharides are determined by frontal analysis. By employing a simple approximation [K.-I. Kasai et al. (1986) J. Chromatogr. 376, 33-47] these parameters can be used to rationally design columns with properties appropriate for zonal affinity chromatography. The affinity for binding of the Lea-active oligosaccharide lacto-N-fucopentaose II (LNF II) by the anti-Lea antibody CO-514 doubles for each 8 degrees C downward shift in temperature between 37 and 4 degrees C. By zonal chromatography, Lea- or Leb-active oligosaccharides are recovered from a complex mixture of milk oligosaccharides containing more than a 20-fold molar excess of structurally similar but antigenically distinct oligosaccharides. The capacity for preparative isolation of an oligosaccharide increases in a linear fashion with the amount of antibody loaded on the solid support. The monoclonal antibodies used in these studies are products of hybridomas derived from mice immunized with human colorectal carcinoma cell lines [M. Blaszczyk et al. (1984) Arch. Biochem. Biophys. 233, 161-168]. The experiments establish that affinity chromatography applied to mixtures of oligosaccharides released by enzymatic or chemical cleavage of glycoconjugates may simplify the task of isolating and characterizing biologically interesting target antigens of monoclonal antibodies.     

A new non-equilibrium enzyme linked immunosorbent assay for a glycogen-derived urinary tetrasaccharide

The tetrasaccharide, Glc1-6Glc1-4Glc1-4Glc, denoted (Glc)₄, is a limit dextrin formed by amylolytic degradation of glycogen. In order to evaluate the possible clinical importance of (Glc)₄ excretion as an indicator of certain physiological and pathological conditions, we have developed a new rapid and inexpensive immunoassay using a monoclonal antibody raised against (Glc)₄ glycosidically-linked to a carrier protein. As the antibody is highly specific, it can be used to measure native (Glc)₄ directly, without the chemical reduction step required in previous methods. A new type of non-equilibrium ELISA inhibition test was developed based on the capacity of free (Glc)₄ to decrease initial rates of antibody binding to (Glc)₄-coated microtiter wells. The method is highly reproducible and is as sensitive and accurate as the gas chromatography method or radioimmunoassay used previously.Abbreviations (Glc)₄ Glc1-6Glc1-4Glc1-4Glc - KLH keyhole limpet hemacyanin - BSA bovine serum albumin - PEG polyethylene glycol     

A two-component signal-transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae

Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin-binding protein (PBP) variants with decreased affinities for β-lactam antibiotics. Cefotaxime-resistant laboratory mutants, selected after several steps on increasing concentrations of this β-lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, claR and claH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal-transducing proteins). Two hydrophobic peptide regions divided the histidine kinase ClaH into two putative domains: an N-terminal extracelluiar sensor part, and an intracelluiar C-terminal domain with the conserved His-226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime-resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime-resistant mutants were each located in the C-terminal half of ClaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated claH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal-transducing system cia is involved in early steps of competence regulation.     

Age-related nonrandom chromosomal abnormalities in human low-grade astrocytomas

We report a cytogenetic investigation of 55 low-grade astrocytomas in 52 patients, 15 children and 37 adults. In addition to numerical aberrations such as trisomy 7 and gonosomal losses, we found structural and/or numerical aberrations of chromosome 1 in eight astrocytomas. There was a striking difference between the rearranged chromosomes in pediatric and adult patients. Whereas the pediatric tumors revealed monosomies 1p with accompanying trisomy 1q, the astrocytomas in adults showed partial or complete monosomies 1q.     

Back to log phase: σS as a global regulator in the osmotic control of gene expression in Escherichia coli

It is now well established that the σ^S subunit of RNA polymerase is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-inducible genes in Escherichiacoli. In this review, more recent findings will be summarized that demonstrate that σ^S also acts as a global regulator for the osmotic control of gene expression, and actually does so in exponentially growing cells. Thus, many σ^S-dependent genes are induced during entry into stationary phase as well as in response to osmotic upshift. K⁺ glutamate, which accumulates in hyperosmotically stressed cells, seems to specifically stimulate the activity of σ^S-containing RNA polymerase at σ^S-dependent promoters. Moreover, osmotic upshift results in an elevated cellular σ^S level similar to that observed in stationary-phase cells. This increase is the result of a stimulation of rpoS translation as well as an inhibition of the turnover of σ^S, which in exponentially growing non-stressed cells is a highly unstable protein. Whereas the RNA-binding protein HF-I, previously known as a host factor for the replication of phage Qβ RNA, is essential for rpoS translation, the recently discovered response regulator RssB, and ClpXP protease, have been shown to be required for σ^S degradation. The finding that the histone-like protein H-NS is also involved in the control of rpoS translation and σ^S turnover, sheds new light on the function of this protein in osmoregulation. Finally, preliminary evidence suggests that additional stresses, such as heat shock and acid shock, also result in increased cellular σ^S levels in exponentially growing cells. Taken together, σ^S function is clearly not confined to stationary phase. Rather, σ^S may be regarded as a sigma factor associated with general stress conditions.     

Continuous Synthesis of (R)-(+)-Benzaldehydecyanohydrin Using (R)-Oxynitrilase in Lyotropic Liquid Crystals

The possibility of using the enzyme (R)-Oxynitrilase in a biphasic lyotropic liquid crystal/dibutylether system has been demonstrated. This reaction system is applicable for the continuous production of (R)-benzaldehydecyanohydrin in a fixed bed reactor. The optical purity was between 94 and 96% ee and independent of the flow rate. The space time yield was maximal (2650 g/(1*d)) at a flow rate of 1.6 ml/min.