Human immune cells express ppMCH mRNA and functional MCHR1 receptor

The ubiquitously expressed Na(+)/H(+) exchanger (NHE1) plays an important role in the regulation of the intracellular pH. Induction of NHE activity by phorbol esters and inhibition of growth factor-mediated stimulation of the NHE by protein kinase C (PKC) inhibitors suggest an implication of PKCs in the regulation of the NHE. Expression of PKC isotype-specific dominant negative and constitutively active mutants or downregulation of PKC by isotype-specific antisense oligonucleotides revealed that stimulation by epidermal growth factor (EGF) or phorbol ester of the NHE in NIH3T3 cells is a PKC(alpha)-specific effect. Elevation of cytoplasmic calcium by a Ca(2+) ionophore or thapsigargin causes a growth factor-independent stimulation of the NHE predominantly mediated by calcium/calmodulin kinase II. It is concluded that in NIH3T3 cells overexpressing the EGF receptor (EGFR6 cells), EGF requires cPKC(alpha) for the activation of the NHE, while calcium/calmodulin-dependent kinases are essential in thapsigargin induced stimulation of the NHE.     

Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree (Euonymus europaeus L.)

A small 45 amino acid residue antifungal polypeptide was isolated from the bark of spindle tree (Euonymus europaeus L.). Though the primary structure of this so-called E. europaeus chitin-binding protein or Ee-CBP is highly similar to the hevein domain, it distinguishes itself from most previously identified hevein-type antimicrobial peptides (AMP) by the presence of two extra cysteine residues that form an extra disulfide bond. Due to these five disulfide bonds Ee-CBP is a remarkably stable protein. Agar diffusion and microtiterplate assays demonstrated that Ee-CBP is a potent antimicrobial protein. IC₅₀-values as low as 1 μg/ml were observed for the fungus Botrytis cinerea. Comparative assays further demonstrated that Ee-CBP is a stronger inhibitor of fungal growth than Ac-AMP2 from Amaranthus caudatus seeds, which is considered one of the most potent antifungal hevein-type plant proteins.     

Biochemical, molecular and structural analysis of multiple thaumatin-like proteins from the elderberry tree (Sambucus nigra L.)

Thaumatin-like proteins (TLPs) were isolated and characterized from fruits and leaves of elderberry (Sambucus nigra) and their corresponding genes cloned. In addition, the developmental regulation and induction of the different TLPs was followed in some detail. Ripening berries accumulated a fruit-specific TLP during the final stages of maturation. This fruit-specific TLP had no antifungal activity and was devoid of beta-glucanase activity. Leaves constitutively expressed a TLP that closely resembled the fruit-specific homologue. Treatment with jasmonate methyl ester induced two additional TLPs in leaves but did not induce or enhance the expression of TLPs in immature berries. In contrast to jasmonate methyl ester, both ethephon and garlic extract induced the expression of a TLP in unripe berries that normally do not express any TLP. Sequence analysis and molecular modeling indicated that all elderberry thaumatin-like proteins share a high sequence similarity with group-5 pathogenesis-related proteins. However, the proteins encoded by the different sequences differed from each other in isoelectric point and the distribution of the charges on the surface of the molecule.     

Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins

Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.     

A murine gene with circadian expression revealed by transposon insertion: self-sustained rhythmicity in the liver and the photoreceptors

We have previously identified in some mouse strains (e.g. BALB/c, DBA/2) a murine Intracisternal A-particle (IAP) transposable element specifically expressed in the liver. This IAP sequence is inserted within a gene, mCCR4/m. nocturnin, the sequence of which is related to the circadian Xenopus nocturnin gene. Here we show, using real-time quantitative RT-PCR, that both the IAP sequence and the m. nocturnin gene display strong circadian expression in the liver, with peak abundance after dusk. Circadian oscillations of m. nocturnin RNA are maintained in mice without the IAP insertion (e.g. CBA/J, 129/sv), are free-running under constant light and dark conditions, and persist upon food and water privation, demonstrating that m. nocturnin is a circadian gene. In situ hybridization analyses (in 129/sv mice) further show circadian expression of m. nocturnin also in the retina, precisely at the level of the photoreceptors, a result consistent with the previously described circadian expression of the Xenopus gene. These results strengthen the strong conservation of the nocturnin gene with the identification of a functional mouse ortholog of the Xenopus gene, and demonstrate the reciprocal influence of nearby genes on the expression of transposable elements via "position effects".     

Regulation of yeast actin behavior by interaction of charged residues across the interdomain cleft

His(73) participates in the regulation of the nucleotide binding cleft conformation in yeast actin. Earlier molecular dynamics studies suggested that Asp(184) interacts with His(73) thereby stabilizing a "closed-cleft" G-actin. However, beta-actin in the open-cleft state shows a closer interaction of His(73) with Asp(179) than with Asp(184). We have thus assessed the relative importance of Asp(184) and Asp(179) on yeast actin stability and function. Neutral substitutions at 184 or 179 alone had little adverse effect on the monomer and polymerization behavior of actin. Arg or His at 184 in H73E actin partially rescued the monomeric properties of H73E actin, as demonstrated by near-normal thermostability and wild-type (WT)-like protease digestion patterns. ATP exchange was still considerably faster than with WT-actin although slower than that of H73E alone. However, polymerization of H73E/D184R and H73E/D184H is worse than with H73E alone. Conversely, D179R rescued all monomeric properties of H73E to near WT values and largely restored polymerization rate and filament thermostability. These results and new simulations of G-actin in the "open" state underscore the importance of the His(73)-Asp(179) interaction and suggest that the open and not the closed state of yeast actin may be favored in the absence of the methyl group of His(73).     

Stereoselective microbial dehalorespiration with vicinal dichlorinated alkanes

The suspected carcinogen 1,2-dichloroethane (1,2-DCA) is the most abundant chlorinated C(2) groundwater pollutant on earth. However, a reductive in situ detoxification technology for this compound does not exist. Although anaerobic dehalorespiring bacteria are known to catalyze several dechlorination steps in the reductive-degradation pathway of chlorinated ethenes and ethanes, no appropriate isolates that selectively and metabolically convert them into completely dechlorinated end products in defined growth media have been reported. Here we report on the isolation of Desulfitobacterium dichloroeliminans strain DCA1, a nutritionally defined anaerobic dehalorespiring bacterium that selectively converts 1,2-dichloroethane and all possible vicinal dichloropropanes and -butanes into completely dechlorinated end products. Menaquinone was identified as an essential cofactor for growth of strain DCA1 in pure culture. Strain DCA1 converts chiral chlorosubstrates, revealing the presence of a stereoselective dehalogenase that exclusively catalyzes an energy-conserving anti mechanistic dichloroelimination. Unlike any known dehalorespiring isolate, strain DCA1 does not carry out reductive hydrogenolysis reactions but rather exclusively dichloroeliminates its substrates. This unique dehalorespiratory biochemistry has shown promising application possibilities for bioremediation purposes and fine-chemical synthesis.     

Strain-specific ureolytic microbial calcium carbonate precipitation

During a study of ureolytic microbial calcium carbonate (CaCO(3)) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO(3) crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected. Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (K(m)) and maximum hydrolysis rates (V(max)) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium.