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A structural model of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata deduced from density measurements at different osmotic pressures.

Density measurements made on cholinergic synaptic vesicles from the electric organs of Torpedo marmorata at different osmotic pressures are consistent with the following structural model of the vesicle. The particle behaves like a sphere 80-100 nm in diameter bounded by a semi-permeable membrane. The bulk of its soluble constituents are in true solution at physiological osmolalities. The limiting membrane is approximately 4-5 nm thick, suggesting that it contains large areas of phospholipid bilayer exposed to its bathing medium. The limiting membrane takes up about 26% (v/v) of the particle, a further 34% (v/v) of which is osmotically active water and 31% (v/v) hydrated core material at 800 mosmol/1. The buoyant density of the membrane is 1.132 g . cm-3. The density of the hydrated core material is approximately 1.05 g . cm-3. The membrane is selectively permeable to small molecules when subjected to hypo-osmotic stress. It is proposed that this occurs by the formation of small transient pores in the lipid bilayer of the membrane, which are induced by stretching caused by the osmotic pressure change.

Acetylcholine

Structural models of the evolutionarily conservative central domain of silk-moth chorion proteins.

Silk-moth chorion proteins belong to a small number of families: A, B, C, Hc-A and Hc-B. The central domain is an evolutionarily conservative region in each family, of variable length and composition between families. This domain shows clear 6-fold periodicities for various amino acid residues, e.g. glycine. The periodicities, together with the well-documented prevalence of beta-sheet and beta-turn secondary structure of chorion proteins, strongly support a structural model in which four-residue beta-strands alternate with beta-turns, forming a compact antiparallel, probably twisted beta-sheet. Conformational analysis, aided by interactive graphics refinement and recent experimental findings, further suggest that this structure consists of beta-strands, alternating with I' and II' beta-turns, and apparently forms the basis for the molecular and supramolecular assembly of chorion.

Amino Acid Sequence

A proposed structural model of endothelin.

We have assessed the hydropathic nature, secondary structure, and flexibility of endothelin and report a testable model for the structure of this 21-amino acid vasoconstrictor peptide. The proposed model consists of a rigid (disulfide bonded), hydrophilic amino-terminal half of the molecule with two turns, and an extended hydrophobic sheet structure comprising the C-terminal half of the molecule. The extended structure may be stabilized by intermolecular hydrogen bonding, leading to dimers or higher order aggregates, and is hydrophobic enough to partition into an organic solvent such as ether, as long as the molecule can keep its hydrophilic amino terminus in an aqueous phase. To test this model, partitioning measurements were made on bioactive synthetic endothelin. It was found to partition at the interface of an ether/water system in a concentration- and surface area-dependent manner. A further indication as to the amphipathic nature of the molecule is that microcrystals of synthetic endothelin tend to grow at an organic-aqueous interface in a two-phase system.

Amino Acid Sequence

A tree-structure model for analyzing laboratory test costs.

A method of costing clinical laboratory tests is described in which total laboratory costs are apportioned into 3 components: an 'organizational overhead cost' to describe the cost of running an organization; a 'cost per episode' to describe the costs incurred per request; and a 'cost per test' to describe the actual analytical costs of tests performed. A tree-like structural model is described which allows for distribution of this 'cost per test' component to the contributing tests and the calculation of the true 'cost per test'. The implementation of this model on a computer spreadsheet has been achieved. When combined with the other cost components, the true total costs of laboratory testing can be determined for any particular test. The model also allows calculation of the incremental and marginal costs of a test and permits optimization of laboratory test frequencies and work-load to minimize laboratory analytical costs.

Algorithms

How wide is the widening scope of psychoanalysis and how solid is its structural model? Some concerns and observations.

The author advances the thesis that in the past 35 years there has been a relatively silent but nonetheless significant movement within the mainstream of American psychoanalysis toward a more "modest" position. This movement has been stimulated from different sources, sometimes with diverse goals and different programs. One determinant was the reaction to the post-World War II euphoria in regard to psychoanalysis and its possible therapeutic powers. Another element has been the ongoing consolidation of our knowledge and understanding of the ego-psychological, structural-model approach to analytic theory and technique, an approach which emphasizes both intrapsychic conflict and compromise formations. A consequence of this more modest position has been a greater appreciation of the limitations of psychoanalysis as well as the significance of those limitations. This more realistic appraisal of psychoanalysis may not have encouraged the widening scope of the indications for analysis, but the enhanced understanding of its limitations offers the promise of more effective psychoanalytic work in areas that have not been considered ideal for the so-called "traditional" analysis. It is suggested that more sophisticated approaches in the analysis of resistance and character, of "conflict" (in distinction to "diagnoses"), together with a more applicable understanding of the psychoanalytic process, can all contribute to a deepening, if not necessarily widening, of our psychoanalytic endeavors.

Humans

Structure model of core proteins in photosystem I inferred from the comparison with those in photosystem II and bacteria; an application of principal component analysis to detect the similar regions between distantly related families of proteins.

A principal component analysis based on the physico-chemical properties of amino acid residues is developed to assign similar regions between distantly related families of proteins, taking account of the species diversities in respective families. The most important advantage of this analysis should be that it reflects different physico-chemical properties and thus can predict more detailed structural properties, including the transmembrane helices, than the hydropathy analysis. Its first application reconfirms the similarity between the core proteins of photosynthetic reaction center in purple bacteria and those of photosystem II, indicating that the low percentage of identical amino acid residues estimated previously between them is due to much allowance for amino acid substitutions in purple bacteria. The application of this analysis to the core proteins of photosystem I reveals that any of these proteins includes two domains, each showing high similarity to the amino acid sequences of core proteins in photosystem II and purple bacteria. A core structure model of A1 and A2 proteins folded into four layers of sheets of transmembrane helices is proposed to provide a molecular basis for the electron pathway suggested by spectroscopic experiments as well as for the interaction sites with plastocyanin, 9 kDa protein and LHC proteins.

Amino Acid Sequence

Drosophila mitochondrial DNA: conserved sequences in the A + T-rich region and supporting evidence for a secondary structure model of the small ribosomal RNA.

The sequence of a segment of the Drosophila virilis mitochondrial DNA (mtDNA) molecule that contains the A + T-rich region, the small rRNA gene, the tRNA(f-met), tRNA(gln), and tRNA(ile) genes, and portions of the ND2 and tRNA(val) genes is presented and compared with the corresponding segment of the D. yakuba mtDNA molecule. The A + T-rich regions of D. virilis and D. yakuba contain two correspondingly located sequences of 49 and 276/274 nucleotides that appear to have been conserved during evolution. In each species the replication origin of the mtDNA molecule is calculated to lie within a region that overlaps the larger conserved sequence, and within this overlap is found a potential hairpin structure. Substitutions between the larger conserved sequences of the A + T-rich regions, the small mt-rRNA genes, and the ND2 genes are biased in favor of transversions, 71-97% of which are A----T changes. There is a 13.8 times higher frequency of nucleotide differences between the 5' halves than between the 3' halves of the D. virilis and D. yakuba small mt-rRNA genes. Considerations of the effects of observed substitutions and deletion/insertions on possible nucleotide pairing within the small mt-rRNA genes of D. virilis and D. yakuba strongly support the secondary structure model for the Drosophila small mt-rRNA that we previously proposed.

Adenine

Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels.

Charybdotoxin (ChTX), a protein present in the venom of the scorpion Leiurus quinquestriatus var. hebraeus, has been purified to homogeneity by a combination of ion-exchange and reversed-phase chromatography. Polyacrylamide gel electrophoresis, amino acid analysis, and complete amino acid sequence determination of the pure protein reveal that it consists of a single polypeptide chain of 4.3 kDa. Purified ChTX is a potent and selective inhibitor of the approximately 220-pS Ca2+-activated K+ channel present in GH3 anterior pituitary cells and primary bovine aortic smooth muscle cells. The toxin reversibly blocks channel activity by interacting at the external pore of the channel protein with an apparent Kd of 2.1 nM. The primary structure of ChTX is similar to a number of neurotoxins of diverse origin, which suggests that ChTX is a member of a superfamily of proteins that modify ion-channel activities. On the basis of this similarity, the three-dimensional structure of ChTX has been modeled from the known crystal structure of alpha-bungarotoxin. These studies indicate that ChTX is useful as a probe of Ca2+-activated K+-channel function and suggest that the proposed tertiary structure of ChTX may provide insight into the mechanism of channel block.

Amino Acid Sequence

Molecular genetics of the VDAC ion channel: structural model and sequence analysis.

The voltage-dependent anion-selective channel of the outer mitochondrial membrane provides a unique system in which to study the molecular basis of voltage gating of ion flow. We have cloned and sequenced a cDNA coding for this protein in yeast. From the derived amino acid sequence, we have generated a preliminary model for the secondary structure of the protein which suggests that the protein forms a "beta-barrel" type structure. Comparison of the VDAC amino acid sequence with that of the bacterial porins has indicated that the two classes of molecules appear to be unrelated.

Amino Acid Sequence

An initial slope method for model structure: independent estimation of the elimination rate constant of a metabolite.

A model structure-independent method for calculating the true elimination rate constant of a primary metabolite is presented. It does not require direct metabolite administration and uses data on drug and metabolite blood (plasma) concentrations after a bolus drug input. The method has been tested and compared with the moment method and the area function method using errorless and errant data simulated on the basis of one- and two-compartment models of the metabolite kinetics. In contrast to known methods the proposed method provided exact estimates of the elimination rate constant in the case of errorless data of both one- and two-compartment models. However the estimates are sensitive to random errors in the concentration data.

Mathematical Computing

Structured model for cell growth and enzyme production by recombinant Escherichia coli.

A structured cell model has been developed to describe the cultivation of recombinant Escherichia coli K12 with multicopy plasmid under the control of a lambda PR-promoter and a temperature-sensitive lambda cI 857 repressor. The model, based on measurements of a batch culture in a stirred tank reactor, allows statements to be made on the time variation of intracellular processes. Based on cell regulation, the substrate transfer into the cell was considered to be the rate-limiting step for substrate utilization. The model describes substrate utilization, cell growth, and product formation by means of a system of time-dependent, coupled, and partly non-linear differential equations. The solution of these equations allows calculation of the time variation of the concentrations of substrates (glucose and amino acids), dissolved oxygen and cells in the broth as a function of time and the process parameters.

Cell Division

Structural model of vestibular effects on the blood pressure.

The model described in the present work is a structural presentation of certain aspcets of the vesibular effects on the blood pressure. The model comprises the joint action of the otolith system and the semicircular canals, gangl. Scarpe, the vestibular nuclei, the vasomotor centre and the nuclei of n. vagus. The direct transfer of information both from gangl. Scarpe to the vasoregulating centre and from nucl. Schwalbe to the vagal nuclei, as well as its polysynaptic transmission, are considered. The model examines a case when only two factors, namely decrease in the heart rate controlled by n. vagus and decrease in the heart tone controlled by the vasoregulating centre, determine the effect of blood pressure drop after vestibular stimulation. The structure of the model is flexible and permits its elaboration by including additional factors for the blood pressure drop.

Blood Pressure

Structural modeling and functional characterization of a novel gain-of-function TLR8 variant causing severe inflammatory syndrome.

With the increasing use of genetic sequencing to investigate inborn errors of immunity, rare variants are frequently identified, yet their clinical relevance often remains uncertain. Establishing pathogenicity requires a multidisciplinary approach that integrates genetic, structural, functional, and clinical data. Here, we used such a strategy to investigate a previously unreported hemizygous missense variant - alanine (A) to threonine (T) at residue 518 - in Toll-like receptor 8 (TLR8), identified in 2 male siblings with recurrent infections and systemic inflammation, characterized by a proinflammatory immune signature and B cell dysregulation. Functional studies showed that the TLR8 A518T variant enhanced NF-κB activation and increased secretion of proinflammatory cytokines compared with WT TLR8 upon stimulation, consistent with a gain-of-function effect. Protein degradation and turnover assays revealed reduced abundance of the mutant TLR8 protein due to faster turnover and increased proteasomal degradation. Computational modeling predicted enhanced structural stabilization of the active TLR8 homodimer interface via additional water-mediated hydrogen bonds introduced by the A518T substitution. Together, these findings integrating structural modeling with functional assays identify a novel TLR8 ligand-specific gain-of-function mutation resulting in complex immunopathology in 2 siblings.

Humans

[Small-angle X-ray-scattering investigation and structural-model study of the fatty-acid synthetase from pig liver (author's transl)].

The structure of the fatty acid synthetase from pig liver was studied on models based upon structural and functional properties selected from pertinent results available from numerous investigations carried out with fatty acid synthetases from this and other sources. When comparing small-angle X-ray-scattering curves calculated with these models and curves obtained from small-angle X-ray-scattering experiments carried out with the pig-liver enzyme, we tried to select a model which would lead to an acceptable correlation between the calculated and the experimental curves and at the same time fulfil the known structural and functional requirements. The comparison of the curves was started with a model of low complexity. The observed discrepancy, together with arguments from the structural and the functional properties, helped decide which is the next most reasonable model to be considered. This procedure was repeated for five models of increasing complexity. In the model which led to the best fit the multienzyme complex is composed of two halves in an assymetric conformation including hollow spaces. This highly anisotropic model would imply that the two halves change their conformation each time a synthetic cycle is completed and that the growing fatty acid is handed over from one half to the other.

Animals

Application of structural model of glucose-insulin relations to assess beta-cell function and insulin sensitivity.

A structural mathematical model of glucose-insulin relationships based on known quantitative responses of the major organs involved with glucose metabolism has been computed. Different degrees of beta-cell function and insulin sensitivity can be included, and the effect of their interaction assessed (i) in a steady state, basal homeostasis after an overnight fast and (ii) in response to a glucose infusion. By comparing a patient's basal plasma glucose and insulin (or C-peptide) concentrations with the predictions of a basal homeostatic model, the degree of impairment of beta-cell function and insulin sensitivity can be assessed. Similarly, the plasma glucose and insulin (or C-peptide) concentrations after a continuous glucose infusion can also be compared with predictions from the model to estimate beta-cell function and insulin sensitivity. These assessments of pathophysiology can be applied to data from individual patients or to patient populations.

Blood Glucose

Manganese superoxide dismutase from Thermus thermophilus. A structural model refined at 1.8 A resolution.

The structure of Mn(III) superoxide dismutase (Mn(III)SOD) from Thermus thermophilus, a tetramer of chains 203 residues in length, has been refined by restrained least-squares methods. The R-factor [formula: see text] for the 54,056 unique reflections measured between 10.0 and 1.8 A (96% of all possible reflections) is 0.176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P4(1)2(1)2 cell. The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 203) is an alpha + beta structure including a three-stranded sheet. Features that may be important for the folding and function of this MnSOD include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position 30 that distorts the helix near the first Mn ligand; and (3) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the beta-sheet. Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions. The tetramer possesses 222 symmetry but is held together by only two types of interfaces. The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/monomer) and incorporates 17 trapped or structural solvents. The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain. Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry. One of the axial ligands is solvent; the four protein ligands are His28, His83, Asp166 and His170. Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp132A, Trp168A, Tyr183A, Tyr172B, Tyr173B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr36 and His32 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence

Structural modeling of the human erythrocyte bisphosphoglycerate mutase.

Using the crystallographic structure of yeast monophosphoglycerate mutase (MPGM) as a framework we constructed a three-dimensional model of the homologous human erythrocyte bisphosphoglycerate mutase (BPGM). The modeling procedure consisted of substituting 117 amino acid residues and positioning 19 C-terminal residues (unresolved in the X-ray structure) by empirical methods, followed by energy minimization. Among several differences in the active site region the most significant appears to be the replacement of Ser11 in MPGM by Gly in BPGM. The C-terminal segment, which contains mainly basic amino acids, lines the cavity of the active site. The seven amino acid residues, which have been shown to be essential for the three catalytic functions of the human BPGM, interact with the amino acids in the protein core, near the active site. In addition, a cluster of several positively charged residues, particularly arginines, has been identified at the entrance of the active site; this cluster may serve as a secondary binding site for polyanionic substrates or cofactors, as required by a two-binding-site model of the catalytic activities. This model is in agreement with recent studies of an inactive BPGM variant substituent at an Arg position situated in this positively charged cluster. The position of Cys20 in the model constructed suggests that this residue is responsible for inactivation of the enzyme by sulfhydryl reagents. Subunit interfaces have also been constructed for BPGM by analogy with MPGM and suggest that, in addition to the known dimerization of BPGM, tetramerization may occur under certain conditions.

Amino Acid Sequence