Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.     

Sphingolipid trafficking and protein sorting in epithelial cells

Sphingolipids represent a minor, but highly dynamic subclass of lipids in all eukaryotic cells. They are involved in functions that range from structural protection to signal transduction and protein sorting, and participate in lipid raft assembly. In polarized epithelial cells, which display an asymmetric apical and basolateral membrane surface, rafts have been proposed as a sorting principle for apical resident proteins, following their biosynthesis. However, raft-mediated trafficking is ubiquitous in cells. Also, sphingolipids per se, which are strongly enriched in the apical domain, are subject to sorting in polarity development. Next to the trans Golgi network, a subapical compartment called SAC or common endosome appears instrumental in regulating these sorting events.     

The leucine-rich repeat protein PRELP binds perlecan and collagens and may function as a basement membrane anchor

PRELP (proline arginine-rich end leucine-rich repeat protein) is a heparin-binding leucine-rich repeat protein in connective tissue extracellular matrix. In search of natural ligands and biological functions of this molecule, we found that PRELP binds the basement membrane heparan sulfate proteoglycan perlecan. Also, recombinant perlecan domains I and V carrying heparan sulfate bound PRELP, whereas other domains without glycosaminoglycan substitution did not. Heparin, but not chondroitin sulfate, inhibited the interactions. Glycosaminoglycan-free recombinant perlecan domain V and mutated domain I did not bind PRELP. The dissociation constants of the PRELP-perlecan interactions were in the range of 3-18 nm as determined by surface plasmon resonance. As expected, truncated PRELP, without the heparin-binding domain, did not bind perlecan. Confocal immunohistochemistry showed that PRELP outlines basement membranes with a location adjacent to perlecan. We also found that PRELP binds collagen type I and type II through its leucine-rich repeat domain. Electron microscopy visualized a complex with PRELP binding simultaneously to the triple helical region of procollagen I and the heparan sulfate chains of perlecan. Based on the location of PRELP and its interaction with perlecan heparan sulfate chains and collagen, we propose a function of PRELP as a molecule anchoring basement membranes to the underlying connective tissue.     

Cre recombinase expression can result in phenotypic aberrations in plants

The cre recombinase gene was stably introduced and expressed in tomato, petunia and Nicotiana tabacum. Some plants expressing the cre gene driven by a CaMV 35S promoter displayed growth retardation and a distinct pattern of chlorosis in their leaves. Although no direct relation can be proven between the phenotype and cre expression, aberrant phenotypes always co-segregate with the transgene, which strongly suggests a correlation. The severity of the phenotype does not correlate with the level of steady-state mRNA in mature leaves, but with the timing of cre expression during organogenesis. The early onset of cre expression in tomato is correlated with a more severe phenotype and with higher germinal transmission frequencies of site-specific deletions. No aberrant phenotype was observed when a tissue-specific phaseolin promoter was used to drive the cre gene. The data suggest that for the application of recombinases in plants, expression is best limited to specific tissues and a short time frame.[12pt] Abbreviations: bar, the phosphinotricin acetyltransferase gene; CAM, chloramphenicol resistance gene; Ds 5 & Ds 3, borders of the Ds transposable element from maize forming a functional transposable element that embodies the interjacent DNA; gus, the -glucoronidase gene; gus-int, the gus gene interrupted by a plant intron; hpt, the hygromycin phosphotransferase gene; nptII, the neomycin phosphotransferase gene; ORI, bacterial origin for plasmid replication in Escherichia coli of plasmid p15A     

Molecular phylogeny and phylogeography of free-living Bryozoa (Cupuladriidae) from both sides of the Isthmus of Panama

Genetic data were used to identify Recent species of free-living bryozoans (Cupuladriidae) from both sides of the Isthmus of Panama, and to examine their phylogenetic relationships, species richness, and population structures. An approximately 480bp fragment of the 16S mitochondrial rRNA gene was sequenced from 182 individuals from Panama, the Gulf of Mexico, and El Salvador. Ten haplotype groups (Cupuladria 4, 5, and 6; Discoporella 1, 2, 3A, 3B, 3C, 7, and 8) were identified. Genetic distances between haplotype groups (3.2-26.5%; K2P+Gamma) were 1-2 orders of magnitude greater than within groups (0.1-1.4%). Seven of the haplotype groups represent morphologically distinct species; Discoporellas 3A-C appear to be cryptic species. Phylogenetic analyses identified two pairs of transisthmian sister clades. An average divergence rate derived from other taxa suggests that Cupuladrias 4 and 5 diverged approximately 7Ma, a Discoporella 7 clade diverged from a 3A-C clade approximately 11Ma, and the 3A-C clade radiated approximately 6-4Ma; these events all predated final closure of the isthmus? 3Ma. The Caribbean side of the isthmus, with 5 species, is only marginally richer in cupuladriids than the Pacific side, with 4, but has greater phylogenetic depth. The Caribbean retains lineages stemming from a New World Miocene radiation that are not represented in the eastern Pacific; extant eastern Pacific cupuladriids share most recent common ancestry with only two of the Caribbean lineages. Species in the eastern Pacific tend to show shallow population structures, with high levels of gene flow between geographically separate populations, whereas Caribbean species tend to show deeper populations structures, with indications of restricted gene flow between Bocas del Toro/Gulf of Mosquitos and Costa Arriba/San Blas. The population structures derive from Pleistocene histories and may be of limited value in interpreting the macroevolutionary pattern, as our results provide no evidence of speciation on either side of the isthmus following closure in the late Pliocene.     

Protected peptide p-nitroanilides by solid-phase synthesis

Peptide p-nitroanilides (peptide p> NAs) have found wide application as chromogenic substrates. An improved SPPS method to synthesize rapidly and in good yield a broad range of peptide pNAs under mild conditions will be presented here. To obtain a suitable carrier, the (4-aminophenyl)aminocarbonyloxy derivatives of Wang resin and Sasrin were synthesized. SPPS employing Fmoc/tBu-based protection followed by acidolytic cleavage yielded the (protected) peptide p-aminoanilide which was oxidized with sodium perborate in acetic acid to yield the(protected) peptide pNA. Side-chain protectionproved to be advantageous. Acid-labile peptide pNAs such as the proteasome substrate Z-Glu(OtBu)-Ala-Leu-pNA thus can be obtaineddirectly. The behavior of Cys, Met, Tyr and Trp being susceptible towards oxidation was studied more closely.     

Use of length heterogeneity PCR and fatty acid methyl ester profiles to characterize microbial communities in soil

In length heterogeneity PCR (LH-PCR) a fluorescently labeled primer is used to determine the relative amounts of amplified sequences originating from different microorganisms. Labeled fragments are separated by gel electrophoresis and detected by laser-induced fluorescence with an automated gene sequencer. We used LH-PCR to evaluate the composition of the soil microbial community. Four soils, which differed in terms of soil type and/or crop management practice, were studied. Previous data for microbial biomass, nitrogen and carbon contents, and nitrogen mineralization rates suggested that the microbial characteristics of these soils were different. One site received two different treatments: no-till and conventional till perennial ryegrass. The other sites were no-till continuous grass plots at separate locations with different soil types. Community composition was characterized by assessing the natural length heterogeneity in eubacterial sequences amplified from the 5' domain of the 16S rRNA gene and by determining fatty acid methyl ester (FAME) profiles. We found that LH-PCR results were reproducible. Both methods distinguished the three sites. The most abundant bacterial community members, based on cloned LH-PCR products, were members of the beta subclass of the class Proteobacteria, the Cytophaga-Flexibacter-Bacteriodes group, and the high-G+C-content gram-positive bacterial group. Strong correlations were found between LH-PCR results and FAME results. We found that the LH-PCR method is an efficient, reliable, and highly reproducible method that should be a useful tool in future assessments of microbial community composition.