Degradation of Host Sphingomyelin Is Essential for Leishmania Virulence

In eukaryotes, sphingolipids (SLs) are important membrane components and powerful signaling molecules. In Leishmania, the major group of SLs is inositol phosphorylceramide (IPC), which is common in yeast and Trypanosomatids but absent in mammals. In contrast, sphingomyelin is not synthesized by Leishmania but is abundant in mammals. In the promastigote stage in vitro, Leishmania use SL metabolism as a major pathway to produce ethanolamine (EtN), a metabolite essential for survival and differentiation from non-virulent procyclics to highly virulent metacyclics. To further probe SL metabolism, we identified a gene encoding a putative neutral sphingomyelinase (SMase) and/or IPC hydrolase (IPCase), designated ISCL (Inositol phosphoSphingolipid phospholipase C-Like). Despite the lack of sphingomyelin synthesis, L. major promastigotes exhibited a potent SMase activity which was abolished upon deletion of ISCL, and increased following over-expression by episomal complementation. ISCL-dependent activity with sphingomyelin was about 20 fold greater than that seen with IPC. Null mutants of ISCL (iscl⁻) showed modest accumulation of IPC, but grew and differentiated normally in vitro. Interestingly, iscl⁻ mutants did not induce lesion pathology in the susceptible BALB/c mice, yet persisted indefinitely at low levels at the site of infection. Notably, the acute virulence of iscl⁻ was completely restored by the expression of ISCL or heterologous mammalian or fungal SMases, but not by fungal proteins exhibiting only IPCase activity. Together, these findings strongly suggest that degradation of host-derived sphingomyelin plays a pivotal role in the proliferation of Leishmania in mammalian hosts and the manifestation of acute disease pathology.     

Study of response of Swiss Webster mice to light subunit of mushroom tyrosinase

The light subunit of mushroom, Agaricus bisporus, tyrosinase (LSMT), has been identified as an extrinsic component of the enzyme. Its function is unknown, but it can cross an epithelial cell layer, which suggests that it can be absorbed by the intestine. A similar capability has been demonstrated for the HA-33 component of the progenitor toxin from Clostridium botulinum, which is the closest structural homolog of LSMT. Unlike HA-33, LSMT appears to be non-immunogenic as shown by preliminary tests in Swiss Webster mice. We investigated the immunogenicity and histopathology of LSMT in mice to determine its safety in vivo. LSMT did not evoke generation of antibodies after prolonged periods of intraperitoneal administration. Histopathological observations confirmed the absence of responses in organs after twelve weekly administrations of LSMT. We found that LSMT is not toxic and is less immunogenic than the C. botulinum HA-33 protein, which supports further research and development for pharmaceutical application.     

Changes in protein expression during honey bee larval development

Background  

The honey bee (Apis mellifera), besides its role in pollination and honey production, serves as a model for studying the biochemistry of development, metabolism, and immunity in a social organism. Here we use mass spectrometry-based quantitative proteomics to quantify nearly 800 proteins during the 5- to 6-day larval developmental stage, tracking their expression profiles.     

Severe asthma and the omalizumab option

Atopic diseases and asthma are increasing at a remarkable rate on a global scale. It is now well recognized that asthma is a chronic inflammatory disease of the airways. The inflammatory process in many patients is driven by an immunoglobulin E (IgE)-dependent process. Mast cell activation and release of mediators, in response to allergen and IgE, results in a cascade response, culminating in B lymphocyte, T lymphocyte, eosinophil, fibroblast, smooth muscle cell and endothelial activation. This complex cellular interaction, release of cytokines, chemokines and growth factors and inflammatory remodeling of the airways leads to chronic asthma. A subset of patients develops severe airway disease which can be extremely morbid and even fatal. While many treatments are available for asthma, it is still a chronic and incurable disease, characterized by exacerbation, hospitalizations and associated adverse effects of medications. Omalizumab is a new option for chronic asthma that acts by binding to and inhibiting the effects of IgE, thereby interfering with one aspect of the asthma cascade reviewed earlier. This is a humanized monoclonal antibody against IgE that has been shown to have many beneficial effects in asthma. Use of omalizumab may be influenced by the cost of the medication and some reported adverse effects including the rare possibility of anaphylaxis. When used in selected cases and carefully, omalizumab provides a very important tool in disease management. It has been shown to have additional effects in urticaria, angioedema, latex allergy and food allergy, but the data is limited and the indications far from clear. In addition to decreasing exacerbations, it has a steroid sparing role and hence may decrease adverse effects in some patients on high-dose glucocorticoids. Studies have shown improvement in quality of life measures in asthma following the administration of omalizumab, but the effects on pulmonary function are surprisingly small, suggesting a disconnect between pulmonary function, exacerbations and quality of life. Anaphylaxis may occur rarely with this agent and appropriate precautions have been recommended by the Food and Drug Administration (FDA). As currently practiced and as suggested by the new asthma guidelines, this biological agent is indicated in moderate or severe persistent allergic asthma (steps 5 and 6).     

Hepatitis C Virus Core and Envelope Proteins Do Not Suppress the Host's Ability To Clear a Hepatic Viral Infection

Several hepatitis C virus (HCV) proteins have been shown in vitro to interact with host cellular components that are involved in immune regulation. However, there is a paucity of data supporting the relevance of these observations to the in vivo situation. To test the hypothesis that such an interaction suppresses immune responses, we studied a line of transgenic C57BL/6 mice that express the HCV core and envelope proteins in the liver. The potential effects of these proteins on the hepatic immune response were evaluated by challenging these mice with a hepatotropic adenovirus. Both transgenic and nontransgenic mice developed similar courses of infection and cleared the virus from the liver by 28 days postinfection. Both groups of mice mounted similar immunoglobulin G (IgG), IgG2a, interleukin-2, and tumor necrosis factor alpha responses against the virus. Additionally, BALB/c mice were able to clear infection with recombinant adenovirus that does or does not express the HCV core and envelope 1 proteins in the same manner. These data suggest that HCV core and envelope proteins do not inhibit the hepatic antiviral mechanisms in these murine experimental systems and thus favor a model in which HCV circumvents host responses through a mechanism that does not involve general suppression of intrahepatic immune responses.     

Gene selection for oligonucleotide array: an approach using PM probe level data

MOTIVATION: Analysis of oligonucleotide array data, especially to select genes of interest, is a highly challenging task because of the large volume of information and various experimental factors. Moreover, interaction effect (i.e. expression changes depend on probe effects) complicates the analysis because current methods often use an additive model to analyze data. We propose an approach to address these issues with the aim of producing a more reliable selection of differentially expressed genes. The approach uses the rank for normalization, employs the percentile-range to measure expression variation, and applies various filters to monitor expression changes. RESULTS: We compare our approach with MAS and Dchip models. A data set from an angiogenesis study is used for illustration. Results show that our approach performs better than other methods either in identification of the positive control gene or in PCR confirmatory tests. In addition, the invariant set of genes in our approach provides an efficient way for normalization.     

Site and mechanism of action of dynorphin A-(1-13) and N-methyl-D-aspartate on ACTH release in fetal sheep

Dynorphin A (Dyn A) stimulates the release of ACTH in fetal sheep, a response that involves N-methyl-D-aspartate (NMDA) receptors but not the secretogogues corticotropin-releasing hormone or arginine vasopressin. We now find that neither Dyn A-(1-13) (0.5 mg/kg, i.v.) nor NMDA (4 mg/kg, i.v.) elicits ACTH release in postnatal lambs. This led us to hypothesize that Dyn A-(1-13) and NMDA might act to release placental ACTH. However, the ability of Dyn A-(1-13), NMDA, and the kappa-opioid receptor agonist U-50488H (1 mg/kg, i.v.) to release ACTH was lost after either fetal hypophysectomy (n = 4) or hypothalamo-pituitary disconnection (n = 4). These results indicate that neither the placenta nor the fetal pituitary is the site of action for these agonists and suggest a hypothalamic or suprahypothalamic site of action. Furthermore, the release of ACTH by Dyn A-(1-13) and NMDA was abolished after pretreatment with indomethacin, suggesting that they might cause the release of a prostanoid, possibly from the placenta, that subsequently acts at the hypothalamus or serves as a permissive factor in the action of Dyn A-(1-13) and NMDA at the hypothalamus.     

Infection of human T lymphocytes with varicella-zoster virus: an analysis with viral mutants and clinical isolates

Varicella-zoster virus (VZV) disseminates in the body in peripheral blood mononuclear cells during chickenpox. Up to 1 in 10,000 mononuclear cells are infected during the viremic phase of the disease. We developed an in vitro system to infect human mononuclear cells with VZV by using umbilical cord blood. In this system, 3 to 4% of T cells were infected with VZV. VZV mutants unable to express certain genes, such as open reading frame 47 (ORF47) or ORF66, were impaired for growth in T cells, while other mutants showed little difference from parental virus. VZV unable to express ORF47 was even more impaired for spread from umbilical cord blood cells to melanoma cells in vitro. Early-passage clinical isolates of VZV infected T cells at a similar rate to the Oka vaccine strain; however, the clinical isolates were more efficient in spreading from infected T cells to melanoma cells. This in vitro system for infecting human T cells with VZV should be useful for identifying cellular and viral proteins that are important for virus replication in T cells and for the spread of virus from T cells to other cells.     

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.     

A novel amidase (half-amidase) for half-amide hydrolysis involved in the bacterial metabolism of cyclic imides

A novel amidase involved in bacterial cyclic imide metabolism was purified from Blastobacter sp. strain A17p-4. The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (K(m) = 6.2 mM; k(cat) = 5.76 s(-1)) and glutaramic acid (K(m) = 2.8 mM; k(cat) = 2.23 s(-1)). However, the substrates of known amidases such as short-chain (C(2) to C(4)) aliphatic amides, long-chain (above C(16)) aliphatic amides, amino acid amides, aliphatic diamides, alpha-keto acid amides, N-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme. Based on its high specificity toward half-amides, the enzyme was named half-amidase. This half-amidase exists as a monomer with an M(r) of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.