Covalent immobilization of avidin on glassy carbon electrodes as the basis for multivalent biosensors.

One of the most crucial steps for the successful construction of a biosensor is the appropriate and reproducible coupling of the biological part (e.g. enzyme, antibody) to the inorganic moiety of the device (e.g. electrode, microchip). In this paper three methods of immobilization of avidin to a glassy carbon electrode are described. Depending on the type of immobilization, avidin may lose its biological activity as determined by an enzyme immunoassay, using biotinylated reagents. If avidin is covalently bound to the glassy carbon electrode via the bridge molecule 4.4'-diaminodiphenylamine, the biological activity is retained. About 1.5 pmol of avidin can be bound to the electrode (3 mm in diameter), resulting in a nearly complete monolayer of protein.     

Inside-out integrin signalling.

Integrins are expressed by virtually all cells and play key roles in a range of cellular processes. Changes in the integrin surface repertoire provide a means of altering the strength and ligand preferences of cell adhesion. Recent research has examined the affinity modulation of integrins, a rapid and versatile mechanism of cell adhesion regulation. Studies with a prototype, alpha IIb beta 3, indicate that intracellular events influence the conformation and ligand-binding affinity of the extracellular domain of integrins. This 'inside-out' signal transduction appears to be mediated through the integrin cytoplasmic domains. In addition, in some cases affinity modulation of integrins may be cell-type specific. The clarification of the mechanisms of integrin affinity modulation should help explain rapid changes in cell adhesion that occur during cell migration, aggregation and the cell cycle.     

Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability.

In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within alpha-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the alpha-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions. All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (< or = 0.4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133-->Ala made alpha-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino- and carboxy-terminal domains changed by 3.6 degrees. This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.     

Monitoring and control of biotechnological production processes by Bio-FET-FIA-sensors

Summary Single and multisensor field effect transistors (FET) with a pH-sensitive Si/SiO₂/Si₃N₄/Ta₂O₅-gate and reference electrode (for single sensor) were developed and used for manufacturing the following biological (Bio)-FETs: for glucose analysis, glucose oxidase-FET (GOD-FET); for urea analysis, urease-FET; and for cephalosporin C analysis, cephalosporinase-FET. The GOD-FETs were integrated into flow injection analysis (FIA) of the Eppendorf variables analyser (EVA) system and used for monitoring the glucose concentration in microbial cultivation and production processes with recombinant Escherichia coli K12 MF, recombinant E. coli JM103, Saccharomyces cerevisiae H620, and Candida boidinii. Urease-FET-FIA was used to monitor the urea concentration in a simulated cultivation of Cephalosporium acremonium and urease-FET-FIA and GOD-FET-FIA for the monitoring of urea and glucose concentrations in simulated S. cerevisiae cultivations.     

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.     

Transcription from the adenovirus major late promoter uses redundant activating elements

The adenovirus major late promoter (MLP) has been analyzed by constructing recombinant viral genomes containing mutations in possible promoter elements. Single base pair changes in the TATA box had no effect on viral replication, and MLP expression, as measured by the accumulation of late mRNAs, was at wild type levels. However, a double mutation in the TATA box reduced viral replication and MLP expression, demonstrating that the TATA box is important, although not essential, for maximal activity in virus. Primer extension analysis showed that the mRNAs were initiated at the correct position. A mutation in the CAAT box was viable, and had only minor effects on MLP expression. However, this mutation when coupled to a single mutation in the TATA box, severely reduced viral replication and expression from the MLP. Similarly, a viable mutation in the UPE, shown previously to abolish binding of USF, coupled to a single mutation in the TATA box was lethal. These results suggest that both USF and the CAAT box binding factor CP1 can interact with TFIID to effect activation, and thus that the mechanism of activation is functionally redundant.     

The effect of the length of direct repeats and the presence of palindromes on deletion between directly repeated DNA sequences in bacteriophage T7.

The frequency of genetic deletion between directly repeated DNA sequences in bacteriophage T7 was measured as a function of the length of the direct repeat. The non-essential ligase gene (gene 1.3) of bacteriophage T7 was interrupted with pieces of synthetic DNA bracketed by direct repeats of various lengths. Deletion of these 76 bp long inserts was too low to be measured when the direct repeats were less than 6 bp long. However, the frequency of deletion of inserts with longer direct repeats increased exponentially as the length of the repeats increased from 8 to 20 bp. When inverted repeats (palindromes) were designed in the midst of the insert there was essentially no increase in deletion frequency between 10 bp direct repeats. But, the same palindromic sequences increased the deletion frequency between 5 bp direct repeats by at least two orders of magnitude. Thus, in this system homology at the endpoints is a more important determinant of deletion frequency than is the presence of palindromes between the direct repeats.     

Eliminating primers from completed polymerase chain reactions with exonuclease VII.

Single-stranded oligonucleotide primers can be efficiently removed after PCR using E.coli exonuclease VII. Even only a few molecules of double stranded PCR product are unaffected by a treatment which eliminates 20 picomoles of primer in the presence of 500 ng of denatured genomic DNA. Exonuclease VII treatment is rapid and could simplify complicated multistep PCR protocols.