The helical content of the YscP molecular ruler determines the length of the Yersinia injectisome

The length of the Yersinia injectisome needle is determined by the protein YscP, which could act as a molecular ruler. The analysis of the correlation between the size of YscP and the needle length in seven wild-type strains of Yersinia enterocolitica reinforced this hypothesis but hinted that the secondary structure of YscP might influence needle length. Hence, 11 variants of YscP₅₁₅ were generated by multiple Pro or Gly substitutions. The needle length changed in inverse function of the helical content, indicating that not only the number of residues but also their structure controls length. Taking the secondary motifs into account, Pro/Gly-variants were subjected to in silico modelling to simulate the extension of YscP upon needle growth. The calculated lengths when the helical content is preserved correlated strikingly with the measured needle length, with a constant difference of ∼29 nm, which corresponds approximately to the size of the basal body. These data support the ruler model and show that the functional ruler has a helical structure.     

Evaluation of GABA Production and Probiotic Activities of Enterococcus faecium BS5

Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of...     

Site-Directed Mutations in the Lanthipeptide Mutacin 1140

The oral bacterium Streptococcus mutans, strain JH1140, produces the antibiotic mutacin 1140. Mutacin 1140 belongs to a group of antibiotics called lanthipeptides. More specifically, mutacin 1140 is related to the epidermin type A(I) lanthipeptides. Mutagenesis experiments of this group of lanthipeptides have been primarily restricted to the posttranslationally modified meso-lanthionine and 3-methyllanthionine residues. Site-directed mutagenesis of the core peptide of mutacin 1140 was performed using the suicide vector pVA891. Substitutions of the N-terminal residue, the charged residue in the hinge region, and residues in ring A and intertwined rings C and D were investigated. A truncation and insertion of residues in ring A and intertwined rings C and D were also performed to determine whether or not they would alter the antimicrobial activity of the producing strain. Bioassays revealed that five of 14 mutants studied had improved antimicrobial activity against the indicator strain Micrococcus luteus ATCC 10240. MICs against Streptococcus mutans UA159, Streptococcus pneumoniae ATCC 27336, Staphylococcus aureus ATCC 25923, Clostridium difficile UK1, and Micrococcus luteus ATCC 10240 were determined for three mutacin 1140 variants that had the most significant increases in bioactivity in the M. luteus bioassay. This mutagenesis study of the epidermin group of lanthipeptides shows that antimicrobial activity can be significantly improved.     

How Does Circadian Rhythm Impact Salt Sensitivity of Blood Pressure in Mice? A Study in Two Close C57Bl/6 Substrains

BackgroundMouse transgenesis has provided the unique opportunity to investigate mechanisms underlying sodium kidney reabsorption as well as end organ damage. However, understanding mouse background and the experimental conditions effects on phenotypic readouts of engineered mouse lines such as blood pressure presents a challenge. Despite the ability to generate high sodium and chloride plasma levels during high-salt diet, observed changes in blood pressure are not consistent between wild-type background strains and studies.MethodsThe present work was designed in an attempt to determine guidelines in the field of salt-induced hypertension by recording continuously blood pressure by telemetry in mice submitted to different sodium and potassium loaded diets and changing experimental conditions in both C57BL/6N and C57BL/6J mice strain (Normal salt vs. Low salt vs. High-salt/normal potassium vs. High salt/low potassium, standard vs. modified light cycle, Non-invasive tail cuff blood pressure vs. telemetry).ResultsIn this study, we have shown that, despite a strong blood pressure (BP) basal difference between C57BL/6N and C57BL/6J mice, High salt/normal potassium diet increases BP and heart rate during the active phase only (dark period) in the same extent in both strains. On the other hand, while potassium level has no effect on salt-induced hypertension in C57BL/6N mice, high-salt/low potassium diet amplifies the effect of the high-salt challenge only in C57BL/6J mice. Indeed, in this condition, salt-induced hypertension can also be detected during light period even though this BP increase is lower compared to the one occurring during the dark period. Finally, from a methodological perspective, light cycle inversion has no effect on this circadian BP phenotype and tail-cuff method is less sensitive than telemetry to detect BP phenotypes due to salt challenges.ConclusionsTherefore, to carry investigations on salt-induced hypertension in mice, chronic telemetry and studies in the active phase are essential prerequisites.     

O6-methylguanine-DNA-methyltransferase (MGMT) gene therapy targeting haematopoietic stem cells: studies addressing safety issues

As haematopoietic stem cell gene therapy utilizing O(6)-methylguanine-DNA-methyltransferase has reached the clinical stage, safety-related questions become increasingly important. These issues concern insertional mutagenesis of viral vectors, the acute toxicity of pre-transplant conditioning protocols and in vivo selection regimens as well as potential genotoxic side effects of the alkylating drugs administered in this context. To address these questions, we have investigated toxicity-reduced conditioning regimens combining low-dose alkylator application with sublethal irradiation and have analysed their influence on engraftment and subsequent selectability of transduced haematopoietic stem cells. In addition, a strategy to monitor the acute and long-term genotoxic effects of drugs with high guanine-O(6) alkylating potential, such as chloroethylnitrosoureas or temozolomide is introduced. For this purpose, assays were implemented which allow an assessment of the generation and fate of primary drug-induced adducts as well as their long-term effect on chromosomal integrity at the single cell level.     

Interaction of soluble CD163 with activated T lymphocytes involves its association with non-muscle myosin heavy chain type A

CD163 is a monocyte/macrophage-specific scavenger receptor that undergoes ectodomain shedding upon an inflammatory stimulus. Soluble CD163 (sCD163) actively inhibits lymphocyte proliferation, but to date exactly how it interacts with these cells has remained elusive. We screened T lymphocytes and endothelial cells for proteins binding to sCD163. In both cell types a high affinity binding protein was detected. Partial sequencing of the protein revealed sequence identity to a non-muscle myosin heavy chain type A. Employing labelled sCD163 we found little specific binding of sCD163 to the extracellular domains of T lymphocytes and human umbilical vein endothelial cells (HUVEC). In activated T lymphocytes we demonstrated specific binding of sCD163 to intracellular structures as well as the presence of the native protein within the cell after co-incubation with purified sCD163. Furthermore, we developed a novel ELISA for highly specific detection of sCD163-myosin complexes. These complexes were present in activated T lymphocytes after incubation with shed sCD163. Co-localization of sCD163 and cellular myosin in T lymphocytes was further confirmed by fluorescence microscopy. Our results suggest that sCD163 associates with cellular myosin, thereby possibly modulating the cells' response to an inflammatory stimulus.     

Only the soluble form of the scavenger receptor CD163 acts inhibitory on phorbol ester-activated T-lymphocytes,whereas membrane-bound protein has no effect

We have studied the expression of XIAP, cIAP-1 and cIAP-2 in fetal rat hepatocytes and its possible regulation by pro-apoptotic stimuli (transforming growth factor-β (TGF-β)) and survival signals (epidermal growth factor (EGF)). The three forms of inhibitor of apoptosis proteins (IAPs) are expressed in fetal hepatocytes and only cIAP-1, but not XIAP or cIAP-2, is cleaved during TGF-β-induced apoptosis. The pan-caspase inhibitor Z-VAD.fmk blocked this effect, which indicates that cIAP-1 is a caspase substrate. EGF plays a dual role in the regulation of IAPs expression. On one hand, it increases cIAP-1 and cIAP-2 basal expression and, on the other hand, it blocks the cleavage of cIAP-1 by caspases induced by TGF-β.     

Loss of S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface expression,a polarized microfilament system,and diminished responsiveness to chemoattractants in vitro

The S100A9 (MRP14) protein is abundantly expressed in myeloid cells and has been associated with various inflammatory diseases. The S100A9-deficient mice described here were viable, fertile, and generally of healthy appearance. The myelopoietic potential of the S100A9-null bone marrow was normal. S100A8, the heterodimerization partner of S100A9 was not detectable in peripheral blood cells, suggesting that even a deficiency in both S100A8 and S100A9 proteins was compatible with viable and mature neutrophils. Surprisingly, the invasion of S100A9-deficient leukocytes into the peritoneum and into the skin in vivo was indistinguishable from that in wild-type mice. However, stimulation of S100A9-deficient neutrophils with interleukin-8 in vitro failed to provoke an up-regulation of CD11b. Migration upon a chemotactic stimulus through an endothelial monolayer was markedly diminished in S100A9-deficient neutrophils. Attenuated chemokinesis of the S100A9-deficient neutrophils was observed by using a three-dimensional collagen matrix migration assay. The altered migratory behavior was associated with a microfilament system that was highly polarized in unstimulated S100A9-deficient neutrophils. Our data suggest that loss of the calcium-binding S100A9 protein reduces the responsiveness of the neutrophils upon chemoattractant stimuli at least in vitro. Alternative pathways for neutrophil emigration may be responsible for the lack of any effect in the two in vivo models we have investigated so far.     

Copurification of P6, MRP8, and MRP14 from Human Granulocytes and Separation of Individual Proteins

A method is described for purification of P6, MRP8, and MRP14, three calcium-binding proteins assigned to the S100 protein family. The purification procedure included preparation of human granulocytes, ammonium sulfate precipitation, and anion-exchange chromatography and resulted in the copurification of P6, MRP8, and MRP14. Individual proteins were separated by either preparative isoelectric focusing or preparative SDS–PAGE. The procedure was carried out in the course of 4 days and yielded several milligrams of essentially pure P6, MRP8, and MRP14 in either native or denatured form.     

Monolayer formation of human osteoblastic cells on vertically aligned multiwalled carbon nanotube scaffolds

Monolayer formation of SaOS‐2 (human osteoblast‐like cells) was observed on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds without purification or functionalization. The VACNT were produced by a microwave plasma chemical vapour deposition on titanium surfaces with nickel or iron as catalyst. Cell viability and morphology studies were evaluated by LDH (lactate dehydrogenase) release assay and SEM (scanning electron microscopy), respectively. The non‐toxicity and the flat spreading with monolayer formation of the SaOs‐2 on VACNT scaffolds surface indicate that they can be used for biomedical applications.