Two cytosolic protein families implicated in lipid-binding: main structural and functional features

1. According to the important biological role of fatty acids and phospholipids in cell membranes, two cytosolic proteins implicated in their binding and transport in brain were considered, namely: Fatty Acid-Binding Protein and basic 21 kDa protein. 2. They were reviewed as well as their related protein families. 3. Although the two protein groups do not present significant sequence homologies, they share several similar properties and might thus be implicated in common physiological functions.     

Mitotic activity in non-neoplastic melanocytes in vivo as determined by histochemical, autoradiographic, and electron microscope studies

Mitotic figures were demonstrated in the differentiated melanocytes of normal epidermal and nonepidermal tissues without the presence of external stimuli. These dividine melanocytes were present in human and mouse skin, mouse hair, chick feathers, and embryonic chick retinal pigment epithelium. In normal adult human epidermis, dividing melanocytes, though rare, were found in the nonstimulated areas. L-3,4-dihydroxyphenylalanine reaction on the melanocytes during mitosis demonstrated activity of the melanin-forming enzyme, tyrosinase, and ultrastructural studies demonstrated the characteristic melanosomes in variour stages of maturation. Other ultrastructural characteristics of the melanocytes during mitosis, except for the Golgi apparatus, which was smaller and less complex, were similar to those seen in well-differentiated nondividing melanocytes. Autoradiographic studies of thymidine incorporation into mouse skin indicated that 0.7% of epidermal melanocytes, when slightly stimulated, are in the S phase. Thus, in vivo differentiation of non-neoplastic melanocytes (to produce pyrosinase and melanosomes) does not preclude their replication by mitotic division.     

Tilting of helix I and ligand-induced changes in the lactose permease determined by site-directed chemical cross-linking in situ

The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of lactose permease, each with a single Cys residue, were co-expressed, and cross-linking was studied. The proximity of paired Cys residues in helices I (positions 11, 14, 15, 18, 25, 28, 29, or 32) and VII (positions 227, 231, 232, 234, 235, 238, 239, 241, 242, 245, or 246) was studied by using homobifunctional thiol-specific chemical linkers of different lengths and chemical properties. The results demonstrate that Cys residues on one face of the periplasmic half of helix I (positions 32, 29, 28, or 25) cross-link to Cys residues on one face of the periplasmic half of helix VII (242 or 245). In contrast, no cross-linking is evident with paired Cys residues in the cytoplasmic halves of helices I (positions 11, 14, 15, or 18) and VII (positions 227, 230, 231, 232, 234, 235, 238, or 239). The results indicate that helices I and VII are in close proximity only at their periplasmic halves. Ligand binding decreases cross-linking efficiency of the Cys pair 28/245 or 25/242 with N, N'-o-phenylenedimaleimide (rigid 6 A) and increases efficiency with N,N'-p-phenylenedimaleimide (rigid 10 A) or 1,6-bismaleimidohexane (flexible 16 A), indicating that the inter-thiol distance is about 6 A in the absence of ligand and that ligand binding increases the distance up to 10 A. The inter-thiol distance for Cys pairs 29/245 or 32/245 is less than 6 A in the absence of ligand, and in the presence of ligand, distance increases to between 6 and 10 A. Taken together, the results indicate that ligand binding induces a translational or scissors-like rigid body movement of helix I and/or VII at the periplasmic interface between the helices.     

Conformational changes in conantokin-G induced upon binding of calcium and magnesium as revealed by NMR structural analysis

The apo- and metal-bound solution conformations of synthetic conantokin-G (con-G, G1Egamma gammaL5Q gamma NQgamma 10LIRgamma K15SN-CONH2, gamma = gamma-carboxyglutamic acid), an antagonist of N-methyl-D-aspartate receptor-derived neuronal ion channels, have been examined by one- and two-dimensional 1H NMR at neutral pH. A complete structure for the Mg2+-loaded peptide was defined by use of distance geometry calculations and was found to exist as an alpha-helix that spans the entire peptide. The alpha-helical nature of Mg2+/con-G was also supported by the small values (<5.5 Hz) of the 3JHNalpha coupling constants measured for amino acid residues 3-5, 8, 9, and 11-16, and the small values (<4 ppb/K) of the temperature coefficients observed for the alphaNH protons of residues 5-17. This conformation contrasted with that obtained for apo-con-G, which was nearly structureless in solution. Docking of Mg2+ into con-G was accomplished by use of the genetic algorithm/molecular dynamics simulation method, employing the NMR-derived Mg2+-loaded structure for initial coordinates in the midpoint calculations. For the 3 Mg2+/con-G model, it was found that binding of one Mg2+ ion is stabilized by oxygen atoms from three gamma-carboxylates of Gla3, Gla4, and Gla7; another Mg2+ is coordinated by two oxygen atoms, one from each of the gamma-carboxylates of Gla7; and a third metal ion through three donor oxygen atoms of gamma-carboxylates from Gla10 and Gla14. As shown from direct metal binding measurements to mutant con-G peptides, these latter two Gla residues probably stabilized the tightest binding Mg2+ ion. Circular dichroism studies of these same peptide variants demonstrated that all Gla residues contribute to the adoption of the Mg2+-dependent alpha-helical conformation in con-G. The data obtained in this investigation provide a molecular basis for the large conformational alteration observed in apo-con-G as a result of divalent cation binding and allow assessment of the roles of individual Gla residues in defining certain of the structure-function properties of con-G.     

The helix-hinge-helix structural motif in human apolipoprotein A-I determined by NMR spectroscopy

The conformation of a synthetic peptide of 46 residues from apoA-I was investigated by fluorescence, CD, and 2D NMR spectroscopies in lipid-mimetic environments. ApoA-I(142-187) is mainly unstructured in water but helical in SDS or dodecylphosphocholine (DPC), although the peptide only associates with DPC at approximately the critical micellar concentration. Solution structures of apoA-I(142-187) were determined by distance geometry calculations based on 450 (in DPC-d38) or 397 (in SDS-d25) NOE-derived distance restraints, respectively. Backbone RMSDs for superimposing the two helical regions 146-162 and 168-182 are 0.98 +/- 0.22 (2.38 +/- 0.20) and 1.99 +/- 0.42 (2.02 +/- 0.21) A in DPC (SDS), respectively. No interhelical NOE was found, suggesting that helix-helix interactions between the two helical domains in apoA-I(142-187) are unlikely. Similar average, curved helix-hinge-helix structures were found in both SDS and DPC micelles with the hydrophobic residues occupying the concave face, indicating that hydrophobic interactions dominate. Intermolecular NOESY experiments, performed in the presence of 50% protonated SDS, confirm that the two amphipathic helices and Y166 in the hinge all interact with the micelle. The involvement of Y166 in lipid binding is supported by fluorescence spectroscopy as well. On the basis of all the data above, we propose a model for the peptide-lipid complexes wherein the curved amphipathic helix-hinge-helix structural motif straddles the micelle. The peptide-aided signal assignment achieved for apoA-I(122-187) (66mer) and apoA-I suggests that such a structural motif is retained in the longer peptide and most likely in the intact protein.     

Synthesis of structural analogues of leukotriene B4 and their receptor binding activity

Structural analogues of leukotriene B4 (LTB4) were designed based on the plausible conformation of LTB4 (1). Joining C-7-C-9 of the conformer A or B into an aromatic ring system led to the discovery of benzene analogues 2, 4 and 6a. Joining C-4-C-9 of the conformer C or D into an aromatic ring system led to the discovery of analogues 3, 5 and 7. The compounds examined in this study were evaluated as to their inhibition of [3H] LTB4 binding to human neutrophils, and by a secondary intact human neutrophil functional assay for agonist/antagonist activity. The first analogues prepared, compounds 2-7, demonstrated moderate potency in the LTB4 receptor binding assay. The modification of these compounds by the introduction of another substituent into the aromatic ring produced a marked increase in receptor binding (28c, IC50 = 0.020 microM; 38c, IC50 = 0.020 microM; 52a, IC50 = 0.020 microM; 52b, IC50 = 0.018 microM). Most of these structural analogues of LTB4 demonstrated agonist activity. Of the analogues prepared in this study, only compound 57 demonstrated weak LTB4 receptor antagonist activity, at 10 microM.     

Characterization of striped bass growth hormone receptors by disulfide-bond reduction and cross-linking studies

Growth hormone (GH) receptors were analyzed in striped bass (Morone saxatilis) by addition of disulfide-bond reducing agents to radioreceptor assays and by cross-linking both striped bass and coho salmon (Oncorhynchus kisutch) crude membrane preparations to radiolabeled hormone. Dithiothreitol (DTT) caused a dose-dependent increase in specific binding of 125I-tilapia (Oreochromis mossambicus) GH to striped bass membrane preparations. Maximal enhancement of 3.4-fold was obtained with 1 mM DTT and 0.03 trypsin inhibitor units/ml of aprotinin. Addition of N-ethylmaleimide (NEM), which binds covalently to free sulfhydryl groups, decreased specific binding. Scatchard analysis of striped bass membrane preparations indicated a single class of GH receptors. Addition of DTT with aprotinin increased GH-binding site concentration from 278 to 507 fmol/mg, while the dissociation constant of 0.56 nM remained unchanged. Cross-linking 125I-tilapia GH to striped bass hepatic membrane preparations and 125I-salmon GH to coho salmon membrane preparations yielded two to three specifically labeled proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Endoglycosidase H treatment was without effect on specifically labeled proteins from either species. Following digestion with N-glycosidase F, relative molecular weights of specifically labeled 125I-GH complexes were reduced, suggesting that hepatic GH-binding proteins in striped bass and salmon are N-linked glycoproteins.     

Three tRNA binding sites in rabbit liver ribosomes and role of the intrinsic ATPase in 80S ribosomes from higher eukaryotes

Three tRNA binding sites have been found in organisms of all domains (former kingdoms) with only one exception: Four binding sites have been reported for cytoplasmic 80S ribosomes from rabbit liver. Therefore, the issue was reconsidered, and the data revealed that rabbit liver ribosomes contain three tRNA binding sites, underlining the universal character of this ribosomal feature. Furthermore, a first analysis of the role of the ribosome intrinsic ATPase was performed. This ATPase is found in ribosomes of higher eukarya but not in lower eukarya such as yeast or ribosomes of the domains archea and bacteria. The results suggest that the intrinsic ATPase fulfills the same function as the essential third elongation factor EF-3, an ATPase in higher fungi (yeast etc.), that facilitates the release of the deacylated tRNA from the E site.     

Towards a functional neuroanatomy of conscious perception and its modulation by volition: implications of human auditory neuroimaging studies

Conscious sensory perception and its modulation by volition are integral to human mental life. Functional neuroimaging techniques provide a direct means of identifying and characterizing in vivo the systems-level patterns of brain activity associated with such mental functions. In a series of positron emission tomography activation experiments, we and our colleagues have examined a range of normal and abnormal auditory states that, when contrasted, provide dissociations relevant to the question of the neural substrates of sensory awareness. These dissociations include sensory awareness in the presence and absence of external sensory stimuli, the transition from sensory unawareness to awareness (or vice versa) in the presence of sensory stimuli, and sensory awareness with and without volition. The auditory states studied include hallucinations, mental imagery, cortical deafness modulated by attention, and hearing modulated by sedation. The results of these studies highlight the distributed nature of the functional neuroanatomy that is sufficient, if not necessary, for sensory awareness. The probable roles of unimodal association (as compared with primary) cortices, heteromodal cortices, limbic/paralimbic regions and subcortical structures (such as the thalamus) are discussed. In addition, interactions between pre- and post-rolandic regions are examined in the context of top-down, volitional modulation of sensory awareness.     

Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance

The present study determines the affinity of cholera toxin for the ganglioside series GM1, GM2, GM3, GD1A, GD1B, GT1B, asialo GM1, globotriosyl ceramide, and lactosyl ceramide using real time biospecific interaction analysis (surface plasmon resonance, SPR). SPR shows that cholera toxin preferably binds to gangliosides in the following sequence: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1. The measured binding affinity of cholera toxin for the ganglioside sequence ranges from 4.61 x 10-12 M for GM1 to 1.88 x 10-10 M for asialo GM1. The picomolar values obtained by surface plasmon resonance are similar to Kd values determined with whole-cell binding assays. Both whole-cell assays and SPR measurements on synthetic membranes are higher than free solution measurements by several orders of magnitude. This difference may be caused by the effects of avidity and charged lipid head-groups, which may play a major role in the binding between cholera toxin, the receptor, and the membrane surface. The primary difference between free solution binding studies and surface plasmon resonance studies is that the latter technique is performed on surfaces resembling the cell membrane. Surface plasmon resonance has the further advantage of measuring apparent kinetic association and dissociation rates in real time, providing direct information about binding events at the membrane surface.     

Serotonin modulation of low-affinity ouabain binding in rat brain determined by quantitative autoradiography

Previous results showed that Na+/K+-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na+/K+-ATPase activity and [3H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which alpha isoform is involved in the Na+/K+-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [3H]ouabain binding to rat brain slices was employed at different [3H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [3H]ouabain binding was sensitive to serotonin 10(-3) M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [3H]ouabain concentrations, strongly suggests the participation of the alpha2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K+ handling by the glial alpha2 isoform of the pump.     

Interview decisions as determined by competency and attitude similarity.

Asked 51 college students to assume that they worked for a large company and that the president had asked them to evaluate a candidate for a position as a vice president. The target's dossier and information concerning 10 of his attitudes were given to Ss as stimuli for the evaluation. 3 levels of the target's competency and 2 levels of attitude similarity between the S and the target were varied in a 3 * 2 design to examine their effects on subsequent job recommendations and suggested salaries. Similarity tended to influence the recommendation and significantly influenced salary. Competency significantly influenced both the recommendation and salary. Implications for industry are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Osteocalcin expression in young and aged dental pulps as determined by RT-PCR

OBJECTIVE: To determine the effect of intramuscular gold and oral hydroxychloroquine (HCQ) on the lipid profile of patients with rheumatoid arthritis (RA). METHOD: A prospective randomised clinical trial of 12 months' duration was performed in 100 RA patients. Data on clinical and laboratory parameters of disease activity, and fasting serum lipid samples was collected at baseline and at three monthly intervals over one year. RESULTS: The expected second line response was seen with no significant difference in efficacy between the groups at 12 months. The HCQ group had a significant overall improvement in their lipid profile while there was a trend for lipid profiles in the gold group to worsen. CONCLUSIONS: HCQ is an effective second line agent that has beneficial effects on serum lipids. This should be taken into account when choosing a disease modifying anti-rheumatic drug in patients who suffer from RA and who have significant cardiovascular risk factors.     

Promotion of met-tRNAiMet binding to ribosomes by yIF2, a bacterial IF2 homolog in yeast

Delivery of the initiator methionine transfer RNA (Met-tRNAiMet) to the ribosome is a key step in the initiation of protein synthesis. Previous results have indicated that this step is catalyzed by the structurally dissimilar translation factors in prokaryotes and eukaryotes-initiation factor 2 (IF2) and eukaryotic initiation factor 2 (eIF2), respectively. A bacterial IF2 homolog has been identified in both eukaryotes and archaea. By using a combination of molecular genetic and biochemical studies, the Saccharomyces cerevisiae IF2 homolog is shown to function in general translation initiation by promoting Met-tRNAiMet binding to ribosomes. Thus, the mechanism of protein synthesis in eukaryotes and prokaryotes is more similar than was previously realized.     

Subfamily of olfactory receptors characterized by unique structural features and expression patterns

The complex chemospecificity of the olfactory system is probably due to the large family of short-looped, heptahelical receptor proteins expressed in neurons widely distributed throughout one of the several zones within the nasal neuroepithelium. In this study, a subfamily of olfactory receptors has been identified that is characterized by distinct structural features as well as a unique expression pattern. Members of this receptor family are found in mammals, such as rodents and opossum, but not in lower vertebrates. All identified subtypes comprise an extended third extracellular loop that exhibits amphiphilic properties and contains numerous charged amino acids in conserved positions. Olfactory sensory neurons expressing these receptor types are segregated in small clusters on the tip of central turbinates, thus representing a novel pattern of expression for olfactory receptors. In mouse, genes encoding the new subfamily of receptors were found to be harbored within a small contiguous segment of genomic DNA. Based on species specificity as well as the unique structural properties and expression pattern, it is conceivable that the novel receptor subfamily may serve a special function in the olfactory system of mammals.     

Increasing the moment arm of the tibialis anterior induces structural and functional adaptation: implications for tendon transfer

Previous studies on the functional effects of tendon transfer have not examined possible muscle adaptation following transfer. The purpose of the present study was to test the hypothesis that muscle adapts to increased moment arm and excursion such that joint torque is maintained near normal levels. The moment arm and excursion of the tibialis anterior (TA) were increased by releasing the TA from its retinacular restraint at the ankle joint in growing (4-week-old) rabbits. Twelve weeks post-release, in vivo TA force during hopping was smaller in released compared with control rabbits, compensating for the increased moment arm, and thus TA torque at the ankle joint was not significantly different between groups. Physiological cross-sectional area was smaller, and the number of sarcomeres in series was larger, in the released TA compared with the control TA. These adaptations may result from chronically decreased in vivo TA force production, and chronically increased TA excursion, respectively. In addition, these adaptations were consistent with the smaller in vivo force for the released TA. Comparisons between control and sham-operated rabbits showed no significant differences for in vivo TA force, torque, or muscle architecture. Thus, muscle appears capable of adapting to increased moment arm and excursion such that joint torque is maintained near normal levels. These findings have important implications for tendon transfer procedures that increase the moment arm and/or excursion of the released muscle.