Structural basis for endothelial nitric oxide synthase binding to calmodulin

The enzyme nitric oxide synthase (NOS) is exquisitely regulated in vivo by the Ca(2+) sensor protein calmodulin (CaM) to control production of NO, a key signaling molecule and cytotoxin. The differential activation of NOS isozymes by CaM has remained enigmatic, despite extensive research. Here, the crystallographic structure of Ca(2+)-loaded CaM bound to a 20 residue peptide comprising the endothelial NOS (eNOS) CaM-binding region establishes their individual conformations and intermolecular interactions, and suggests the basis for isozyme-specific differences. The alpha-helical eNOS peptide binds in an antiparallel orientation to CaM through extensive hydrophobic interactions. Unique NOS interactions occur with: (i). the CaM flexible central linker, explaining its importance in NOS activation; and (ii). the CaM C-terminus, explaining the NOS-specific requirement for a bulky, hydrophobic residue at position 144. This binding mode expands mechanisms for CaM-mediated activation, explains eNOS deactivation by Thr495 phosphorylation, and implicates specific hydrophobic residues in the Ca(2+) independence of inducible NOS.     

Comparison of the structural and physical properties of human hair eumelanin following enzymatic or acid/base extraction

Eumelanin was isolated from a sample of black, Indonesian human hair using three different published procedures: two different acid/base extractions and an enzymatic extraction. The morphology and spectroscopic properties of the isolated pigments differ significantly. The acid/base procedures both yield an amorphous material, while enzymatic extraction yields ellipsoidal melanosomes. Amino acid analysis shows that there is protein associated with the isolated pigments, accounting for 52, 40 and 14% of the total mass for the two acid/base extractions and the enzymatic extraction, respectively. The amino acid compositions do not correlate with those of keratin or tyrosinase. Metal elemental analysis shows that the acid/base extraction removes a majority of many metal ions bound to the pigment. Chemical degradation analysis by KMnO4/H+ and H2O2/OH- indicates significant differences between the pigments isolated by acid/base and enzymatic extraction. After correction for the protein mass in the two pigments, the lower yields of both pyrrole-2,3,5-tricarboxylic acid and pyrrole-2,3-dicarboxylic acid, eumelanin degradation products, indicate acid/base extraction modifies the chemical structure of the melanin, consistent with the result of Soluene solubilization assay. While the optical absorption spectra of the bulk pigments are similar, the spectra of the molecular weight less than 1000 mass fractions differ significantly. The data clearly indicate that pigment obtained from human hair by acid/base extraction contains significant protein, exhibits destruction of the melanosome, and possesses altered molecular structure. The acid/base extracted hair melanin is not representative of the natural material and is a poor model system for studying the physical and biological properties of melanins. The enzymatically extracted hair melanin, on the contrary, retains the morphology of intact melanosomes and is an excellent source of human melanin.     

Temporal contribution of body movement to very long-term heart rate variability in humans

A newly developed, very long-term ( approximately 7 days) ambulatory monitoring system for assessing beat-to-beat heart rate variability (HRV) and body movements (BM) was used to study the mechanism(s) responsible for the long-period oscillation in human HRV. Data continuously collected from five healthy subjects were analyzed by 1) standard auto- and cross-spectral techniques, 2) a cross-Wigner distribution (WD; a time-frequency analysis) between BM and HRV for 10-s averaged data, and 3) coarse-graining spectral analysis for 600 successive cardiac cycles. The results showed 1) a clear circadian rhythm in HRV and BM, 2) a 1/f (beta)-type spectrum in HRV and BM at ultradian frequencies, and 3) coherent relationships between BM and HRV only at specific ultradian as well as circadian frequencies, indicated by significant (P < 0.05) levels of the squared coherence and temporal localizations of the covariance between BM and HRV in the cross-WD. In a single subject, an instance in which the behavioral (mean BM) and autonomic [HRV power >0.15 Hz and mean heart rate (HR)] rhythmicities were dissociated occurred when the individual had an irregular daily life. It was concluded that the long-term HRV in normal humans contained persistent oscillations synchronized with those of BM at ultradian frequencies but could not be explained exclusively by activity levels of the subjects.     

Morphologic differentiation of HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori VacA sensitivity

Phorbol 12-myristate 13-acetate (PMA) induces differentiation of human leukemic HL-60 cells into cells with macrophage-like characteristics and enhances the susceptibility of HL-60 cells to the Helicobacter pylori VacA toxin (de Bernard, M., Moschioni., M., Papini, E., Telford, J. L., Rappuoli, R., and Montecucco, C. (1998) FEBS Lett. 436, 218-222). We examined the mechanism by which HL-60 cells acquire sensitivity to VacA, in particular, looking for expression of RPTPbeta, a VacA-binding protein postulated to be the VacA receptor (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693-36699). PMA induced expression of RPTPbeta mRNA and protein as determined by RNase protection assay and indirect immunofluorescence studies, respectively. Vitamin D(3) and interferon-gamma, which stimulate differentiation of HL-60 cells into monocyte-like cells, also induced VacA sensitivity and expression of RPTPbeta mRNA, whereas 1. 2% Me(2)SO and retinoic acid, which stimulated the maturation of HL-60 into granulocyte-like cells, did not. RPTPbeta antisense oligonucleotide inhibited induction of VacA sensitivity and expression of RPTPbeta. Double immunostaining studies also indicated that newly expressed RPTPbeta colocalized with VacA in PMA-treated HL-60 cells. In agreement with these data, BHK-21 cells, which are insensitive to VacA, when transfected with the RPTPbeta cDNA, acquired VacA sensitivity. All data are consistent with the conclusion that acquisition of VacA sensitivity by PMA-treated HL-60 cells results from induction of RPTPbeta, a protein that functions as the VacA receptor.     

Bacteriological Assessment of Healthcare-Associated Pneumonia Using a Clone Library Analysis

Background

The causative pathogens of healthcare-associated pneumonia (HCAP) remain controversial, and the use of conventional cultivation of sputum samples is occasionally inappropriate due to the potential for oral bacterial contamination. It is also sometimes difficult to determine whether methicillin-resistant Staphylococcus aureus (MRSA) is a true causative pathogen of HCAP.

Methods

We evaluated the bacterial diversity in bronchoalveolar lavage fluid (BALF) using molecular and cultivation methods in 82 HCAP patients. BALF specimens were obtained from the lesions of pneumonia using bronchoscopy. The bacterial flora was analyzed according to the clone library method using amplified fragments of the 16S ribosomal RNA gene with universal primers. In addition, sputum cultures and the above specimens were assessed.

Results

Eighty (97.6%) of the 82 BALF samples obtained from the patients with HCAP showed positive polymerase chain reaction results. The predominant phylotypes detected in the BALF in this study included bacteria common in cases of community- and hospital-acquired pneumonia. In addition, the phylotypes of streptococci and anaerobes were detected in 19 (23.2%) and 8 (9.8%) cases, respectively. In particular, phylotypes of streptococci were highly detected among the patients 75 of age or older. Staphylococcus aureus was cultured in 23 (28.0%) cases using conventional cultivation methods and detected in only 6 (7.3%) cases as predominant phylotypes according to the clone library method.

Conclusions

The clone library analysis of BALF in the HCAP patients detected heterogeneous bacteria and a high incidence of streptococci compared with that observed using cultivation methods. In addition, the results of our study may indicate a lower incidence of MRSA than previously expected in HCAP patients.     

Co-operative Bmp- and Fgf-signaling inputs convert skin wound healing to limb formation in urodele amphibians

Urodele amphibians have remarkable organ regeneration capability, and their limb regeneration capability has been investigated as a representative phenomenon. In the early 19th century, nerves were reported to be an essential tissue for the successful induction of limb regeneration. Nerve substances that function in the induction of limb regeneration responses have long been sought. A new experimental system called the accessory limb model (ALM) has been established to identify the nerve factors. Skin wounding in urodele amphibians results in skin wound healing but never in limb induction. However, nerve deviation to the wounded skin induces limb formation in ALM. Thus, nerves can be considered to have the ability to transform skin wound healing to limb formation. In the present study, co-operative Bmp and Fgf application, instead of nerve deviation, to wounded skin transformed skin wound healing to limb formation in two urodele amphibians, axolotl (Ambystoma mexicanum) and newt (Pleurodeles waltl). Our findings demonstrate that defined factors can induce homeotic transformation in postembryonic bodies of urodele amphibians. The combination of Bmp and Fgf(s) may contribute to the development of novel treatments for organ regeneration.     

Functional expression of α7 nicotinic acetylcholine receptors in human periodontal ligament fibroblasts and rat periodontal tissues

Tobacco smoking is the main risk factor associated with chronic periodontitis, but the mechanisms that underlie this relationship are largely unknown. Recent reports proposed that nicotine plays an important role in tobacco-related morbidity by acting through the nicotinic acetylcholine receptors (nAChRs) expressed by non-neuronal cells. The aim of this study was to investigate whether α7 nAChR was expressed in periodontal tissues and whether it functions by regulating IL-1β in the process of periodontitis. In vitro, human periodontal ligament (PDL) cells were cultured with 10⁻¹² M of nicotine and/or 10⁻⁹ M of alpha-bungarotoxin (α-Btx), a α7 nAChR antagonist. The expression of α7 nAChR and IL-1β in PDL cells and the effects of nicotine/α-Btx administration on their expression were explored. In vivo, an experimental periodontitis rat model was established, and the effects of nicotine/α-Btx administration on expression of α7 nAChR and development of periodontitis were evaluated. We found that α7 nAChR was present in human PDL cells and rat periodontal tissues. The expressions of α7 nAChR and IL-1β were significantly increased by nicotine administration, whereas α-Btx treatment partially suppressed these effects. This study was the first to demonstrate the functional expression of α7 nAChR in human PDL cells and rat periodontal tissues. Our results may be pertinent to a better understanding of the relationships among smoking, nicotine, and periodontitis.     

A Highlights from MBoC Selection: Involvement of p114-RhoGEF and Lfc in Wnt-3a– and Dishevelled-Induced RhoA Activation and Neurite Retraction in N1E-115 Mouse Neuroblastoma Cells

The Wnt-induced planar cell polarity (PCP) signaling pathway is essential for polarized cell migration and morphogenesis. Dishevelled (Dvl) and its binding protein Daam1 mediate RhoA activation in this pathway. WGEF, a member of the Rho-guanine nucleotide exchange factor (Rho-GEF) family, was shown to play a role in Wnt-induced RhoA activation in Xenopus embryos. However, it has remained unknown which member(s) of a Rho-GEF family are involved in Wnt/Dvl-induced RhoA activation in mammalian cells. Here we identified p114-RhoGEF and Lfc (also called GEF-H1) as the Rho-GEFs responsible for Wnt-3a–induced RhoA activation in N1E-115 mouse neuroblastoma cells. We screened for Rho-GEF–silencing short-hairpin RNAs (shRNAs) that are capable of suppressing Dvl-induced neurite retraction in N1E-115 cells and found that p114-RhoGEF and Lfc shRNAs, but not WGEF shRNA, suppressed Dvl- and Wnt-3a–induced neurite retraction. p114-RhoGEF and Lfc shRNAs also inhibited Dvl- and Wnt-3a–induced RhoA activation, and p114-RhoGEF and Lfc proteins were capable of binding to Dvl and Daam1. Additionally, the Dvl-binding domains of p114-RhoGEF and Lfc inhibited Dvl-induced neurite retraction. Our results suggest that p114-RhoGEF and Lfc are critically involved in Wnt-3a– and Dvl-induced RhoA activation and neurite retraction in N1E-115 cells.