Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: II. Cloning and expression in Escherichia coli

The genes for the large and small subunits of anthranilate synthase (trpE and trpG, respectively) have been cloned from Pseudomonas aeruginosa PAC174 into E. coli by R-prime formation with the broad-host- range plasmid R68.44. Sequential subcloning into plasmid vectors reduced the active Pseudomonas DNA fragment to a length of 3.1 kb. We obtained evidence that this region contains the promoter for its own expression and retains a vestigial regulatory response to tryptophan scarcity or excess.      

High affinity specific [3H] (+)PN 200-110 binding to dihydropyridine receptors associated with calcium channels in rat cerebral cortex and heart

The binding properties of the 1,4-dihydropyridine calcium channel antagonist, [³H](⁺)PN 200-110, were studied in rat cerebral cortical and cardiac homogenates (37°C, Krebs phosphate buffer). Specific binding of [³H](⁺)PN 200-110 was saturable, reversible, and of high affinity (K_d values are 35 and 64 pM for the cerebral cortex and heart, respectively). In parallel studies with [³H](⁺)PN 200-110, the dissociation constant of [³H]nitrendipine was 10–12 times higher. Substituted dihydropyridine calcium channel antagonists and agonists competitively inhibited specific [³H](⁺)PN 200-110 binding, but d-cis diltiazem enhanced and verapamil incompletely inhibited [³H](⁺)PN 200-110 binding in both the cerebral cortex and the heart. The effects of diltiazem and verapamil on [³H](⁺)PN 200-110 binding were due mainly to alterations in the dissociation constant (K_d), without alterations in the binding density (B_max). The new [³H](⁺)PN 200-110 receptor binding assay is remarkable for its low degree of nonspecific binding as compared to [³H]nitrendipine at physiological temperatures. [³H(⁺)PN 200-110 is a useful ligand for the further analysis of the dihydropyridine binding sites associated with calcium channels.     

Schiff and pseudo-Schiff reagents: the reactions and reagents of Hugo Schiff,including a classification of various kinds of histochemical reagents used to detect aldehydes

During the 1860’s, Hugo Schiff studied many reactions between amines and aldehydes, some of which have been used in histochemistry, at times without credit to Schiff. Much controversy has surrounded the chemical structures and reaction mechanisms of the compounds involved, but modern analytical techniques have clarified the picture. I review these reactions here. I used molecular modeling software to investigate dyes that contain primary amines representing eight chemical families. All dyes were known to perform satisfactorily for detecting aldehydes in tissue sections. The models verified the correct chemical structures at various points in their reactions and also determined how decolorization occurred in those with “leuco” forms. Decolorization in the presence of sulfurous acid can occur by either adduction or reduction depending on the dye. The final condensation product with aldehyde was determined to be either a C-sulfonic acid adduct on the carbonyl carbon atom or an aminal at the same atom. Based on the various outcomes, I have placed the dyes and their reactions into five categories. Because Hugo Schiff studied the reactions between aldehydes and amines with and without various acids or alcohol, it is only proper to call each of them Schiff reactions that used various types of Schiff reagents.     

Three new cardenolides from the milkweeds Asclepias eriocarpa and A. Labriformis

Asclepias eriocarpa and A. labriformis contain three new cardenolides, the structures of which have been partially assigned by their spectral properties and comparison with the known cardenolides of A. curassavica. They include labriformin (C₃₁H₃₉O₁₀NS), labriformidin (C₂₉H₃₁O₁₁) and eriocarpin.     

Phosphate groups as substrate determinants for casein kinase I action

Phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase has been shown to enhance subsequent phosphorylation by casein kinase I (Flotow, H., and Roach, P. J. (1989) J. Biol. Chem. 264, 9126-9128). In the present study, synthetic peptides based on the sequences of the four phosphorylated regions in muscle glycogen synthase were used to probe the role of substrate phosphorylation in casein kinase I action. With all four peptides, prior phosphorylation significantly stimulated phosphorylation by casein kinase I. A series of peptides was synthesized based on the NH2-terminal glycogen synthase sequence PLSRTLS7VSS10LPGL, in which phosphorylation at Ser7 is required for modification of Ser10 by casein kinase I. The spacing between the P-Ser and the acceptor Ser was varied to have 1, 2, or 3 intervening residues. The peptide with a 2-residue spacing (-S(P)-X-X-S-) was by far the best casein kinase I substrate. When the P-Ser residue at Ser7 was replaced with P-Thr, the resulting peptide was still a casein kinase I substrate. However, substitution of Asp or Glu residues at Ser7 led to peptides that were not phosphorylated by casein kinase I. Phosphorylation of one of the other peptides showed that Thr could also be the phosphate acceptor. From these results, we propose that there are substrates for casein kinase I for which prior phosphorylation is a critical determinant of protein kinase action. In these instances, an important recognition motif for casein kinase I appears to be -S(P)/T(P)-Xn-S/T- with n = 2 much more effective than n = 1 or n = 3. Thus, casein kinase I may be involved in hierarchal substrate phosphorylation schemes in which its activity is controlled by the phosphorylation state of its substrates.     

Macromolecular changes caused by formalin fixation and antigen retrieval

Although the mechanics of formalin fixation and antigen retrieval have been studied extensively and reviewed periodically, little attention has been directed toward conformational changes in target molecules. Formaldehyde changes the shape of tissue molecules by appending small hydroxymethyl groups to them. These adducts, in turn, can react with other tissue molecules to form crosslinks, or they can participate in a variety of reactions during tissue processing, including formation of imines, ethoxymethyl adducts, and further crosslinks. Under the influence of alcohol dehydration, fixed DNA may fragment and form a variety of depurination products. The situation becomes even more complex with short fixation times because under these conditions, the dehydrating agent used for tissue processing denatures macromolecules in other ways, most notably through rearrangement of molecular shape to move hydrophobic realms outward and hydrophilic areas inward (hydrophobic inversions). How tissue molecules are modified affects the outcome of immunohistochemical staining and prospects for restoration of antigenicity. Immunoreacitivity may be compromised because epitopes are either sterically hidden, but otherwise unaffected, or they have been altered more directly. Enzyme-based retrieval methods are best suited for the former because they literally snip the molecule apart to reveal the portions of interest. Heat-induced retrieval with buffers can demodify affected epitopes by removing adducts and breaking crosslinks. The choice of temperature and pH is usually critical for optimal retrieval. Effective temperatures are directly related to the strength of bonds-higher temperatures are needed to break stronger bonds. The pH of the retrieval solution determines the charge on the tissue molecule; the goal is to create a charge that causes the demodified molecule to assume a near natural conformation. Rational use of these concepts should lead to better control of immunohistochemical reactions.     

Dyes from a twenty-first century perspective

In June 2008, the Biological Stain Commission sponsored A Seminar on Dyes and Staining the purpose of which was twofold: first, to show that very useful information applicable to biomedical dyes and staining is available from unrelated disciplines and second, to summarize modern thinking on how dyes, solvents, and tissues interact to produce selective staining. In this introduction to the papers from the symposium, we acknowledge that biomedical dye research has declined as newer technologies have gained importance. We should point out, however, that dyes and staining still are vitally important. Moreover, needs abound for innovative studies concerned with dye analysis, synthesis, and mode of action. Concepts and tools from unrelated fields hold promise for significant breakthroughs in many areas of interest.     

Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages

Background  

Exported proteases are commonly associated with virulence in bacterial pathogens, yet there is a paucity of information regarding their role in Mycobacterium tuberculosis. There are five genes (mycP1-5) present within the genome of Mycobacterium tuberculosis H37Rv that encode a family of secreted, subtilisin-like serine proteases (the mycosins). The gene mycP1 (encoding mycosin-1) was found to be situated 3700 bp (four ORF's) from the RD1 deletion region in the genome of the attenuated vaccine strain M. bovis BCG (bacille de Calmette et Guérin) and was selected for further analyses due to the absence of expression in this organism.     

Epigenetic reprogramming in the mammalian embryo: struggle of the clones

During preimplantation development in mammals, distinct epigenetic marks on oocyte and sperm DNA are remodeled to an embryonic pattern. A recent study examining global methylation of repetitive elements in various mammals showed that the reprogramming that occurs during normal preimplantation development is aberrant in cloned embryos.