Diversity and evolutionary patterns of immune genes in free-ranging Namibian leopards (Panthera pardus pardus)

The genes of the major histocompatibility complex (MHC) are a key component of the mammalian immune system and have become important molecular markers for fitness-related genetic variation in wildlife populations. Currently, no information about the MHC sequence variation and constitution in African leopards exists. In this study, we isolated and characterized genetic variation at the adaptively most important region of MHC class I and MHC class II-DRB genes in 25 free-ranging African leopards from Namibia and investigated the mechanisms that generate and maintain MHC polymorphism in the species. Using single-stranded conformation polymorphism analysis and direct sequencing, we detected 6 MHC class I and 6 MHC class II-DRB sequences, which likely correspond to at least 3 MHC class I and 3 MHC class II-DRB loci. Amino acid sequence variation in both MHC classes was higher or similar in comparison to other reported felids. We found signatures of positive selection shaping the diversity of MHC class I and MHC class II-DRB loci during the evolutionary history of the species. A comparison of MHC class I and MHC class II-DRB sequences of the leopard to those of other felids revealed a trans-species mode of evolution. In addition, the evolutionary relationships of MHC class II-DRB sequences between African and Asian leopard subspecies are discussed.     

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.     

Mutations in ACY1, the gene encoding aminoacylase 1, cause a novel inborn error of metabolism

N-terminal acetylation of proteins is a widespread and highly conserved process. Aminoacylase 1 (ACY1; EC 3.5.14) is the most abundant of the aminoacylases, a class of enzymes involved in hydrolysis of N-acetylated proteins. Here, we present four children with genetic deficiency of ACY1. They were identified through organic acid analyses using gas chromatography-mass spectrometry, revealing increased urinary excretion of several N-acetylated amino acids, including the derivatives of methionine, glutamic acid, alanine, leucine, glycine, valine, and isoleucine. Nuclear magnetic resonance spectroscopy analysis of urine samples detected a distinct pattern of N-acetylated metabolites, consistent with ACY1 dysfunction. Functional analyses of patients' lymphoblasts demonstrated ACY1 deficiency. Mutation analysis uncovered recessive loss-of-function or missense ACY1 mutations in all four individuals affected. We conclude that ACY1 mutations in these children led to functional ACY1 deficiency and excretion of N-acetylated amino acids. Questions remain, however, as to the clinical significance of ACY1 deficiency. The ACY1-deficient individuals were ascertained through urine metabolic screening because of unspecific psychomotor delay (one subject), psychomotor delay with atrophy of the vermis and syringomyelia (one subject), marked muscular hypotonia (one subject), and follow-up for early treated biotinidase deficiency and normal clinical findings (one subject). Because ACY1 is evolutionarily conserved in fish, frog, mouse, and human and is expressed in the central nervous system (CNS) in human, a role in CNS function or development is conceivable but has yet to be demonstrated. Thus, at this point, we cannot state whether ACY1 deficiency has pathogenic significance with pleiotropic clinical expression or is simply a biochemical variant. Awareness of this new genetic entity may help both in delineating its clinical significance and in avoiding erroneous diagnoses.     

Generation of Neutralizing Human Monoclonal Antibodies against Parvovirus B19 Proteins

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 μg/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 μg/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.     

The periplasmic chaperone SurA exploits two features characteristic of integral outer membrane proteins for selective substrate recognition

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins. Thus, SurA may be specialized for the maturation of outer membrane proteins. We have characterized the substrate specificity of SurA based on its natural, biologically relevant substrates by screening cellulose-bound peptide libraries representing outer membrane proteins. We show that two features are critical for peptide binding by SurA: specific patterns of aromatic residues and the orientation of their side chains, which are found more frequently in integral outer membrane proteins than in other proteins. For the first time this sufficiently explains the capability of SurA to discriminate between outer membrane protein and non-outer membrane protein folding intermediates. Furthermore, peptide binding by SurA requires neither an active PPIase domain nor the presence of proline, indicating that the observed substrate specificity relates to the chaperone function of SurA. Finally, we show that SurA is capable of associating with the outer membrane. Together, our data support a model in which SurA is specialized to interact with non-native periplasmic outer membrane protein folding intermediates and to assist in their maturation from early to late outer membrane-associated steps.     

Invasion of Endothelial Cells by Tissue-invasive M3 Type Group A Streptococci Requires Src Kinase and Activation of Rac1 by a Phosphatidylinositol 3-Kinase-independent Mechanism

Streptococcus pyogenes can cause invasive diseases in humans, such as sepsis or necrotizing fasciitis. Among the various M serotypes of group A streptococci (GAS), M3 GAS lacks the major epithelial invasins SfbI/PrtF1 and M1 protein but has a high potential to cause invasive disease. We examined the uptake of M3 GAS into human endothelial cells and identified host signaling factors required to initiate streptococcal uptake. Bacterial uptake is accompanied by local F-actin accumulation and formation of membrane protrusions at the entry site. We found that Src kinases and Rac1 but not phos pha tidyl ino si tol 3-kinases (PI3Ks) are essential to mediate S. pyogenes internalization. Pharmacological inhibition of Src activity reduced bacterial uptake and abolished the formation of membrane protrusions and actin accumulation in the vicinity of adherent streptococci. We found that Src kinases are activated in a time-de pend ent manner in response to M3 GAS. We also demonstrated that PI3K is dispensable for internalization of M3 streptococci and the formation of F-actin accumulations at the entry site. Furthermore, Rac1 was activated in infected cells and accumulated with F-actin in a PI3K-independent manner at bacterial entry sites. Genetic interference with Rac1 function inhibited streptococcal internalization, demonstrating an essential role of Rac1 for the uptake process of streptococci into endothelial cells. In addition, we demonstrated for the first time accumulation of the actin nucleation complex Arp2/3 at the entry port of invading M3 streptococci.Streptococcus pyogenes or group A streptococcus (GAS)² is an important human pathogen that causes localized infections of the respiratory tract and the skin but also severe invasive disease, sepsis, and toxic shock-like syndrome. Group A streptococci, although traditionally viewed as extracellular pathogens, are able to adhere to and invade into several eukaryotic cell types (1–5).Localized S. pyogenes infections may lead to dissemination of bacteria through the vascular system, resulting in bacteremia and sepsis. For evasion of the vascular system, S. pyogenes may directly interact with the endothelium, which lines the inner surface of blood vessels. M3 type streptococci are, besides the M1 and M28 strains, most commonly associated with invasive GAS infections (6) and have been shown to be internalized into human umbilical vein endothelial cells (HUVEC) in vitro (7).S. pyogenes can express several invasins, but only the signal transduction pathways of two streptococcal factors, SfbI/prtF1 and M1 protein, respectively, have been studied in more detail. Both invasins trigger bacterial uptake by binding to soluble fibronectin, which acts as a bridging molecule and induces the clustering of host integrins, which in turn activates host signaling pathways. In the case of M1-mediated internalization, activation of PI3K, ILK, paxillin, and focal adhesion kinase has been shown, which promotes actin polymerization-based zipper-like bacterial uptake into epithelial cells (8–10). In contrast to this, caveolae were shown to act as entry port for SfbI-expressing S. pyogenes (11), a mechanism distinct from the zipper-like uptake mechanism employed by strains expressing M1 protein (12). SfbI/protein F1-expressing streptococci form a focal complex-like structure that consists of focal adhesion kinase, Src kinases, paxillin, and Rho GTPases, resulting in uptake of the bacteria (13). However, a requirement for PI3K activation, which in turn induced paxillin phosphorylation, was recently shown for M1-mediated as well as SfbI-mediated invasion (10). In contrast, M3 streptococci do not express these two well characterized invasins (14), the mechanism by which M3 streptococci are able to trigger entry into human endothelial cells is still poorly understood, and no information is currently available concerning host cell signaling factors involved in this process.In this study, we characterized the intracellular signals governing internalization of SfbI/prtF1/M1-negative M3 GAS into primary endothelial cells. We found an essential role for host cell protein-tyrosine kinases (PTKs) and identified Src family PTKs to play an essential role during the uptake process. In contrast to the already characterized receptor-mediated bacterial invasion strategies, which rely on PI3K activation, internalization of M3 GAS is PI3K-independent. In addition to Src family PTKs, the GTPase Rac1 was identified as an important factor for M3 S. pyogenes internalization. Rac1 was found to be activated in response to bacterial internalization, and genetic interference with Rac1 function significantly reduced uptake. Rac1 as well as the actin nucleation complex Arp2/3 was found to accumulate at streptococcal entry ports, strengthening the important role of this GTPase for uptake of M3 type streptococci into human endothelial cells.     

Isoaspartate-containing amyloid precursor protein-derived peptides alter efficacy and specificity of potential beta-secretases

Neuritic plaques of Alzheimer patients are composed of multiple protein components. Among them, the amyloid beta-peptides (Abeta) 1-40/42 and further N- and C-terminally modified fragments of Abeta are highly abundant. Most prominent are the isoaspartate (isoAsp)-Abeta peptides and pyroglutamyl (pGlu)-Abeta. While pGlu-Abeta can only be formed from an N-terminal glutamate by glutaminyl cyclase, spontaneous isoAsp-isomerization cannot occur at an N-terminal aspartate of peptides. This means that isoAsp-Abeta formation must precede proteolysis of the amyloid precursor protein (APP). Abeta generation from APP by beta- and gamma-secretases initiates the amyloid peptide aggregation and deposition process. Two aspartate proteases have been identified as secretases: BACE-1 (beta-site amyloid precursor protein cleaving enzyme) and the intramembrane gamma-secretase multiprotein complex. However, recent evidence supports more than one beta-secretase initiating this cascade. Formation of Abeta1-40/42 was predominantly studied by expression of mutated human APP sequences in cell culture and transgenic animals, generating Abeta fragments that did not contain such multiple posttranslational modifications as in Alzheimer's disease. This prompted us to investigate the catalytic turnover of Asp- or isoAsp-containing APP-derived peptide sequences by BACE-1 and cathepsin B, another potential beta-secretase. While cathepsin B is more effective than BACE-1 in processing the Asp-containing peptide derivatives, only cathepsin B can cleave the isoAsp-containing peptides, which occurs with high catalytic efficiency.