The binding of human milk lactoferrin to immunoglobulin A

To define the step at which translational initiation factor IF1 exercises its stimulation, initial rate kinetic analyses of 30 S initiation complex formation were carried out in the presence and absence of this factor. It was shown that, without affecting the affinity of the ribosomes either for the initiator tRNA or for the poly(AUG) used as template, IF1 increases approximately 2.5-fold the limiting Vmax of the 'pre-ternary complex'----ternary complex transition which represents the rate-limiting step in 30 S initiation complex formation. This kinetic effect titrates with the 30 S ribosomal subunit which must therefore represent the target of IF1 action.     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.     

Role of UV-inducible proteins in repair of various wild-type Escherichia coli cells

3 wild-type strains of E. coli, namely K12 AB2497, B/r WP2 and 15 555-7v proficient in excision and post-replication repair, differ markedly in their UV resistance. To elucidate this difference, the influence was investigated of induction by application of inducing fluence (IF) before lethal fluence (LF) on repair processes after LF. In cells distinguished by low UV resistance (E. coli 15 555-7; E. coli B/r WP2), dimer excision was less complete in cultures irradiated with IF + LF than in cultures irradiated with LF only. The highly resistant E. coli K12 AB2497 performed complete excision both after IF + LF or after LF alone. All 3 types of cell survived better after IF + LF than after LF only. Because, in most strains so far investigated, the application of IF reduced dimer excision and increased survival, dimer excision per se does not appear important for survival.We conclude that the rate and completeness of dimer excision can serve as a measure of efficiency of the excision system whose action is necessary for repair of another lesion. Cells of all investigated strains could not resume DNA replication and died progressively when irradiated with LF and post-incubated with chloramphenicol (LF CAP⁺). Thus, it appears that inducible proteins are necessary for repair in all wild-type E. coli cells give with potentially lethal doses of UV irradiation.     

Monoclonal antibody against the poly-γ-d-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge

Background

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT.

Methods

To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K_d values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed.

Results

Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen.

General significance

Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.     

Expression of integrins α3β1 and α5β1 and GlcNAc β1,6 glycan branching influences metastatic melanoma cell migration on fibronectin

Acquisition of metastatic potential is accompanied by changes in cell surface N-glycosylation. One of the best-studied changes is increased expression of N-acetylglucosaminyltransferase V enzyme (GnT-V) and its products, β1,6-branched N-linked oligosaccharides, observed in the tumorigenesis of many cancers. In this study we demonstrate that during the transition from the vertical growth phase (VGP) (WM793 cell line) to the metastatic stage (WM1205Lu line), β1,6 glycosylation of melanoma cell surface proteins increases as a consequence of elevated expression of the GnT-V-encoding Mgat-5 gene. Treatment with swainsonine led to reduced cell motility on fibronectin in both cell lines; the effect was stronger in metastatic cells, probably due to the higher content of GlcNAc β1,6-branched glycans on the main fibronectin receptors – integrins α5β1 and α3β1. Our results show that GlcNAc β1,6 N-glycosylation of cell surface receptors, which increases with the aggressiveness of melanoma cells, is an important factor influencing melanoma cell migration.     

Negative regulation of the hepatic fibrogenic response by suppressor of cytokine signaling 1

Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFNγ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFNγ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFNγ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in HSC activation. Liver fibrosis was induced in Socs1^−/−Ifng^−/− mice with dimethylnitrosamine or carbon tetrachloride. Ifng^−/− and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1^−/−Ifng^−/− mice showed elevated serum ALT levels and increased liver fibrosis compared to Ifng^−/− mice. The latter group showed higher ALT levels and fibrosis than C57BL/6 controls. The livers of SOCS1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. SOCS1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from SOCS1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of SOCS1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFNγ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability.     

昆虫病原线虫Heterorhabditis beicherriana LF品系与Bt HBF-18菌株混用对华北大黑鳃金龟幼虫的防治效果

【目的】明确昆虫病原线虫 Heterorhabditis beicherriana LF品系(LF)与苏云金芽孢杆菌 Bacillus thuringiensis HBF-18菌株(Bt HBF-18)混用后对华北大黑鳃金龟 Holotrichia oblita 幼虫的致病力的协同增效作用,为该害虫的防治提供新的技术措施。【方法】在室内测定了LF在不同使用剂量、不同环境温度及不同土壤湿度条件下对华北大黑鳃金龟7-10日龄幼虫的致病力;通过室内生测测定了Bt HBF-18对LF存活的影响,以及Bt HBF-18与LF两者混用后对7-10日龄华北大黑鳃金龟幼虫的防治效果;同时通过室外盆栽试验测定了两者混用对华北大黑鳃金龟幼虫的防治效果。【结果】华北大黑鳃金龟幼虫死亡率随LF施用剂量和处理时间的增加而升高,其中,侵染期线虫(infective juveniles, IJs)800 IJs/100 μL及以上剂量处理7 d后幼虫死亡率达到了100%;25℃为该线虫侵染的最适宜环境温度;适宜土壤湿度范围为14%~20%,湿度过低或过高都会显著影响其侵染效率。室内生测结果表明, Bt HBF-18处理9 d对华北大黑鳃金龟幼虫的致死中浓度(LC 50 )为 1.44× 10^8 CFU/g土,此浓度对LF的存活基本没有影响。另外,室内生测和室外盆栽试验结果均表明,将LF与Bt HBF-18混用能显著提高对华北大黑鳃金龟幼虫的防治效果,混用后具有不同程度的加成或协同增效作用。室内生测试验中LC 50 Bt+200 IJs/100 μL LF混用处理3 d后,较单独LF和Bt HBF-18处理幼虫死亡率分别提高了约43.07%和36.05%,具有显著的协同增效作用;室外盆栽试验中1/2 LC 50 Bt+1 000 IJs/mL LF, LC 50 Bt+1 000 IJs/mL LF和1/2 LC 50 Bt+1 500 IJs/mL LF均具有协同增效作用,其中1/2 LC 50 Bt+1 500 IJs/mL LF增效作用最佳,较单独LF和Bt HBF-18处理幼虫死亡率分别提高了约38.89%和80.55%。【结论】将昆虫病原线虫LF与Bt HBF-18混用对华北大黑鳃金龟幼虫的防治具有加成或协同增效作用。     

Structure of a complex of human lactoferrin N-lobe with pneumococcal surface protein a provides insight into microbial defense mechanism

Human lactoferrin, a component of the innate immune system, kills a wide variety of microorganisms including the Gram positive bacteria Streptococcus pneumoniae. Pneumococcal surface protein A (PspA) efficiently inhibits this bactericidal action. The crystal structure of a complex of the lactoferrin-binding domain of PspA with the N-lobe of human lactoferrin reveals direct and specific interactions between the negatively charged surface of PspA helices and the highly cationic lactoferricin moiety of lactoferrin. Binding of PspA blocks surface accessibility of this bactericidal peptide preventing it from penetrating the bacterial membrane. Results of site-directed mutagenesis, in vitro protein binding assays and isothermal titration calorimetry measurements corroborate that the specific electrostatic interactions observed in the crystal structure represent major associations between PspA and lactoferrin. The structure provides a snapshot of the protective mechanism utilized by pathogens against the host's first line of defense. PspA represents a major virulence factor and a promising vaccine candidate. Insights from the structure of the complex have implications for designing therapeutic strategies for treatment and prevention of pneumococcal diseases that remain a major public health problem worldwide.