The group B streptococcal beta and pneumococcal Hic proteins are structurally related immune evasion molecules that bind the complement inhibitor factor H in an analogous fashion

Complement evasion by different mechanisms is important for microbial virulence and survival in the host. One strategy used by pathogenic bacteria is to bind the soluble complement inhibitor factor H (fH) to their surfaces. In group B streptococci and pneumococci, fH binding has been shown to be mediated by the surface proteins beta and Hic, respectively. We showed previously that Hic binds to the middle region of fH and protects the pneumococcus from opsonophagocytosis. As the beta protein and Hic are structurally closely related, we wanted to compare the fH binding characteristics of these two proteins. By using direct binding assays with radiolabeled proteins and surface plasmon resonance analysis we show that both beta and Hic bind to the short consensus repeats 8-11 and 12-14 in the middle region of fH. Peptide mapping analysis suggested that the fH-binding sites on beta and Hic were composed of discontinuous and partially homologous sequences. Thus, the bacterial virulence proteins use multiple binding sites on fH to secure high avidity. Also, the functionally active sites on fH are thereby left free to inhibit C3b deposition and opsonophagocytosis. These results reveal the evolutionary conservation of an analogous immune evasion strategy in different types of pathogenic streptococci. Importantly, the respective virulence factors could be exploited in the development of protein-based vaccines against these pathogens.     

Interdomain contact regions and angles between adjacent short consensus repeat domains

The short consensus repeat domain (SCR, complement control protein module, sushi-domain) is a structural unit found in multiple adjacent copies in more than 40 human proteins. Each bead-like domain is composed of approximately 60 residues and the adjacent domains are connected in a head-to-tail fashion with linkers that consist of two to 12 amino acid residues. Based on experimentally determined structures the neighbouring SCR domains interact with each other at the so-called hinge or interdomain contact region. The functions mediated by the SCR domains have been studied using mutagenesis but the possible effects of the mutations on the hinge regions and interdomain angles have not been analysed. In this study, the linker and three loops in conserved locations were found to be responsible for the interdomain contact regions of all the solved experimental structures. The interdomain contact regions were identified in sequences of 140 human SCR domain pairs, and distinct hydrophobic and charge features were found in different subsets of SCR proteins and functional domains. To compare the possible associations of the interdomain contact region characteristics to the interdomain orientations all the experimentally solved SCR structures were subjected to a uniform calculation of tilt, twist, and skew angles that define the interdomain orientation. The twist and skew angles were found to have a linear correlation and the spatial location of one loop of the N-terminal domain (N#1) was found to have an effect on the skew angle. Thus, we describe location of the interdomain contact regions in primary structures of SCR domains and report that the orientation of adjacent SCR domains is not random and depends partially on the interdomain contact regions. On the basis of these results, mutations within the interdomain contact regions and subsequent loss-of-function effects caused by changes in the interdomain orientation can be avoided in mutagenesis studies.     

Ring nitrogen-substituted non-steroidal estrogens: pyridine and pyrimidine analogs of the phenol in deoxyhexestrol experience resonance constraints on preferred ligand conformation

To develop compounds selective for estrogen receptor beta (ERbeta), we substituted hydroxypyridine and pyrimidine heteroaryl groups for the characteristic phenol ring of non-steroidal estrogens. The unexpectedly low affinity showed by some of these compounds is ascribed, in part, to a resonance-enforced conformational constraint that prevents their optimal accommodation in the ER ligand binding pocket.     

Impact of mobile clinical analyzers on disaster medicine: a lesson from crush syndrome in the 1995 Hanshin-Awaji earthquake

The Great Hanshin-Awaji earthquake caused many people to develop crush syndrome. Analysis of these patients revealed that the severity is not related to their hemodynamics but to hematocrit, base deficits, and potassium concentrations soon after their extrications. In the disaster site, these parameters can only be measured using whole-blood samples by mobile instruments. The present study was made to evaluate the possibility of uses of a mobile measuring device, ABL77, for the triage of crush syndrome patients in disaster sites. Heparin-added blood samples and serum samples were collected from patients admitted to Senshu Critical Care Medical Center. Blood gases, electrolytes, and hematocrit of the heparin-added blood samples were measured using ABL77 and compared with those measured using the stationary device ABL725. Potassium concentrations of the heparin-added blood samples measured by the ABL77 were compared with those of the serum samples measured by the stationary EA06T. Significant correlations were observed between the measurements. We conclude that the ABL77 was satisfactorily compatible with stationary devices in the hospital. Medical institutions should have simplified, mobile measuring devices as a precaution against disasters, so that they can get ready to take appropriate action promptly.     

Expression and characterization of biologically active human Fas ligand produced in CHO cells

We describe an expression system for high-yield production of recombinant soluble human FasL (rsh-FasL) in CHO cells. After one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 μg/L × 10⁶ cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures. The protein secreted into growth medium was purified by immunoaffinity. The rsh-FasL thus obtained was further fractionated by gel filtration and a form of approx 140 kDa was isolated and characterized. Mass spectral analysis yielded a main peak of 28,321.15 Da, while, although to a lesser extent, dimeric and trimeric forms were also detected according to the described oligomerized state of native FasL. Our procedure permits consistent production of biologically active rsh-FasL as shown in tests on FasL-sensitive cells and in in vitro binding assays. S. Zappitelli and L. D’Alatri contributed equally to this work.     

Complement C3b/C3d and cell surface polyanions are recognized by overlapping binding sites on the most carboxyl-terminal domain of complement factor H

Factor H (FH) is a potent suppressor of the alternative pathway of C in plasma and when bound to sialic acid- or glycosaminoglycan-rich surfaces. Of the three interaction sites on FH for C3b, one interacts with the C3d part of C3b. In this study, we generated recombinant constructs of FH and FH-related proteins (FHR) to define the sites required for binding to C3d. In FH, the C3d-binding site was localized by surface plasmon resonance analysis to the most C-terminal short consensus repeat domain (SCR) 20. To identify amino acids of FH involved in binding to C3d and heparin, we compared the sequences of FH and FHRs and constructed a homology-based molecular model of SCR19-20 of FH. Subsequently, we created an SCR15-20 mutant with substitutions in five amino acids that were predicted to be involved in the binding interactions. These mutations reduced binding of the SCR15-20 construct to both C3b/C3d and heparin. Binding of the wild-type SCR15-20, but not the residual binding of the mutated SCR15-20, to C3d was inhibited by heparin. This indicates that the heparin- and C3d-binding sites are overlapping. Our results suggest that a region in the most C-terminal domain of FH is involved in target recognition by binding to C3b and surface polyanions. Mutations in this region, as recently reported in patients with familial hemolytic uremic syndrome, may lead to indiscriminatory C attack against self cells.     

The Chapman-Enskog procedure for an age-structured population model: initial, boundary and corner layer corrections

We consider a mathematical model of an age-structured population of some fisheries (for example, anchovies, sardines or soles). Two time scales are involved in the problem: the fast time scale for the migration dynamics and the slow time scale for the demographic process. At a first step, we study the so called 'aggregated' system by means of the semigroups theory. Then, we study the asymptotic behaviour of the model by using the Chapman-Enskog procedure. In particular, we study initial, boundary and corner layer effects in order to obtain the exact initial and boundary conditions the approximated solution has to satisfy.     

Ultrastructure of the vegetative cell ofBrassica napus pollen with particular reference to microbodies

Summary The ultrastructure of the vegetative cell ofBrassica napus tricellular pollen grains, just before anthesis with standard chemical fixation, is reported. The vegetative cell may be regarded as a highly differentiated and metabolically active fat-storage cell. It contains many mitochondria with a well developed internal membrane system, starchless plastids, microbodies, lipid bodies, dictyosomes and numerous vesicles thought to originate from the dictysomes. Rough endoplasmic reticulum organized in stacks of cisternae is also spatially associated with certain organelles, mainly lipid bodies, microbodies and plastids. There are also randomly distributed polyribosome areas. The microbodies are mainly polymorphic in shape and are often observed in contact with lipid bodies. The above spatial relationship implies that the microbodies may have a glyoxysomal function. In the late period of vegetative cell maturation, the microbodies are probably involved in the process of glyconeogenesis in which the conversion of lipid reserves to sugar takes place.Abbreviations VC vegetative cell - VN vegetative nucleus - SC sperm cell - M mitochondria - MB microbodies - L lipid body - P plastid - D dictyosomes     

Y402H polymorphism of complement factor H affects binding affinity to C-reactive protein

Complement factor H (FH) is an important regulator of the alternative complement pathway. The Y402H polymorphism within the seventh short consensus repeat of FH was recently shown to be associated with age-related macular degeneration, the most common cause of irreversible blindness in the Western world. We examined the effects of this polymorphism on various FH functions. FH purified from sera of age-related macular degeneration patients homozygous for the FH(402H) variant showed a significantly reduced binding to C-reactive protein (CRP), an acute phase protein, as compared with FH derived from unaffected controls homozygous for the FH(402Y) variant. Strongly reduced binding to CRP was also observed with a recombinant fragment of FH (short consensus repeat 5-7) containing the same amino acid change. Because the interaction of CRP and FH promotes complement-mediated clearance of cellular debris in a noninflammatory fashion, we propose that the reduced binding of FH(402H) to CRP could lead to an impaired targeting of FH to cellular debris and a reduction in debris clearance and enhanced inflammation along the macular retinal pigmented epithelium-choroid interface in individuals with age-related macular degeneration.     

Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types

To fully understand self-renewal and pluripotency and their regulation in human embryonic stem cells (hESCs), it is necessary to generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes. Genes expressed in hESCs using plasmid vectors, however, are subject to silencing. Moreover, hESCs have a low plating efficiency when dissociated to single cells, making creation of subcloned lines inefficient. In addition to overexpression experiments, it is important to perform loss-of-function studies, which can be achieved rapidly using RNA interference (RNAi). We report stable long-term expression of enhanced green fluorescent protein (eGFP) in hESCs using a lentiviral vector, and establishment of an eGFP-expressing subline (RG6) using manual dissection. To demonstrate the efficacy of RNAi in hESCs, an RNAi expression vector was used to achieve reduced expression of eGFP in hESCs. To evaluate the role of OCT4 in the regulation of hESC self-renewal and differentiation, a vector expressing a hairpin RNA targeting endogenous expression of OCT4 was constructed. In a novel experiment in hESCs, the OCT4 cDNA sequence was cloned into an expression vector to allow for the transient upregulation of OCT4 in hESCs. The ability to manipulate levels of OCT4 above and below enodogenous levels allows the determination of OCT4 function in hESCs. Specifically, reduced expression of OCT4 in hESCs promoted upregulation of markers indicative of mesoderm and endoderm differentiation, and elevated levels of OCT4 in hESCs promoted upregulation of markers indicative of endoderm derivatives. Thus, both upregulation and downregulation of Oct4 in hESCs results in differentiation, but with patterns distinct from parallel experiments in mice.