Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome

Atypical hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. Disease-associated mutations have been described in the genes encoding the complement regulators complement factor H, membrane cofactor protein, factor B, and factor I. In this study, we show in two independent cohorts of aHUS patients that deletion of two closely related genes, complement factor H-related 1 (CFHR1) and complement factor H-related 3 (CFHR3), increases the risk of aHUS. Amplification analysis and sequencing of genomic DNA of three affected individuals revealed a chromosomal deletion of approximately 84 kb in the RCA gene cluster, resulting in loss of the genes coding for CFHR1 and CFHR3, but leaving the genomic structure of factor H intact. The CFHR1 and CFHR3 genes are flanked by long homologous repeats with long interspersed nuclear elements (retrotransposons) and we suggest that nonallelic homologous recombination between these repeats results in the loss of the two genes. Impaired protection of erythrocytes from complement activation is observed in the serum of aHUS patients deficient in CFHR1 and CFHR3, thus suggesting a regulatory role for CFHR1 and CFHR3 in complement activation. The identification of CFHR1/CFHR3 deficiency in aHUS patients may lead to the design of new diagnostic approaches, such as enhanced testing for these genes.     

Sequence-Specific Size, Structure, and Stability of Tight Protein Knots

Approximately 1% of known protein structures display knotted configurations in their native fold, but the function of these configurations is not understood. It has been speculated that the entanglement may inhibit mechanical protein unfolding or transport, e.g., as in cellular threading or translocation processes through narrow biological pores. Protein knot manipulation, e.g., knot tightening and localization, has become possible in single-molecule experiments. Here, we investigate tight peptide knot (TPK) characteristics in detail by pulling selected 3₁ and 4₁-knotted peptides using all-atom molecular dynamics computer simulations. We find that the 3₁- and 4₁-TPK lengths are typically Δl ≈ 47± 4 Å and 69 ± 4 Å, respectively, for a wide range of tensions (0.1 nN ≲ F ≲ 1.5 nN). The 4₁-knot length is in agreement with recent atomic force microscopy pulling experiments. Calculated TPK radii of gyration point to a pore diameter of ∼20 Å, below which a translocated knotted protein might get stuck. TPK characteristics, however, may be sequence-specific: we find a different size and structural behavior in polyglycines, and, strikingly, a strong hydrogen bonding and water trapping capability of hydrophobic TPKs. Water capture and release is found to be controllable by the tightening force in a few cases. These mechanisms result in a sequence-specific “locking” and metastability of TPKs, which might lead to a blocking of knotted peptide transport at designated sequence positions. We observe that macroscopic tight 4₁-knot structures are reproduced microscopically (“figure of eight” versus the “pretzel”) and can be tuned by sequence, in contrast to mathematical predictions. Our findings may explain a function of knots in native proteins, challenge previous studies on macromolecular knots, and prove useful in bio- and nanotechnology.     

Aeromonas salmonicida subsp. salmonicida in the light of its type-three secretion system

Aeromonas salmonicida subsp. salmonicida is an important pathogen in salmonid aquaculture and is responsible for the typical furunculosis. The type-three secretion system (T3SS) is a major virulence system. In this work, we review structure and function of this highly sophisticated nanosyringe in A. salmonicida. Based on the literature as well as personal experimental observations, we document the genetic (re)organization, expression regulation, anatomy, putative functional origin and roles in the infectious process of this T3SS. We propose a model of pathogenesis where A. salmonicida induces a temporary immunosuppression state in fish in order to acquire free access to host tissues. Finally, we highlight putative important therapeutic and vaccine strategies to prevent furunculosis of salmonid fish.     

The co-chaperone SGT of <Emphasis Type="Italic">Leishmania donovani</Emphasis> is essential for the parasite's viability

Molecular chaperone proteins play a pivotal role in the protozoan parasite Leishmania donovani, controlling cell fate and ensuring intracellular survival. In higher eukaryotes, the so-called co-chaperone proteins are required for client protein recognition and proper function of chaperones, among them the small glutamine-rich tetratricopeptide repeat proteins (SGT) which interact with both HSP70 and HSP90 chaperones. An atypical SGT homolog is found in the L. donovani genome, encoding a protein lacking the C-terminal glutamine-rich region, normally typical for SGT family members. The gene is expressed constitutively during the life cycle and is essential for survival and/or growth of the parasites. LdSGT forms large, stable complexes that also include another putative co-chaperone, HSC70 interacting protein (HIP). The gene product forms cytoplasmic clusters, matching the subcellular distribution of HIP and partly that of the major cytoplasmic chaperones, HSP70 and HSP90, reflecting a direct molecular interaction with both chaperones.     

Multitrophic influences on egg distribution in a specialized leaf beetle at multiple spatial scales

Egg distribution in herbivorous beetles can be affected by bottom-up (host plant), and by top-down factors (parasitoids and predators), as well as by other habitat parameters. The importance of bottom-up and top-down effects may change with spatial scale.

In this study, we investigated the influence of host plant factors and habitat structure on egg distribution in the leaf beetle Cassida canaliculata Laich. (Coleoptera: Chrysomelidae), a monophagous herbivore on Salvia pratensis L. (Lamiales: Lamiaceae), on four spatial scales: individual host plant, microhabitat, macrohabitat, and landscape. At the individual host plant scale we studied the correlation between egg clutch incidence and plant size and quality. On all other scales we analyzed the relationship between the egg clutch incidence of C. canaliculata and host plant percentage cover, host plant density, and the surrounding vegetation structure. Vegetation structure was examined as herbivores might escape egg parasitism by depositing their eggs on sites with vegetation factors unfavorable for host searching parasitoids.

The probability that egg clutches of C. canaliculata were present increased with an increasing size, percentage cover, and density of the host plant on three of the four spatial scales: individual host plant, microhabitat, and macrohabitat. There was no correlation between vegetation structure and egg clutch occurrence or parasitism on any spatial scale. A high percentage of egg clutches (38–56%) was parasitized by Foersterella reptans Nees (Hymenoptera: Tetracampidae), the only egg parasitoid, but there was no relationship between egg parasitism and the spatial distribution of egg clutches of C. canaliculata on any of the spatial scales investigated. However, we also discuss results from a further study, which revealed top-down effects on the larval stage.     

A family of oligopeptide transporters is required for growth of Candida albicans on proteins

The human fungal pathogen Candida albicans can use proteins as the sole source of nitrogen for growth. The secretion of aspartic proteinases, which have been shown to contribute to virulence of C. albicans, allows the fungus to digest host proteins to produce peptides that must be taken up into the cell by specific transporters. To understand in more detail how C. albicans utilizes proteins as a nitrogen source, we undertook a comprehensive analysis of oligopeptide transporters encoded in the C. albicans genome. We identified eight OPT genes encoding putative oligopeptide transporters, almost all of which are represented by polymorphic alleles in strain SC5314. Expression of these genes was differentially induced when C. albicans was grown in YCB-BSA medium, which contains bovine serum albumin as the sole nitrogen source. Whereas deletion of single OPT genes in strain SC5314 did not affect its ability to utilize proteins as a nitrogen source, opt123delta triple mutants had a severe growth defect in YCB-BSA which was rescued by reintroduction of a single copy of OPT1, OPT2 or OPT3. In addition, forced expression of OPT4 or OPT5 under control of the ADH1 promoter also restored growth of an opt123delta mutant, demonstrating that at least OPT1-OPT5 encode functional peptide transporters. The various oligopeptide transporters differ in their substrate preferences, as shown by the ability of strains expressing specific OPT genes to grow on peptides of defined length and sequence. We present evidence that in addition to the known role of oligopeptide transporters in the uptake of tetra- and pentapeptides these proteins can also transport longer peptides up to at least eight amino acids in length, ensuring an efficient utilization of the various peptides produced via endoproteolytic digestion of proteins by the secreted aspartic proteinases. As even transporters encoded by polymorphic alleles of a single gene exhibited differences in their efficiency to take up specific peptides, the oligopeptide transporters represent an example for how the evolution of gene families containing differentially expressed and functionally optimized members increases the nutritional versatility and, presumably, the adaptation of C. albicans to different host niches.     

Evaluation by electron microscopy of the integrity of iodinated plasmalipoproteins

Summary Iodination of proteins and lipoproteins is a widely used in vitro labelling procedure in metabolic, autoradiographic and various other studies. However, all available iodination techniques have involved the possible damage to the proteins by self-irradiation, oxidizing agents, the alkaline milieu or by the introduction of iodine into the molecular structure itself. To evaluate the integrity of iodinated lipoproteins, we observed the electron microscopic appearance of normal and iodinated rabbit very low density lipoproteins (VLDL) by negative staining with phosphotungstic acid. Iodination up to a molar iodine/protein ratio of 2.89 did not result in any change of shape, size or aggregating tendency of the particles. No stacks or disk-like particles like those of various hyperlipoproteinemic states were found. We conclude that electron microscopy is a valuable tool in assessing the morphological appearance of lipoprotein iodination, but it should be complemented by other techniques.     

Effects of low frequency electromagnetic fields on the chondrogenic differentiation of human mesenchymal stem cells

Electromagnetic fields (EMF) have been shown to exert beneficial effects on cartilage tissue. Nowadays, differentiated human mesenchymal stem cells (hMSCs) are discussed as an alternative approach for cartilage repair. Therefore, the aim of this study was to examine the impact of EMF on hMSCs during chondrogenic differentiation. HMSCs at cell passages five and six were differentiated in pellet cultures in vitro under the addition of human fibroblast growth factor 2 (FGF‐2) and human transforming growth factor‐β₃ (TGF‐β₃). Cultures were exposed to homogeneous sinusoidal extremely low‐frequency magnetic fields (5 mT) produced by a solenoid or were kept in a control system. After 3 weeks of culture, chondrogenesis was assessed by toluidine blue and safranin‐O staining, immunohistochemistry, quantitative real‐time polymerase chain reaction (PCR) for cartilage‐specific proteins, and a DMMB dye‐binding assay for glycosaminoglycans. Under EMF, hMSCs showed a significant increase in collagen type II expression at passage 6. Aggrecan and SOX9 expression did not change significantly after EMF exposure. Collagen type X expression decreased under electromagnetic stimulation. Pellet cultures at passage 5 that had been treated with EMF provided a higher glycosaminoglycan (GAG)/DNA content than cultures that had not been exposed to EMF. Chondrogenic differentiation of hMSCs may be improved by EMF regarding collagen type II expression and GAG content of cultures. EMF might be a way to stimulate and maintain chondrogenesis of hMSCs and, therefore, provide a new step in regenerative medicine regarding tissue engineering of cartilage. Bioelectromagnetics 32:283–290, 2011. © 2010 Wiley‐Liss, Inc.