Building-block architecture of botulinum toxin complex: Conformational changes provide insights into the hemagglutination ability of the complex

Clostridium botulinum produces the botulinum neurotoxin (BoNT). Previously, we provided evidence for the “building-block” model of botulinum toxin complex (TC). In this model, a single BoNT is associated with a single nontoxic nonhemagglutinin (NTNHA), yielding M-TC; three HA-70 molecules are attached and form M-TC/HA-70, and one to three “arms” of the HA-33/HA-17 trimer (two HA-33 and one HA-17) further bind to M-TC/HA-70 via HA-17 and HA-70 binding, yielding one-, two-, and three-arm L-TC. Of all TCs, only the three-arm L-TC caused hemagglutination. In this study, we determined the solution structures for the botulinum TCs using small-angle X-ray scattering (SAXS). The mature three-arm L-TC exhibited the shape of a “bird spreading its wings”, in contrast to the model having three “arms”, as revealed by transmission electron microscopy. SAXS images indicated that one of the three arms of the HA-33/HA-17 trimer bound to both HA-70 and BoNT. Taken together, these findings regarding the conformational changes in the building-block architecture of TC may explain why only three-arm L-TC exhibited hemagglutination.     

Effect of heat stress or lipopolysaccharide (E. coli) injection on HSP70 levels in the liver and brain of adrenalectomized rats

Hsp70 content (ng Hsp70 μg total protein⁻¹) in the liver and brain of control and adrenalectomized male rats was investigated by Western Blotting after heat stress (40°C) or endotoxin-induced fever (E. coli lipopolysaccharide injection). The increase in rectal temperature was higher after heat stress than after LPS injection. Heat stress affected Hsp70 content of the liver, but not of the brain; however adrenalectomy did not influence any parameter. These results suggest that, under these circumstances, there is no relationship between the hypothalamic–pituitary–adrenal axis and Hsp70 synthesis in liver and brain.     

Vectoring of Hypocrea Nigricans (Hypocreales: Hypocreaceae) by Three Fungivorous Mite Species (Acari: Acaridae)

A bioassay was developed to quantify germination rates of Hypocrea nigricans spores passing through the alimentary canals of the three acarid mite species Tyrophagus putrescentiae, Histiogaster sp. and Schwiebea sp. These mites are suspected of causing damage to mushroom production in Japan. All three species feed on the fungus and excrete fungal spores and fungal particles. Fecal material of the mites was plated on malt agar plates supplemented with 1% CuSO₄ to assess spore germination. Germination rates, and hence the possibility for the mites to disseminate spores of H. nigricans, was so high that these mites may indeed be important vectors of weed fungal spores in indoor mushroom production units.     

Angiotensin II type 2 receptor antagonist reduces bleomycin-induced pulmonary fibrosis in mice

Background

The role of angiotensin II type 2 receptor (AT2) in pulmonary fibrosis is unknown. To evaluate the influence of angiotensin II type 1 receptor (AT1) and AT2 antagonists in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis.

Methods

We examined effects of the AT1 antagonist (AT1A) olmesartan medoxomil (olmesartan) and the AT2 antagonist (AT2A) PD-123319 on BLM-induced pulmonary fibrosis, which was evaluated by Ashcroft''s pathological scoring and hydroxyproline content of lungs. We also analyzed the cellular composition and cytokine levels in bronchoalveolar lavage fluid (BALF).

Results

With olmesartan, the lung fibrosis score and hydroxyproline level were significantly reduced, and lymphocyte and neutrophil counts and tumor necrosis factor (TNF)-α levels in BALF were reduced on day 7. On day 14, macrophage and lymphocyte counts in BALF were reduced, accompanied by a reduction in the level of transforming growth factor (TGF)-β₁. With PD-123319, the lung fibrosis score and hydroxyproline level were reduced. On day 7, macrophage, lymphocyte, and neutrophil counts in BALF were reduced, accompanied by reductions in TNF-α and monocyte chemoattractant protein (MCP)-1 levels. On day 14, macrophage, lymphocyte, and neutrophil counts in BALF were also reduced, accompanied by a reduction in the level of macrophage inflammatory protein (MIP)-2 level but not TGF-β₁.

Conclusion

Both AT1 and AT2 are involved in promoting interstitial pneumonia and pulmonary fibrosis via different mechanisms of action.     

The distribution of Abcc6 in normal mouse tissues suggests multiple functions for this ABC transporter.

We have studied the tissue distribution of Abcc6, a member of the ABC transmembrane transporter subfamily C, in normal C57BL/6 mice. RNase protection assays revealed that although almost all tissues studied contained detectable levels of the mRNA encoding Abcc6, the highest levels of Abcc6 mRNA were found in the liver. In situ hybridization (ISH) demonstrated abundant Abcc6 mRNA in epithelial cells from a variety of tissues, including hepatic parenchymal cells, bile duct epithelia, kidney proximal tubules, mucosa and gland cells of the stomach, intestine, and colon, squamous epithelium of the tongue, corneal epithelium of the eye, keratinocytes of the skin, and tracheal and bronchial epithelium. Furthermore, we detected Abcc6 mRNA in arterial endothelial cells, smooth muscle cells of the aorta and myocardium, in circulating leukocytes, lymphocytes in the thymus and lymph nodes, and in neurons of the brain, spinal cord, and the specialized neurons of the retina. Immunohistochemical analysis using a polyclonal Abcc6 rabbit antibody confirmed the tissue distribution of Abcc6 suggested by our ISH studies and revealed the cellular localization of Abcc6 in the basolateral plasma membrane in the epithelial cells of proximal convoluted tubules in the kidney. Although the function of Abcc6 is unknown, mutations in the human ABCC6 gene result in a heritable disorder of connective tissue called pseudoxanthoma elasticum (PXE). Our results demonstrating the presence of Abcc6 in epithelial and endothelial cells in a variety of tissues, including those tissues affected in PXE patients, suggest a possible role for Abcc6 in the normal assembly of extracellular matrix components. However, the presence of Abcc6 in neurons and leukocytes, two cell populations not associated with connective tissue, also suggests a more complex multifunctional role for Abcc6.     

Evidence that electron-dense bodies in Cyanidium caldarium have an iron-storage role

The acidophilic and thermophilic unicellular red alga, Cyanidium caldarium (Tilden) Geitler, is widely distributed in acidic hot springs. Observation by transmission electron microscopy (TEM) showed that algae grown in Allen's medium contained electron-dense bodies with diameters from 100 to 200 nm. Electron dispersive x-ray analysis indicated that the electron-dense bodies contained high levels of iron, phosphorous, and oxygen; P/Fe ratios were from 1.3 to 2.0. The electron spin resonance (ESR) spectrum of the intact C. caldarium cells showed an isotropic signal at a g value of 2.00. Density-gradient centrifugation of the cell lysate yielded a fraction that included substances showing the isotropic ESR signal. EDTA treatment of this fraction reduced the ESR signal intensity, whereas it increased a signal that is typical of Fe(III)-EDTA. The fact that the isotropic signal dominates the ESR spectrum, together with a previous finding that iron is confined to the electron-dense bodies, led us to conclude that iron in the electron-dense bodies accounts for the isotropic ESR signal. Since the intensity of the ESR signal depends on the amount of iron in the cells, the electron-dense bodies are probably iron storage sites.     

Fluorescent aptasensors based on conformational adaptability of abasic site-containing aptamers in combination with abasic site-binding ligands

Aptamers are nucleic acids that can selectively bind to a variety of targets. Aptamers usually undergo conformational transitions from a flexible or disordered structure into a rigid or ordered structure upon target-binding. This study describes a detection method for l-argininamide (l-Arm) and adenosine based on the conformational adaptability of nucleic acid aptamers. An abasic site (AP site) was formed in the stem and close to the target-binding site of a stem-loop aptamer as an anchoring pocket for a fluorescent ligand. 3,5-Diamino-6-chloro-2-pyrazine carbonitrile (DCPC), which can bind to AP site-containing DNA duplexes by pseudo-base pairing, was utilized as a signaling reporter for the target-binding. The binding of a target to an aptamer induces the tight pairing of bases flanking the AP site, so that DCPC can effectively bind to the stem. The binding of DCPC is accompanied by a significant enhancement of its fluorescence. This new sensing method without an antisense DNA strand was demonstrated by using l-Arm and its aptamer as a model. It was confirmed that the method can sensitively detect l-Arm with a detection limit of 2.1 μM. The proposed method was also applied to adenosine detection, where the reported sequence of an adenosine aptamer was slightly modified. The method based on an AP site-containing aptamer and an AP site-binding ligand was applicable to detection of a target in horse serum.