Integrin conformational regulation: uncoupling extension/tail separation from changes in the head region by a multiresolution approach

Integrin-dependent adhesion and signaling are regulated by conformational changes whose details remain controversial. Crystallography revealed bent shapes for resting and primed integrin ectodomains, whereas large, ligand-induced rearrangements in other constructs suggested extension, "opening," and tail separation. We have used experimental/computed hydrodynamics to discriminate among different alpha(v)beta(3) and alpha(IIb)beta(3) atomic models built on X-ray, NMR, and EM data. In contrast with X-ray structures and EM maps, hydrodynamics indicate that resting integrins are already extended. Furthermore, the hydrodynamics of an alpha(v)beta(3) ectodomain-fibronectin fragment complex support opening via additional head region conformational changes (hybrid domain swing-out), but without tail separation. Likewise, frictional changes induced by priming agents in full-length alpha(IIb)beta(3) correlate well with the swing-out coupled to a simple transmembrane helix shift in an extended, electron tomography-based model. Extension and immediate tail separation are then uncoupled from head region rearrangements following activation, thus underscoring integrins' delicate, finely tuned plasticity.     

A new matrix protein family related to the nacreous layer formation of Pinctada fucata

We have isolated a new matrix protein family (N16) which is specific to the nacreous layer of the Japanese pearl oyster, Pinctada fucata, and have cloned and characterized the cDNAs coding for the components. Analysis of the deduced amino acid sequence revealed that N16 showed no definitive homology with other proteins. The in vitro studies of the crystallization clarified that N16 induced aragonite crystals when fixed on the substrate but inhibited crystal formation without it. The aragonite crystals showed platy morphology different from those formed inorganically, and long intervals of incubation resulted in crystalline layers highly similar to the nacreous layer.     

Regeneration of flowering plants from difficile lily protoplasts by means of a nurse culture

The regeneration of difficile lily protoplasts isolated from suspension cells of the Oriental hybrid lily ( Lilium L.) cultivars Casablanca, Siberia and Acapulco was achieved by using the nurse-culture method. The divided protoplasts grew into colonies with nurse cells that have no regeneration ability, and developed to visible calli on a medium containing picloram. Many plantlets were formed on the calli after transfer of the proliferated calli to hormone-free medium. We were able to transplant the plantlets to soil in pots without acclimatization, and the plantlets grew in a greenhouse until flowering 2 years later.     

Improving the Accuracy of NMR Structures of Large Proteins Using Pseudocontact Shifts as Long-Range Restraints

We demonstrate improved accuracy in protein structure determination for large (>/=30 kDa), deuterated proteins (e.g. STAT4(NT)) via the combination of pseudocontact shifts for amide and methyl protons with the available NOEs in methyl-protonated proteins. The improved accuracy is cross validated by Q-factors determined from residual dipolar couplings measured as a result of magnetic susceptibility alignment. The paramagnet is introduced via binding to thiol-reactive EDTA, and multiple sites can be serially engineered to obtain data from alternative orientations of the paramagnetic anisotropic susceptibility tensor. The technique is advantageous for systems where the target protein has strong interactions with known alignment media.     

Three oxidative metabolites of indole-3-acetic acid from Arabidopsis thaliana

Three metabolites of indole-3-acetic acid (IAA), N-(6-hydroxyindol-3-ylacetyl)-phenylalanine (6-OH-IAA-Phe), N-(6-hydroxyindol-3-ylacetyl)-valine (6-OH-IAA-Val), and 1-O-(2-oxoindol-3-ylacetyl)-beta-d-glucopyranose (OxIAA-Glc), were found by a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based search for oxidative IAA metabolites during the vegetative growth of Arabidopsis. Their structures were confirmed by making a comparison of chromatographic characteristics and mass spectra between naturally occurring compounds and synthetic standards. An incorporation study using deuterium-labeled compounds showed that 6-OH-IAA-Phe and 6-OH-IAA-Val were biosynthesized from IAA-Phe and IAA-Val, respectively, which strongly suggested the formation of these amino acid conjugates of IAA in plants. Both 6-OH-IAA-Phe and 6-OH-IAA-Val were inactive as auxins, as indicated by no significant root growth inhibition in Arabidopsis. Quantitative analysis demonstrated that OxIAA-Glc was present in the largest amount among the metabolites of IAA in Arabidopsis, suggesting that the conversion into OxIAA-Glc represents the main metabolic process regarding IAA in Arabidopsis.     

Prevalence of Mental Illness,Cognitive Disability,and Their Overlap among the Homeless in Nagoya,Japan

Background

While the prevalence of mental illness or cognitive disability is higher among homeless people than the general population in Western countries, few studies have investigated its prevalence in Japan or other Asian countries. The present study conducted a survey to comprehensively assess prevalence of mental illness, cognitive disability, and their overlap among homeless individuals living in Nagoya, Japan.

Methods

Participants were 114 homeless individuals. Mental illness was diagnosed based on semi-structured interviews conducted by psychiatrists. The Wechsler Adult Intelligence Scale-III (WAIS-III, simplified version) was used to diagnose intellectual/ cognitive disability.

Results

Among all participants, 42.1% (95% CI 33.4–51.3%) were diagnosed with a mental illness: 4.4% (95% CI 1.9–9.9%) with schizophrenia or other psychotic disorder, 17.5% (95% CI 11.6–25.6%) with a mood disorder, 2.6% (95% CI 0.9–7.5%) with an anxiety disorder, 14.0% (95% CI 8.8–21.6%) with a substance-related disorder, and 3.5% (95% CI 1.4–8.8%) with a personality disorder. Additionally, 34.2% (95% CI 26.1–43.3%) demonstrated cognitive disability: 20.2% (95% CI 13.8–28.5%) had mild and 14.0% (95% CI 8.8–21.6%) had moderate or severe disability. The percent overlap between mental illness and cognitive disability was 15.8% (95% CI 10.2–23.6%). Only 39.5% (95% CI 26.1–43.3%) of the participants were considered to have no psychological or cognitive dysfunction. Participants were divided into four groups based on the presence or absence of mental illness and/or cognitive disability. Only individuals with a cognitive disability reported a significant tendency toward not wanting to leave their homeless life.

Conclusion

This is the first report showing that the prevalence of mental illness and/or cognitive disability among homeless individuals is much higher than in the general Japanese population. Appropriate support strategies should be devised and executed based on the specificities of an individual’s psychological and cognitive condition.     

Species differences for stereoselective hydrolysis of propranolol prodrugs in plasma and liver

Species differences and substrate specificities for the stereoselective hydrolysis of fifteen O-acyl propranolol (PL) prodrugs were investigated in pH 7.4 Tris-HCl buffer and rat and dog plasma and liver subfractions. The (R)-isomers were preferentially converted to propranolol (PL) in both rat and dog plasma with the exception of isovaleryl-PL in rat plasma, although the hydrolytic activities of prodrugs in rat plasma were 5–119-fold greater than those in dog plasma. The prodrugs with promoieties (C(=O)CH(R)CH₃) based on propionic acid showed marked preference for hydrolysis of the (R)-enantiomers in plasma from both species (R/S ratio 2.5–18.2). On the other hand, the hepatic hydrolytic activities of prodrugs were greater in dog than rat, especially in cytosolic fractions. The hydrolytic activity was predominantly located in microsomes of the liver in rat, while the cytosol also contributed to hepatic hydrolysis in dog. Hepatic microsomal hydrolysis in dog showed a preference for the (R)-isomers except acetyl- and propionyl-PL. Interestingly, in rat liver all types of prodrugs with substituents of small carbon number showed (S)-preference for hydrolysis. The hydrolyses of (R)- and (S)-isomers of straight chain acyl esters in rat liver microsomes were linearly and parabolically related with the carbon number of substituents, respectively, while these relationships were linear for both isomers in dogs. Chirality 9:661–666, 1997. © 1997 Wiley-Liss, Inc.     

Localization of TRH-sensitive sites in rat brain mediating intestinal transit

Stereotaxic microinjection of thyrotropin releasing hormone (TRH) into 16 brain areas revealed that only three sites, the medial septum and the lateral and anterior hypothalami, were sensitive to a 1.0 ug dose in stimulating intestinal transit in anesthetized rats. The medial septum and anterior hypothalamus also responded to 0.1 ug, but not to 0.01 ug, of TRH. Because TRH and its receptors are distributed in these brain areas, the present results suggest a possible role for this peptide in the central regulation of gastrointestinal activity.     

Involvement of oxidative stress in hydroquinone-induced cytotoxicity in catalase-deficient Escherichia coli mutants

Hydroquinone is a benzene-derived metabolite. To clarify whether the reactive oxygen species (ROS) are involved in hydroquinone-induced cytotoxicity, we constructed transformants of Escherichia coli (E. coli) strains that express mammalian catalase gene derived from catalase mutant mice (Cs^b, Cs^c) and the wild-type (Cs^a) using a catalase-deficient E. coli UM255 as a recipient. Specific catalase activities of these tester strains were in order of Cs^a > Cs^c > Cs^b > UM255, and their susceptibility to hydrogen peroxide (H₂O₂) showed UM255 > Cs^b > Cs^c > Cs^a. We found that hydroquinone exposure reduced the survival of catalase-deficient E. coli mutants in a dose-dependent manner significantly, especially in the strains with lower catalase activities. Hydroquinone toxicity was also confirmed using zone of inhibition test, in which UM255 was the most susceptible, showing the largest zone of growth inhibition, followed by Cs^b, Cs^c and Cs^a. Furthermore, we found that hydroquinone-induced cell damage was inhibited by the pretreatment of catalase, ascorbic acid, dimethyl sulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA), and augmented by superoxide dismutase (both CuZnSOD and MnSOD). The present results suggest that H₂O₂ is probably involved in hydroquinone-induced cytotoxicity in catalase-deficient E. coli mutants and catalase plays an important role in protection of the cells against hydroquinone toxicity.     

Teaching high-resolution nuclear magnetic resonance to graduate students in biophysics.

Teaching modern methods of high resolution, multi-dimensional nuclear magnetic resonance (NMR) spectroscopy in the limited time usually available is a challenge because of the breadth and complexity of the subject. Here we present two outlines for lectures on basic principles and biophysical applications of NMR, either in one lecture or in six to twelve lectures. We advocate emphasizing the versatility of NMR, and its numerous applications to biophysical questions. An annotated list of references is provided.