Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.     

1,2,5-Thiadiazolidin-3-one 1,1-dioxide-based heterocyclic sulfides are potent inhibitors of human tryptase

We describe herein the design, synthesis, and in vitro biochemical evaluation of a series of potent, time-dependent inhibitors of the mast cell-derived serine protease tryptase. The inhibitors were readily obtained by attaching various heterocyclic thiols, as well as a basic primary specificity residue P₁, to the 1,2,5-thiadiazolidin-3-one 1,1-dioxide scaffold. The inhibitors were found to be devoid of any inhibitory activity toward a neutral (elastase) or cysteine (papain) protease, however they were also fairly efficient inhibitors of bovine trypsin. The differential inhibition observed with trypsin suggests that enzyme selectivity can be optimized by exploiting differences in the S′ subsites of the two enzymes. The results described herein demonstrate the versatility of the heterocyclic scaffold in fashioning mechanism-based inhibitors of neutral, basic, and acidic (chymo)trypsin-like serine proteases.     

Amino-terminally truncated Abeta peptide species are the main component of cotton wool plaques

Cotton wool plaques (CWPs) are round lesions that lack a central amyloid core. CWPs have been observed in individuals affected by early-onset familial Alzheimer disease (FAD) associated with mutations in the presenilin 1 (PSEN1) gene. Here we present the characterization of the amyloid-beta (Abeta) peptides deposited in the brain of an individual affected by FAD carrying the novel missense (V261I) mutation in the PSEN1 gene. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to determine the Abeta peptide species present in the cerebral and cerebellar cortices, in leptomeningeal vessels, and in CWPs isolated by laser microdissection (LMD). Our results indicate that amino-terminally truncated Abeta peptide species ending at residues 42 and 43 are the main Abeta peptides deposited in brain parenchyma and LMD-CWPs in association with the PSEN1 V261I mutation. Full-length Abeta1-42 and Abeta1-43 peptide species were underrepresented. CWPs were not found to be associated with vessels and did not contain Abeta1-40 peptides, the main component of the vascular deposits. Although Abeta deposits were present mostly in the form of CWPs in the cerebral cortex and as diffuse deposits in the cerebellar cortex, a similar array of amino-terminally truncated Abeta peptide species was seen in both cases. The biochemical data support the concept that parenchymal and vascular amyloid deposits are associated with a different array of Abeta peptide species. The generation and parenchymal deposition of highly insoluble amino-terminally truncated Abeta peptides may play an important role in the pathogenesis of AD and must be taken into consideration in developing new diagnostic and therapeutic strategies.     

A targeted mass spectrometric analysis of phosphatidylinositol phosphate species

The development of a new mass spectrometric lipid profiling methodology permits the identification of cellular phosphatidylinositol monophosphate/phosphatidylinositol bisphosphate/phosphatidylinositol trisphosphate (PIP/PIP2/PIP3) species that includes the fatty acyl composition. Using electrospray ionization mass spectrometry, we were able to resolve and identify 28 PIP and PIP2 compounds as well as 8 PIP3 compounds from RAW 264.7 or primary murine macrophage cell extracts. Analysis of PIP profiles after agonist stimulation of cells revealed the generation of differential PIP3 species and permitted us to propose a novel means for regulation and specificity in signaling through PIP3. This is the first reported identification of intact, cellular PIP3 by mass spectral analysis. The ability to analyze the fatty acyl chain composition of signaling lipids initiates new venues for investigation of the processes by which specific polyphosphoinositide species mediate.     

ELECTRON MICROSCOPY OF LYSOSOME-RICH FRACTIONS FROM RAT LIVER

A preliminary electron microscope study has revealed the presence in lysosome-rich fractions, isolated from rat liver, of hitherto undescribed cytoplasmic particles, called "dense bodies." Approximately 0.37 µ in length, the dense bodies often possess an internal cavity and external membrane. They contain many electron-dense granules 55 to 77 A, or less, in diameter. Such dense bodies are also visible in electron micrographs of parenchymatous cells in liver sections. The correlations between dense bodies and lysosomes are listed, but until pure preparations are available it is not possible to assert that dense bodies and lysosomes are identical.     

Discovery of the catalytic function of a putative 2-oxoacid dehydrogenase multienzyme complex in the thermophilic archaeon Thermoplasma acidophilum

Those aerobic archaea whose genomes have been sequenced possess a single 4-gene operon that, by sequence comparisons with Bacteria and Eukarya, appears to encode the three component enzymes of a 2-oxoacid dehydrogenase multienzyme complex. However, no catalytic activity of any such complex has ever been detected in the Archaea. In the current paper, we have cloned and expressed the first two genes of this operon from the thermophilic archaeon, Thermoplasma acidophilum. We demonstrate that the protein products form an alpha2beta2 hetero-tetramer possessing the decarboxylase catalytic activity characteristic of the first component enzyme of a branched-chain 2-oxoacid dehydrogenase multienzyme complex. This represents the first report of the catalytic function of these putative archaeal multienzyme complexes.     

Saccharomyces cerevisiae SSD1-V confers longevity by a Sir2p-independent mechanism

The SSD1 gene of Saccharomyces cerevisiae is a polymorphic locus that affects diverse cellular processes including cell integrity, cell cycle progression, and growth at high temperature. We show here that the SSD1-V allele is necessary for cells to achieve extremely long life span. Furthermore, addition of SSD1-V to cells can increase longevity independently of SIR2, although SIR2 is necessary for SSD1-V cells to attain maximal life span. Past studies of yeast aging have been performed in short-lived ssd1-d strain backgrounds. We propose that SSD1-V defines a previously undescribed pathway affecting cellular longevity and suggest that future studies on longevity-promoting genes should be carried out in long-lived SSD1-V strains.     

The low density lipoprotein receptor-related protein LRP is regulated by membrane type-1 matrix metalloproteinase (MT1-MMP) proteolysis in malignant cells

We demonstrate that the presentation of LRP and the subsequent uptake of its ligands by malignant cells are both strongly regulated by MT1-MMP. Because LRP is essential for the clearance of multiple ligands, these findings have important implications for many pathophysiological processes including the pericellular proteolysis in neoplastic cells as well as the fate of the soluble matrix-degrading proteases such as MMP-2. MT1-MMP is a key protease in cell invasion and a physiological activator of MMP-2. Cellular LRP consists of a non-covalently associated 515-kDa extracellular alpha-chain (LRP-515) and an 85-kDa membrane-spanning beta-chain, and plays a dual role as a multifunctional endocytic receptor and a signaling molecule. Through the capture and uptake of several soluble proteases, LRP is involved in the regulation of matrix proteolysis. LRP-515 associates with the MT1-MMP catalytic domain and is highly susceptible to MT1-MMP proteolysis in vitro. Similar to MT1-MMP, the metalloproteinases MT2-MMP, MT3-MMP and MT4-MMP also degrade LRP. The N-terminal and C-terminal parts of the LRP-515 subunit are resistant and susceptible, respectively, to MT1-MMP proteolysis. In cells co-expressing LRP and MT1-MMP, the proteolytically competent protease decreases the levels of cellular LRP and releases its N-terminal portion in the extracellular milieu while the catalytically inert protease co-precipitates with LRP. These events implicate MT1-MMP, not only in the activation of MMP-2, but also in the mechanisms that control the subsequent fate of MMP-2 in cells and tissues.