Reversible coordinative bonds in molecular recognition.
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Journal Article↗
Biomedical subjects
Michael Kruppa
Publications and source records attributed to Michael Kruppa.
Two-component signal transduction in human fungal pathogens.
Signal transduction pathways provide mechanisms for adaptation to stress conditions. One of the most studied of these pathways is the HOG1 MAP kinase pathway that in Saccharomyces cerevisiae is used to adapt cells to osmostress. The HOG1 MAPK has also been studied in Candida albicans, and more recently observations on the Hog1p functions have been described in two other human pathogens, Aspergillus fumigatus and Cryptococcus neoformans. The important, but not surprising, concept is that this pathway is used for different yet similar functions in each of these fungi, given their need to adapt to different environmental signals. Current studies of C. albicans focus upon the identification of two-component signal proteins that, in both C. albicans and S. cerevisiae, regulate the HOG1 MAPK. In C. albicans, these proteins regulate cell wall biosynthesis (and, therefore, adherence to host cells), osmotic and oxidant adaptation, white-opaque switching, morphogenesis, and virulence of the organism.
Adaptation, Physiological↗
Enhanced peptide beta-sheet affinity by metal to ligand coordination.
A histidine-coordinating metal complex substituted with methoxypyrrole amino acids (MOPAS) as a peptide beta-sheet binder shows high affinity for a H(2)N-His-Leu-Leu-Val-Phe-OMe pentapeptide in DMSO solution. Within the complex, a beta-sheet conformation is induced into the pentapeptide by weak intra-assembly interactions with the MOPAS units.
Amino Acid Sequence↗
A luminescent receptor with affinity for N-terminal histidine in peptides in aqueous solution.
Crown ethers of suitable size are the perfect artificial host compounds for ammonium ion binding, but the rather low affinity in aqueous solution prevents their use at physiological conditions. We report here the synthesis and properties of a luminescent benzo crown ether with a pendant copper imidodiacetic acid complex, which coordinates with high affinity to histidine. The emission intensity of the benzo crown ether increases significantly in the presence of ammonium ions in methanol. At physiological conditions in buffered water at pH 7.5 these interactions are too weak to be detected. If an ammonium ion and an imidazole moiety are present in the analyte, such as in His-Lys-OMe or His-OMe, high binding affinity in aqueous solution is restored. The binding event is signaled by an increase in emission intensity, which can even be observed with the naked eye. This allows the selective detection of small peptides containing N-terminal histidine or histidine among all other amino acids at physiological conditions.
Copper↗
Sixteen-detector row CT angiography for lower-leg arterial occlusive disease: analysis of section width.
Institutional review board approval and written informed consent from all patients were obtained. Diagnostic accuracy of three reconstructions of 16-detector row computed tomographic (CT) angiography data with different section widths and increments (2.0 and 1.0 mm [CT data set 1], 1.0 and 0.5 mm [CT data set 2], and 0.75 and 0.4 mm [CT data set 3]) was compared with that of digital subtraction angiography (DSA) in 163 arterial segments in 17 patients with occlusive peripheral arterial disease (PAD). Arterial visibility was superior with CT as compared with DSA (P < .008). Sensitivity for stenosis detection did not differ between the CT reconstructions, whereas specificity was significantly improved when CT data set 3 was used (P < .017). Stenosis length did not differ significantly between CT angiography and DSA. Accuracy of stenosis detection was 88.2%, 90.8%, and 96.1% with CT data sets 1, 2, and 3, respectively. CT angiography has excellent diagnostic accuracy in the assessment of lower-leg PAD provided that the thinnest possible section width is used.
Aged↗
The histidine kinases of Candida albicans: regulation of cell wall mannan biosynthesis.
Previously, we have used both biochemical and immunological approaches to determine that the two-component, histidine kinase Chk1p regulates cell wall biosynthesis in Candida albicans. These data were obtained by comparing wild-type cells to a strain of C. albicans deleted in CHK1. The dysregulation of cell wall biosynthesis in the mutant reduces its adherence to human esophageal tissue and results in avirulence. In the current study, we used transmission immune electron microscopy (IEM) to visualize the cell surface of both wild-type (CAF2) and the chk1 mutant (CHK21). IEM was performed using two IgM monoclonal antibodies to either an acid-stable mannan epitope (Mab B6) or to an acid-labile mannan epitope (Mab B6.1). We observed that the cell surface of the CHK21 mutant was more reactive than wild-type cells with Mab B6, while the reactivity of Mab B6.1 was similar for both CAF2 and CHK21. These observations correlate with previous data on the Western blotting of mutant and wild-type cells using the same monoclonal antibodies, i.e., greater activity with Mab B6 than with Mab B6.1. In addition to CHK1, two other histidine kinases (SLN1 and NIK1) have been described in C. albicans. Mutants in both sln1Delta and nik1Delta were compared by Western blotting using Mab B6 and Mab B6.1. Reactivity of each mutant to Mab B6 was similar to that observed with the chk1 mutant; on the other hand, the mannoprotein profiles obtained with Mab B6.1 in all mutants were similar to wild-type cells. We also compared the expression of 29 genes involved in mannan synthesis by reverse transcription-polymerase chain reaction (RT-PCR) and found that expression of a subset of six genes (ALG2, ALG6, ALG8, MNT3, PMT6, KRT2) was upregulated in all histidine kinase mutants, while increased expression of ALG7 was only observed in the sln1 and nik1 mutants, MNN1 was upregulated in the chk1 and nik1 mutants, and MNN4 was upregulated in the nik1Delta. Our data indicate that each of the C. albicans HK proteins may regulate similar functions in cell wall biosynthesis. This activity could be achieved in either a common or parallel, redundant signal transduction pathway(s).
Blotting, Western↗
The two-component signal transduction protein Chk1p regulates quorum sensing in Candida albicans.
Regulation of hyphal morphogenesis in Candida albicans can occur through quorum sensing (QS). A QS signal, farnesol, is produced during high-density growth and inhibits morphogenesis. However, the signal transduction pathway that regulates QS is unknown. Here, we show that a C. albicans mutant lacking Chk1p but not either the Sln1p or the Nik1p histidine kinase is refractory to the inhibitory effect of farnesol both in cell suspension and during the formation of a biofilm. This study is the first to demonstrate a role for a two-component signal transduction protein in QS by a eukaryotic organism.
Biofilms↗
The role of the Candida albicans histidine kinase [CHK1) gene in the regulation of cell wall mannan and glucan biosynthesis.
The human pathogen Candida albicans encodes at least three putative two-component histidine kinase signal transduction proteins, including Chk1p and a response regulator protein (Cssk1p). Strains deleted in CHK1 are avirulent in a murine model of hematogenously disseminated disease. The specific function of Chk1p has not been established, but hyphae of the chk1 mutant exhibit extensive flocculation while yeast forms are less adherent to reconstituted human esophageal tissue, indicating that this protein may regulate cell surface properties. Herein, we analyze glucan, mannan and chitin profiles in strains deleted in chk1 (CHK21) compared to a gene-reconstituted strain (CHK23) and a parental strain CAF2. Total alkali-soluble hexose from the cell wall of the chk1 mutant (strain CHK21) was significantly reduced. Western blots of cell wall extracts from CHK21, CHK23 and CAF2 reacted with a Mab to the acid-stable mannan fraction revealed extensive staining of lower molecular mass species in strain CHK21 only. FACE (fluorophore assisted carbohydrate electrophoresis) was used to characterize the oligosaccharide side chains of beta-eliminated (O-linked), acid-hydrolyzed (acid-labile phosphomannan) and acetolysis (acid-stable mannan) extracted fractions of total mannan. The profiles of O-linked as well as the acid-labile oligosaccharides were similar in both CAF2 and CHK21, but the acid-stable oligosaccharide side chains were significantly truncated. We also characterized the beta-glucan from each strain using NMR, and found that both the degree of polymerization and the ratio of (1-3)/(1-6) linkages was lower in CHK21 relative to wild-type cells. The sensitivity of CHK21 to antifungal drugs and inhibitors was unaffected. In summary, our data have identified a new function for a histidine kinase two-component signal protein in a human pathogenic fungus.
Candida albicans↗
NADH model systems functionalized with Zn(II)-cyclen as flavin binding site-structure dependence of the redox reaction within reversible aggregates.
The relative positions and conformations of the prosthetic group FAD and the cofactor NADH have been remarkably conserved within the structurally diverse group of flavin enzymes. To provide a chemical rational for such an obviously optimal relative disposition of the redox partners for efficient reaction we have synthesized NADH models with Zn(II)-cyclen substituents for reversible flavin binding in water. Altogether, four of these model systems with systematically varying spacer length between the recognition site and the redox active dihydronicotinamide were prepared. The binding of these model systems to riboflavin tetraacetate was confirmed by potentiometric pH titration in water and their reaction with flavin was followed by UV-vis spectroscopy in aqueous media under physiological conditions. The measurements reveal a significant rate enhancement of up to 175 times that of an intermolecular reaction. Moreover, a strong dependence of the reaction rate on the spacer length was observed, which clearly shows that within the dynamic reversible assembly only the optimal relative disposition of the redox partners ensures an efficient redox reaction.
Binding Sites↗