Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins which contain classical monopartite and bipartite nuclear localization sequences (NLSs), and facilitates their transport into the nucleus. To determine the structural basis of the recognition of the two classes of NLSs by mammalian importin-alpha, we co-crystallized an N-terminally truncated mouse receptor protein with peptides corresponding to the monopartite NLS from the simian virus 40 (SV40) large T-antigen, and the bipartite NLS from nucleoplasmin. We show that the monopartite SV40 large T-antigen NLS binds to two binding sites on the receptor, similar to what was observed in yeast importin-alpha. The nucleoplasmin NLS-importin-alpha complex shows, for the first time, the mode of binding of bipartite NLSs to the receptor. The two basic clusters in the NLS occupy the two binding sites used by the monopartite NLS, while the sequence linking the two basic clusters is poorly ordered, consistent with its tolerance to mutations. The structures explain the structural basis for binding of diverse NLSs to the sole receptor protein. (C) 2000 Academic Press.

Synthesis and structure elucidation of kappa-casein (45-87) - A two dimensional nuclear magnetic resonance study

We have shown that 44 amino acid residues N-terminal segment of kappa-casein exhibits considerable a-helical structure. This prompted us to investigate the structures of the remaining segments of kappa-casein. Thus, in this study the chemical synthesis and structure elucidation of the peptide 45-87 amino acid residues of kappa-casein is reported. The peptide was assembled using solid phase peptide synthesis methodology on pam resin, cleaved via HF, freeze dried and, after purification, characterised by mass spectrometry (observed m/z 4929; calculated mit 4929.83). The amino acid sequence of the peptide is: CKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKA Its structure elucidation has been carried out using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques. CD spectrum of the peptide shows it to be a random structure in water but in 30% trifluoroethanol the peptide exhibits considerable structure. The 1D and 2D NMR spectra corroborated the results of CD. The structure elucidation of the peptide using TOCSY and NOESY NMR techniques will be discussed.

Intra- and interspecific divergence in the nuclear sequences of the clock gene period in species of the Drosophila buzzatii cluster

We have measured nucleotide variation in the CLOCK/CYCLE heterodimer inhibition domain (CCID) of the clock X-linked gene period in seven species belonging to the Drosophila buzzatii cluster, namely D. buzzatii, Drosophila koepferae, Drosophila antonietae, Drosophila serido, Drosophila gouveai, Drosophila seriema and Drosophila borborema. We detected that the purifying selection is the main force driving the sequence evolution in period, in agreement with the important role of CCID in clock machinery. Our survey revealed that period provides valuable phylogenetic information that allowed to resolve phylogenetic relationships among D. gouveai, D. borborema and D. seriema, which composed a polytomic clade in preliminary studies. The analysis of patterns of intraspecific variation revealed two different lineages of period in D. koepferae, probably reflecting introgressive hybridization from D. buzzatii, in concordance with previous molecular data.

Mapping cortical change in Alzheimer's disease, brain development, and schizophrenia

This paper describes algorithms that can identify patterns of brain structure and function associated with Alzheimer's disease, schizophrenia, normal aging, and abnormal brain development based on imaging data collected in large human populations. Extraordinary information can be discovered with these techniques: dynamic brain maps reveal how the brain grows in childhood, how it changes in disease, and how it responds to medication. Genetic brain maps can reveal genetic influences on brain structure, shedding light on the nature-nurture debate, and the mechanisms underlying inherited neurobehavioral disorders. Recently, we created time-lapse movies of brain structure for a variety of diseases. These identify complex, shifting patterns of brain structural deficits, revealing where, and at what rate, the path of brain deterioration in illness deviates from normal. Statistical criteria can then identify situations in which these changes are abnormally accelerated, or when medication or other interventions slow them. In this paper, we focus on describing our approaches to map structural changes in the cortex. These methods have already been used to reveal the profile of brain anomalies in studies of dementia, epilepsy, depression, childhood and adult-onset schizophrenia, bipolar disorder, attention-deficit/ hyperactivity disorder, fetal alcohol syndrome, Tourette syndrome, Williams syndrome, and in methamphetamine abusers. Specifically, we describe an image analysis pipeline known as cortical pattern matching that helps compare and pool cortical data over time and across subjects. Statistics are then defined to identify brain structural differences between groups, including localized alterations in cortical thickness, gray matter density (GMD), and asymmetries in cortical organization. Subtle features, not seen in individual brain scans, often emerge when population-based brain data are averaged in this way. Illustrative examples are presented to show the profound effects of development and various diseases on the human cortex. Dynamically spreading waves of gray matter loss are tracked in dementia and schizophrenia, and these sequences are related to normally occurring changes in healthy subjects of various ages. (C) 2004 Published by Elsevier Inc.

Thermal analysis, nuclear magnetic resonance spectroscopy, and impedance spectroscopy of N,N-dimethyl-pyrrolidinium iodide: An ionic solid exhibiting rotator phases

N,N-dimethyl-pyrrolidinium iodide has been investigated using differential scanning calorimetry, nuclear magnetic resonance (NMR) spectroscopy, second moment calculations, and impedance spectroscopy. This pyrrolidinium salt exhibits two solid-solid phase transitions, one at 373 K having an entropy change, Delta S, of 38 J mol(-1) K-1 and one at 478 K having Delta S of 5.7 J mol(-1) K-1. The second moment calculations relate the lower temperature transition to a homogenization of the sample in terms of the mobility of the cations, while the high temperature phase transition is within the temperature region of isotropic tumbling of the cations. At higher temperatures a further decrease in the H-1 NMR linewidth is observed which is suggested to be due to diffusion of the cations. (C) 2005 American Institute of Physics.

Conformational dynamics of thyroid hormones by variable temperature nuclear magnetic resonance: The role of side chain rotations and cisoid/transoid interconversions

H-1 NMR spectra of the thyroid hormone thyroxine recorded at low temperature and high field show splitting into two peaks of the resonance due to the H2,6 protons of the inner (tyrosyl) ring. A single resonance is observed in 600 MHz spectra at temperatures above 185 K. An analysis of the line shape as a function of temperature shows that the coalescence phenomenon is due to an exchange process with a barrier of 37 kJ mol(-1). This is identical to the barrier for coalescence of the H2',6' protons of the outer (phenolic) ring reported previously for the thyroid hormones and their analogues. It is proposed that the separate peaks at low temperature are due to resonances for H2,6 in cisoid and transoid conformers which are populated in approximately equal populations. These two peaks are averaged resonances for the individual H2 and H6 protons. Conversion of cisoid to transoid forms can occur via rotation of either the alanyl side chain or the outer ring, from one face of the inner ring to the other. It is proposed that the latter process is the one responsible for the observed coalescence phenomenon. The barrier to rotation of the alanyl side chain is greater than or equal to 37 kJ mol(-1), which is significantly larger than has previously been reported for Csp(2)-Csp(3) bonds in other Ph-CH2-X systems. The recent crystal structure of a hormone agonist bound to the ligand-binding domain of the rat thyroid hormone receptor (Wagner et al. Nature 1995, 378, 690-697) shows the transoid form to be the bound conformation. The significant energy barrier to cisoid/transoid interconversion determined in the current study combined with the tight fit of the hormone to its receptor suggests that interconversion between the forms cannot occur at the receptor site but that selection for the preferred bound form occurs from the 50% population of the transoid form in solution.

Increased elastic microfibrils and thickening of fibroblastic nuclear lamina in canine cutaneous asthenia

Cutaneous asthenia is a hereditary connective tissue disease, primarily of dogs and cats, resembling Ehlers-Danlos syndrome in man. Collagen dysplasia results in skin hyperextensibility, skin and vessel fragility, and poor wound healing. The purpose of this study was to describe the histological findings in a dog with a collagenopathy consistent with cutaneous asthenia. An 8-month-old crossbreed female dog presented with lacerations and numerous atrophic and irregular scars. The skin was hyperextensible and easily torn by the slightest trauma. Ultrastructurally, the dermis was comprised of elaunin and oxytalan microfibrils. These are immature fibres in which the fibrillar component is increased but elastin is reduced. Collagen fibres were profoundly disorganized. The fibrils had a highly irregular outline and a corroded appearance when viewed in cross-section, and were spiralled and fragmented in a longitudinal view. Dermal fibroblasts displayed a conspicuous thickening of the nuclear lamina. Nuclear lamins form a fibrous nucleoskeletal network of intermediate-sized filaments underlying the inner nuclear membrane. Mutations in lamins or lamin-associated proteins cause a myriad of genetic diseases collectively called laminopathies. Disruption of the nuclear lamina seems to affect chromatin organization and transcriptional regulation of gene expression. A common link among all laminopathies may be a failure of stem cells to regenerate mesenchymal tissue. This could contribute to the connective tissue dysplasia seen in cutaneous asthenia.

The Cholinergic System in Mild Cognitive Impairment and Alzheimer`s Disease: An In Vivo MRI and DTI study

Few studies have investigated in vivo changes of the cholinergic basal forebrain in Alzheimer`s disease (AD) and amnestic mild cognitive impairment (MCI), an at risk stage of AD. Even less is known about alterations of cortical projecting fiber tracts associated with basal forebrain atrophy. In this study, we determined regional atrophy within the basal forebrain in 21 patients with AD and 16 subjects with MCI compared to 20 healthy elderly subjects using deformation-based morphometry of MRI scans. We assessed effects of basal forebrain atrophy on fiber tracts derived from high-resolution diffusion tensor imaging (DTI) using tract-based spatial statistics. We localized significant effects relative to a map of cholinergic nuclei in MRI standard space as determined from a postmortem brain. Patients with AD and MCI subjects showed reduced volumes in basal forebrain areas corresponding to anterior medial and lateral, intermediate and posterior nuclei of the Nucleus basalis of Meynert (NbM) as well as in the diagonal band of Broca nuclei (P < 0.01). Effects in MCI subjects were spatially more restricted than in AD, but occurred at similar locations. The volume of the right antero-lateral NbM nucleus was correlated with intracortical projecting fiber tract integrity such as the corpus callosum, cingulate, and the superior longitudinal, inferior longitudinal, inferior fronto-occipital, and uncinate fasciculus (P < 0.05, corrected for multiple comparisons). Our findings suggest that a multimodal MRI-DTI approach is supportive to determine atrophy of cholinergic nuclei and its effect on intracortical projecting fiber tracts in AD. Hum Brain Mapp 32: 1349-1362, 2011. (C) 2010 Wiley-Liss, Inc.

Brain Structural Variability due to Aging and Gender in Cognitively Healthy Elders: Results from the Sao Paulo Ageing and Health Study

BACKGROUND AND PURPOSE: Several morphometric MR imaging studies have investigated age- and sex-related cerebral volume changes in healthy human brains, most often by using samples spanning several decades of life and linear correlation methods. This study aimed to map the normal pattern of regional age-related volumetric reductions specifically in the elderly population. MATERIALS AND METHODS: One hundred thirty-two eligible individuals (67-75 years of age) were selected from a community-based sample recruited for the Sao Paulo Ageing and Health (SPAH) study, and a cross-sectional MR imaging investigation was performed concurrently with the second SPAH wave. We used voxel-based morphometry (VBM) to conduct a voxelwise search for significant linear correlations between gray matter (GM) volumes and age. In addition, region-of-interest masks were used to investigate whether the relationship between regional GM (rGM) volumes and age would be best predicted by a nonlinear model. RESULTS: VBM and region-of-interest analyses revealed selective foci of accelerated rGM loss exclusively in men, involving the temporal neocortex, prefrontal cortex, and medial temporal region. The only structure in which GM volumetric changes were best predicted by a nonlinear model was the left parahippocampal gyrus. CONCLUSIONS: The variable patterns of age-related GM loss across separate neocortical and temporolimbic regions highlight the complexity of degenerative processes that affect the healthy human brain across the life span. The detection of age-related Ill GM decrease in men supports the view that atrophy in such regions should be seen as compatible with normal aging.