Enhanced cleavage of diaminopimelate-containing isopeptides by leucine aminopeptidase and matrix metalloproteinases in tumors: application to bioadhesive peptides.

We prepared (2S,6S)-Z-Dpm(Z)(OMe) (4) by protease-mediated hydrolysis of (R,R/S,S)-Z-Dpm(Z)(OMe)-OMe (3), converted it to (2S,6S)-Dpm(Z)(OMe) (6) via PCI5 to an NCA intermediate and hydrolysis, protected the amino group with Boc to give (2S,6S)-Boc-Dpm(Z)(OMe) (7), which upon ammonolysis of the Me ester afforded (2S,6S)-Boc-Dpm(Z)(NH2) (8). Hydrogenolysis of 8 and protection with Fmoc gave (2S,6S)-Boc-Dpm(Fmoc)(NH2)(10). Using 10 and SPPS, we prepared three Dpm-containing peptides and their corresponding Lys peptides. Enzymatic studies with mLAP and cLAP showed that the Leu moiety in Ac-Gly-(2S,6S)-Dpm(Leu)(NH2)-Ala (14) was hydrolyzed 68-fold and >1000-fold more rapidly, respectively, than that in Ac-Gly-Lys(Leu)-Ala (12). The enhanced rate of Leu formation from 14 compared to 12 was also observed with homogenates of mouse C3 sarcomas. This homogenate also hydrolyzed Ac-Gly-(2S,6S)-Dpm(Ac-Gly-Pro-Gln-Gly-Leu)(NH2)-Ala (16) to Ac-Gly-(2S,6S)-Dpm(NH2)-Ala (13), Leu and Ac-Gly-Pro-Gln-Gly (17). This implies the side chain is cleaved first by endopeptidases, such as matrix metalloproteinases (MMPs), and then the remaining Leu is cleaved by LAP-like exopeptidases. The rate of liberation of 17 from 16 and the corresponding Lys isopeptide, Ac-Gly-Lys(Ac-Gly-Pro-Gln-Gly-Leu)-Ala (15), was not significantly different. The rate of formation of 13 was faster from 16 than Ac-Gly-Lys-Ala (11) was from 15. Thus, the entire isopeptide side chain can be removed by the cooperative action of LAP-like and MMP-like peptidases present in tumor tissue, which occurs faster in the Dpm peptide 16 than in the Lys peptide 15. The rate of formation of 13 from 16 by lung, liver, and intestine homogenates (from the same C3 tumor-bearing mice) was comparable to or higher than from the tumor homogenates, but the rate by blood was only 4% the value of the tumor homogenates. Analogs of a bioadhesive fragment from the laminin alpha1 chain were prepared by replacing the essential Lys with Dpm(NH2) (20) and Dpm(Leu)(NH2) (21). Both Dpm-containing peptides were active, although considerably weaker than the corresponding Lys peptides 18 and 19, in a cell attachment assay with human fibrosarcoma HT-1080 cells.     

Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30-nm chromatin structure

How a long strand of genomic DNA is compacted into a mitotic chromosome remains one of the basic questions in biology. The nucleosome fibre, in which DNA is wrapped around core histones, has long been assumed to be folded into a 30-nm chromatin fibre and further hierarchical regular structures to form mitotic chromosomes, although the actual existence of these regular structures is controversial. Here, we show that human mitotic HeLa chromosomes are mainly composed of irregularly folded nucleosome fibres rather than 30-nm chromatin fibres. Our comprehensive and quantitative study using cryo-electron microscopy and synchrotron X-ray scattering resolved the long-standing contradictions regarding the existence of 30-nm chromatin structures and detected no regular structure >11 nm. Our finding suggests that the mitotic chromosome consists of irregularly arranged nucleosome fibres, with a fractal nature, which permits a more dynamic and flexible genome organization than would be allowed by static regular structures.     

Synthesis and characterization of Pd(II) and Pt(II) complexes with triazolopyrimidine derivatives: The crystal structure of [Pd2L2Cl4] where L = 5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine

The Pd(II) and Pt(II) complexes with triazolopyrimidine C-nucleosides L¹ (5,7-dimethyl-3-(2′,3′,5′-tri-O-benzoyl-β-d-ribofuranosyl-s-triazolo)[4,3-a]pyrimidine), L² (5,7-dimethyl-3-β-d-ribofuranosyl-s-triazolo[4,3-a]pyrimidine) and L³ (5,7-dimethyl[1,5-a]-s-triazolopyrimidine), [Pd(en)(L¹)](NO₃)₂, [Pd(bpy)(L¹)](NO₃)₂, cis-Pd(L³)₂Cl₂, [Pd₂(L³)₂Cl₄] · H₂O, cis-Pd(L²)₂Cl₂ and [Pt₃(L¹)₂Cl₆] were synthesized and characterized by elemental analysis and NMR spectroscopy. The structure of the [Pd₂(L³)₂Cl₄] · H₂O complex was established by X-ray crystallography. The two L³ ligands are found in a head to tail orientation, with a Pd?Pd distance of 3.1254(17) Å. L¹ coordinates to Pd(II) through N8 and N1 forming polymeric structures. L² coordinates to Pd(II) through N8 in acidic solutions (0.1 M HCl) forming complexes of cis-geometry. The Pd(II) coordination to L² does not affect the sugar conformation probably due to the high stability of the C-C glycoside bond.     

Effect of the Novel Influenza A (H1N1) Virus in the Human Immune System

Background

The pandemic by the novel H1N1 virus has created the need to study any probable effects of that infection in the immune system of the host.

Methodology/Principal Findings

Blood was sampled within the first two days of the presentation of signs of infection from 10 healthy volunteers; from 18 cases of flu-like syndrome; and from 31 cases of infection by H1N1 confirmed by reverse RT-PCR. Absolute counts of subtypes of monocytes and of lymphocytes were determined after staining with monoclonal antibodies and analysis by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from patients and stimulated with various bacterial stimuli. Concentrations of tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-18, interferon (FN)-alpha and of IFN-gamma were estimated in supernatants by an enzyme immunoassay. Infection by H1N1 was accompanied by an increase of monocytes. PBMCs of patients evoked strong cytokine production after stimulation with most of bacterial stimuli. Defective cytokine responses were shown in response to stimulation with phytohemagglutin and with heat-killed Streptococcus pneumoniae. Adaptive immune responses of H1N1-infected patients were characterized by decreases of CD4-lymphocytes and of B-lymphocytes and by increase of T-regulatory lymphocytes (Tregs).

Conclusions/Significance

Infection by the H1N1 virus is accompanied by a characteristic impairment of the innate immune responses characterized by defective cytokine responses to S.pneumoniae. Alterations of the adaptive immune responses are predominated by increase of Tregs. These findings signify a predisposition for pneumococcal infections after infection by H1N1 influenza.     

Effects of radial growth, tree age, climate, and seed origin on wood density of diverse jack pine populations

Several models of the effects of silviculture, radial growth, and tree age on wood density have been developed, but they have rarely considered the roles of diverse seed origins and climate. We developed a model to test the effects of radial growth, tree age, climate, and seed-source origins on wood density in 21 diverse populations of jack pine in a common garden in Petawawa, Ontario, Canada over the last 24 years using a linear mixed-effects model. Although we found significant differences in wood density among diverse seed origins, there were no differences between seed origins having the same ring age and ring width, indicating an indirect effect on wood density of seed-source origin via radial growth. High variation in wood density among trees within the same population and between populations indicated high genetic control of wood density. The climate effect was significant on wood density in all populations, but smaller when radial growth was controlled. Climate effect did not differ significantly among populations. Precipitation in July negatively affected latewood density, whereas precipitation in May in the current year and September of the previous year negatively affected earlywood density. We concluded that a single model of jack pine wood density and radial growth could be used, either controlling for climate effects or not, as the relationship between wood density and radial growth is preserved among the diverse populations, and the climate effect controlling for radial growth in the model was only slight.     

Structure of reconstituted bacterial membrane efflux pump by cryo-electron tomography

Complexes of OprM and MexA, two proteins of the MexA-MexB-OprM multidrug efflux pump from Pseudomonasaeruginosa, an opportunistic Gram-negative bacterium, were reconstituted into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21 nm, separated by lipid bilayers, were obtained at stoichiometry of 1:1 (w/w). Using cryo-electron microscopy and tomography, we showed that these protein layers were composed of MexA-OprM complexes self-assembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers, representing an environment close to that of the native whole pump (i.e. anchored between outer and inner membranes of P. aeruginosa). The MexA-OprM complex appeared as a cylindrical structure in which we were able to identify the OprM molecule and the MexA moiety. MexA molecules have a cylindrical shape prolonging the periplasmic helices of OprM, and widening near the lipid bilayer. The flared part is likely composed of two MexA domains adjacent to the lipid bilayer, although their precise organization was not reachable mainly due to their flexibility. Moreover, the intermembrane distance of 21 nm indicated that the height of the bipartite complex is larger than that of the tripartite AcrA-AcrB-TolC built-up model in which TolC and AcrB are docked into contact. We proposed a model of MexA-OprM taking into account features of previous models based on AcrA-AcrB-TolC and our structural results providing clues to a possible mechanism of tripartite system assembly.     

Unexpected correlations between gene expression and codon usage bias from microarray data for the whole Escherichia coli K-12 genome

Escherichia coli has long been regarded as a model organism in the study of codon usage bias (CUB). However, most studies in this organism regarding this topic have been computational or, when experimental, restricted to small datasets; particularly poor attention has been given to genes with low CUB. In this work, correspondence analysis on codon usage is used to classify E.coli genes into three groups, and the relationship between them and expression levels from microarray experiments is studied. These groups are: group 1, highly biased genes; group 2, moderately biased genes; and group 3, AT-rich genes with low CUB. It is shown that, surprisingly, there is a negative correlation between codon bias and expression levels for group 3 genes, i.e. genes with extremely low codon adaptation index (CAI) values are highly expressed, while group 2 show the lowest average expression levels and group 1 show the usual expected positive correlation between CAI and expression. This trend is maintained over all functional gene groups, seeming to contradict the E.coli-yeast paradigm on CUB. It is argued that these findings are still compatible with the mutation-selection balance hypothesis of codon usage and that E.coli genes form a dynamic system shaped by these factors.     

Enantioselective binding of lambda- and delta-[Ru(bpy)(2)(HPIP)]Cl2 (HPIP=2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline) to the hexanucleotide [d(5'-GTCGAC-3')2

Multidimensional NMR techniques (1D (1)HNMR, 2D DQF (1)H(1)H COSY and 2D (1)H(1)H NOESY), electrospray ionization mass spectrometry (ESI-MS) and electronic spectroscopy, were performed to study the interactions of the enantiomers lambda- and delta-[Ru(bpy)(2)(HPIP)]Cl(2), (HPIP=2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthro-line) with the self complementary hexanucleotide duplex d(5'-GTCGAC-3')(2). The results show that the delta-[Ru(bpy)(2)(HPIP)]Cl(2) binds tightly to the oligonucleotide, by intercalation of the ligand HPIP, between the A5 and C6 base sequence of the same strand, probably through the minor groove. Lambda-enantiomer binds weakly, suggesting groove interactions with the hexanucleotide duplex. ESI-MS spectrometry and UV-vis spectroscopy also confirmed these observations.     

Occurrence and structural characterization of versican-like proteoglycan in human vitreous

Human vitreous gel is a special type of extracellular matrix, in which interpenetrating networks of collagen fibrils and hyaluronan are found. In this study, we report that apart from significant amounts of collagen, hyaluronan and sialylated glycoproteins, it was found that the human vitreous gel also contained low amounts of versican-like proteoglycan. The concentration of versican-like proteoglycan in the whole vitreous is 0.06 mg protein/ml of vitreous gel and represents a small percentage (about 5%) of the total protein content. The versican-like proteoglycan has a molecular mass of 380 kDa, as estimated by gel chromatography. Its core protein is substituted by chondroitin sulphate side chains (average molecular weight 37 kDa), in which 6-sulphated disaccharides predominated. According to the physicochemical data, the number of chondroitin sulphate chains is likely to be 5-7 per molecule. These proteoglycan monomers form large aggregates with endogenous hyaluronan. Versican, which is able to bind lectins via its C-terminal region, may bridge or interconnect various constituents of the extracellular matrix via its terminal domains in order to stabilize large supramolecular complexes at the vitreous, contributing towards the integrity and specific properties of the tissue.     

Compositional and structural alterations of chondroitin and dermatan sulfates during the progression of atherosclerosis and aneurysmal dilatation of the human abdominal aorta

Glycosaminoglycans participate in several biological functions in the arterial wall through their specific structures. They undergo specific compositional and structural modifications during the development of vascular diseases. The present study was performed to determine the variations in the concentration and the structural characteristics of galactosaminoglycans--chondroitin sulfate (CS) and dermatan sulfate (DS)--during the progression of atherosclerosis and aneurysmal dilatation of the human abdominal aorta. The concentration of CS was increased 24% (p < or = 0.05) in atherosclerotic type II aortas, but it was significantly decreased (29%, p < or = 0.05) in atherosclerotic type V aortas and aneurysmal aortas (65%, p < or = 0.01). In contrast, the concentration of DS was almost constant in all stages of arterial disease examined. Significant structural alterations were detected in the disaccharide composition of galactosaminoglycans. The ratio of 6-sulfated to 4-sulfated disaccharides was increased in atherosclerotic type II aortas (4.0 instead of 3.1 in normal aortas) due to the marked increase of CS in this tissue. This ratio was significantly decreased in atherosclerotic type V and aneurysmal aortas (2.1 and 1.6, respectively) due to the significant reduction of CS in the respective tissues. In addition, significant decrease of the oversulfated disaccharides, which are mainly located in DS chains, was recorded in atherosclerotic and aneurysmal aortas. Particularly, deltadi-di(2,6)S were decreased 32% (p < or = 0.01) and 86% (p < or = 0.01) in atherosclerotic type II and V aortas and 88% (p < or = 0.01) in aneurysm. Deltadi-di(2,4)S were increased in atherosclerotic type II aortas (21%, p < or = 0.01), but significantly decreased in atherosclerotic type V (33%, p < or = 0.01) and aneurysmal aortas (56%, p < or = 0.01). The amounts of deltadi-di(4,6)S were not markedly affected in the diseased tissues. These results suggest that the concentration of galactosaminoglycans is differentially affected during the progression of atherosclerosis. Furthermore, the development of vascular disease is associated with specific structural modifications, especially with the significant reduction of particular types of oversulfated disaccharides, which may play essential biological roles in the arterial wall.