Correlation between the structural stability and aggregation propensity of proteins

Protein aggregation, being an outcome of improper protein folding, is largely dependent on the folding kinetics of a protein. Previous studies have reported a positive correlation between the stability of the secondary structural elements of a protein and their rate of folding/unfolding. In this in silico study, the secondary and tertiary structures of proteins a) that form inclusion bodies on overexpression in Escherichia coli, b) that form amyloid fibrils and c) that are soluble on overexpression in E. coli are analyzed for certain features that are known to be associated with structural stability. The study revealed that the soluble proteins seem to have a higher rate of folding (based on contact order) and a lower percentage of exposed hydrophobic residues as compared to the inclusion body forming or amyloidogenic proteins. The soluble proteins also seem to have a more favored helix and strand composition (based on the known secondary structural propensities of amino acids). The secondary structure analyses also reveal that the evolutionary pressure is directed against protein aggregation. This understanding of the positive correlation between structural stability and solubility, along with the other parameters known to influence aggregation, could be exploited in the design of mutations aimed at reducing the aggregation propensity of the proteins.     

Definition of physical integrity of synaptosomes and myelosomes by enzyme osmometry

Isotonic requirements for synaptosomes were shown to vary with the concentration of sucrose or mannitol in the isolation medium, as well as with their differential permeability to polyols and ions. The technique of enzyme osmometry, which permits quantitation of the osmotic integrity in a heterogeneous population, was used to defined the osmotic requirements for synaptosomes and myelosomes in a variety of ionic and non-electrolyte media. Important differences, observed in the rank order of permeability of synaptosomal and myelosomal membranes to electrolyte media, were consistent with the known channel density/electrical activity of the corresponding plasma membranes.     

Correlation between thyroid-follicle fusion and structural modifications of the epithelial cells

Summary Changes in thyroid structure induced by a decrease in TSH or iodide-dependent stimulation were quantified by stereological analysis of light micrographs. Studies were carried out on intact (R5) and hypophysectomized (R 5H) rats receiving 5 g iodide, and on intact rats (R5O) receiving 50 g iodide daily.For R 5H- and R5O-thyroids, the mean parameters of the epithelial cells, height, volume and lateral membrane area, were smaller than those of R 5-thyroids. An inverse shift was observed for the apical membrane area, whereas the peripheral membrane area was unchanged. The number of epithelial cells was similar in each of the three groups; however, the number of follicles was greater in R 5-thyroids, suggesting that follicular fusion occurs in R 5O- and R 5H-thyroids. This was confirmed by direct observation.The present results demonstrate that in adult rats a lack of TSH or an increased iodide diet (insufficient to produce a physiopathological state) induce follicle fusion probably by means of cellular reorganization. This increase in follicle size could be involved in the regulation of thyroid iodine turnover.     

Interaction of ethanol with opiate receptors

Addition of ethanol to rat brain homogenate containing opiate receptors inhibits at a concentration of 50 mM the stereospecific binding of 3H-naloxone at 37 degrees C but not at 0 degree C, with the ID50 being 462 mM under these conditions. The temperature-dependent inhibition of the ligand binding suggests that ethanol does not compete with naloxone for specific binding sites of opiate receptors and changes the structure of lipids in biological membranes. Scatchard's analysis has demonstrated that apart from a decrease in the number of highly affinity binding sites of 3H-naloxone, the total amount of the binding sites remains unchanged both in the presence and absence of ethanol and constitutes 453 and 549 fmol/mg protein. It is assumed that ethanol might interconvert highly and low-affinity binding sites. Analysis of the effect of ethanol on 3H-naloxone binding with opiate receptors contained by synaptic membranes obtained from animals with varying predisposition to voluntary alcoholization has shown that ethanol inhibits to a greater degree ligand binding with membranes obtained from rats predisposed to alcoholization. The possibility of the involvement of receptors in the biochemical mechanisms by which the initial alcoholic motivation is effected is under discussion.     

The molecular structure of opiate receptors

Examples are given which demonstrate that the opiate receptor can be separted from and subtypes by their physical parameters. When the subunit composition of the subtypes are compared, no definite differences are encountered. The data from the literature are also contradictory. This may in part be explained by the fact that the different receptors appear to contain a structurally common high affinity binding site. A possible hypothesis would be that the subtypes differ from each other by the number of subunits.Abbreviations CHAPS 3/cholamidopropyl-dimethylammonio 1-propansulfonate - DAGO D-Ala ²-Me-Phe⁴-Gly-ol⁵-enkephalin - DALA d-Ala²-Leu⁵-enkephalin - DALECK d-Ala²-Leu⁵-enkaphalin chloromethyl ketone - EGF Epidermal growth factor - EKC ethylketocyclazocine - SDS-PAGE Sodiumdodecylsulfate polyacrylamide gel elecctrophoresis Special Issue Dedicated to Dr. Abel Lajtha.     

Characterization of type 2 opiate receptors

Recent evidence suggests that the Type 1 opiate receptor (in rat striatal patches) is a mobile receptor which is able to adopt a mu, delta, or kappa opiate receptor ligand selectivity pattern under appropriate conditions. In this paper, we have investigated such a possibility for Type 2 opiate receptors which are visualized diffusely over rat striatum. Ligand selectivity analysis suggested that the Type 2 opiate binding site is equivalent to a delta opiate receptor. The auto-radiographic distribution of Type 2 opiate binding sites is diffuse over most areas of rat brain. Thus, Type 2 opiate binding sites are different from Type 1 opiate receptors which are very discretely distributed in rat brain. Our results suggest that Type 2 opiate receptors, unlike Type 1 opiate receptors, are receptors locked in a delta-like ligand selectivity conformation.     

Correlation between structural polarity and polar assembly of brain tubulin

The structural polarity of tubulin protofilaments is shown by image processing of tubulin hoops. There is a strict relationship between the shapes of protofilaments and their direction of growth.     

衰老性记忆障碍与脑突触体内游离[Ca^2＋]i的相关性

采用行为观察和生化检测相结合的方法,在过去工作的基础上,研究了１２月龄和１８月龄小鼠学习记忆能力的变化和１８月龄小鼠四个脑区（海马、大脑皮层、四叠体和小脑）交触体内「Ｃａ＾２＋」ｉ的水平,同时还比较了老年记忆保持良好组与记忆障碍组小鼠的脑钙水平。结果表明,随着年龄的增长,小鼠的学习记忆能力显著下降,上述脑区（除大脑皮层外）突触内「Ｃａ＾２＋」ｉ均明显升高,其中老年记忆障碍小鼠脑钙水平升高最为显著。     

Correlation between acetylcholine receptor function and structural properties of membranes

Protein-lipid interactions were studied by using Torpedo californica acetylcholine receptor (AChR) as a model system by reconstituting purified AChR into membranes containing various synthetic lipids and native lipids. AChR function was determined by measuring two activities at 4 degrees C: (1) low to high agonist affinity-state transition of AChR in the presence of an agonist (carbamylcholine) in either membrane fragments or sealed vesicles and (2) ion-gating activity of AChR-containing vesicles in response to carbamylcholine. Sixteen samples were examined, each containing different lipid compositions including phosphatidylcholine, cholesterol, phosphatidic acid, phosphatidylethanolamine, asolectin, neutral lipid depleted asolectin, native lipids, and cholesterol-depleted native lipids. Phosphatidylcholines with different configurations of fatty acyl chains were used. The dynamic structures of these membranes were probed by incorporating spin-labeled fatty acid into AChR-containing vesicles and measuring the order parameters. It was found that both aspects of AChR function were highly dependent on the lipid environment even though carbamylcholine binding itself was not affected. An appropriate membrane fluidity was necessarily required to allow the interconversion between the low and high affinity states of AChR. An optimal fluidity hypothesis is proposed to account for the conformational transition properties of membrane proteins. In addition, the conformational change was only a necessary, but not sufficient, condition for the AChR-mediated ion flux activity. Among membranes in which AChR manifested the affinity-state transition, only those containing both cholesterol and negatively charged phospholipids (such as phosphatidic acid) retained the ion-gating activity.     

Relationship between structural and ionic changes in cardiosclerosis

The parallelism between hypoxic changes in the myocardium revealed by Lie's technique and by fluorescence microscopy and the ionic changes (Na+, K+, Ca2+) was studied in 51 autopsy cases. The Lie technique was positive in 52.6 per cent of the cases of cardiosclerosis with myocardial infarction and in 15 per cent of the cases of cardiosclerosis without infarction, while fluorescent areas were found in all cases of infarction and in 35 per cent of cardiosclerosis cases without infarction. Na+ and K+ were decreased and Ca2+ ions were increased in both ventricles and in both sexes, both in infarctions and in cardiosclerosis cases without infarction. Ionic changes are more probably related to the intensity of cardiosclerosis rather than to age or sex.     

Pregnancy related changes of opiate receptors identified in rat uterine membranes by 3H-naloxone binding

3H-Naloxone was used to demonstrate the presence of specific opiate binding sites in uterine membrane preparations of rats. 3H-Naloxone binding (0.41-27 nM) was found to be rapid, saturable and reversible showing two populations of binding sites with the characteristic of high (KD 2.2 nM; Bmax 46.6 fmol/mg prot.) and low (KD 18.1 nM; Bmax 143.7 fmol/mg prot.) affinity. The number and affinity of the binding sites labelled by 3H-naloxone in the uterus were measured in the rat at mid (14 days), late (21 days) pregnancy and at parturition. The high and low affinity recognition sites labelled by 3H-naloxone showed a consistent reduction during pregnancy and at parturition without changes in the affinity constant. We concluded that pregnancy and parturition are associated with significant changes in the number of the opiate receptors bound in the uterus by 3H-naloxone. This phenomenon which seems to be linked with the several pregnancy-related changes in the levels of endogenous peptides and hormones could be relevant to further explain the pregnancy related changes in pain perception and maternal behavior.     

Striatal opiate receptors: Pre- and postsynaptic localization

Autoradiographic localization of opiate receptors in the rat striatum with specifically bound ³H-diprenorphine reveals relatively small, high density clusters of receptors, a high density band of receptors along the striatal-callosal border, and a lower density of receptors spread over the remainder of the neuropil. Placement of kainate lesions resulted in a 94% loss of receptors in the clusters and a smaller loss in other areas. Removal of the dopamine-containing input to the striatum by placement of medial forebrain bundle lesions or by intrastriatal injection of 6-hydroxydopamine resulted in a greater depletion of receptors in the non-cluster areas compared to cluster areas. In repeated biochemical and autoradiographic studies, no change was found after either decortication or placement of thalamic lesions. It is concluded that the bulk of striatal opiate receptors are localized postsynaptically on intrinsic striatal neurons and their processes with the bulk of the remaining receptors localized to the dopamine-containing axons and terminals.     

Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity

Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defence mechanisms. Thus, when antigen-specific IgG antibodies form antigen/antibody immune complexes the effectively aggregated IgG can activate a wide range of effector systems. Multiple effector mechanisms result from cellular activation mediated through a family of IgG-Fc receptors differentially expressed on leucocytes. It is established that glycosylation of IgG-Fc is essential for recognition and activation of these ligands. IgG antibodies predominate in human serum and most therapeutic antibodies are of the IgG class.The IgG-Fc is a homodimer of N-linked glycopeptide chains comprised of two immunoglobulin domains (Cgamma2, Cgamma3) that dimerise via inter-heavy chain disulphide bridges at the N-terminal region and non-covalent interactions between the C-terminal Cgamma3 domains. The overall shape of the IgG-Fc is similar to that of a "horseshoe" with a majority of the internal space filled by the oligosaccharide chains, only attached through asparagine residues 297.To investigate the influence of individual sugar (monosaccharide) residues of the oligosaccharide on the structure and function of IgG-Fc we have compared the structure of "wild-type" glycosylated IgG1-Fc with that of four glycoforms bearing consecutively truncated oligosaccharides. Removal of terminal N-acetylglucosamine as well as mannose sugar residues resulted in the largest conformational changes in both the oligosaccharide and in the polypeptide loop containing the N-glycosylation site. The observed conformational changes in the Cgamma2 domain affect the interface between IgG-Fc fragments and FcgammaRs. Furthermore, we observed that the removal of sugar residues permits the mutual approach of Cgamma2 domains resulting in the generation of a "closed" conformation; in contrast to the "open" conformation which was observed for the fully galactosylated IgG-Fc, which may be optimal for FcgammaR binding. These data provide a structural rationale for the previously observed modulation of effector activities reported for this series of proteins.