Analysis of distribution of alternating-sign charged clusters on the surface of globular proteins

PURPOSE: In vitro evaluation of three prototype devices designed to trap emboli that are generated by intravascular procedures. MATERIALS AND METHODS: Three prototypes of the safety net were tested in a water flow model. Angioplasty of human endarterectomy specimens was performed upstream of the device (n = 8). In other tests, polyvinyl alcohol particles were injected into the water flow model upstream of the device (n = 15). The safety nets and the effluent that passed through them were examined for embolic particles. A third prototype was tested (n = 5) to evaluate whether the device could be deployed and retrieved as designed. RESULTS: The safety net was able to trap debris released by angioplasty of endarterectomy specimens in every case. Most, but not all, polyvinyl alcohol particles were trapped by the safety net. Four of five devices tested were deployed and retrieved as designed. One became caught in the introducing valve, which prevented deployment. CONCLUSION: The vascular safety net is effective in trapping small volumes of emboli. It can be deployed and retrieved as designed.     

Structural basis for the binding of a globular antifreeze protein to ice

Antifreeze proteins (AFPs) have the unique ability to adsorb to ice and inhibit its growth. Many organisms ranging from fish to bacteria use AFPs to retard freezing or lessen the damage incurred upon freezing and thawing. The ice-binding mechanism of the long linear alpha-helical type I AFPs has been attributed to their regularly spaced polar residues matching the ice lattice along a pyramidal plane. In contrast, it is not known how globular antifreeze proteins such as type III AFP that lack repeating ice-binding residues bind to ice. Here we report the 1.25 A crystal structure of recombinant type III AFP (QAE isoform) from eel pout (Macrozoarces americanus), which reveals a remarkably flat amphipathic ice-binding site where five hydrogen-bonding atoms match two ranks of oxygens on the [1010] ice prism plane in the <0001> direction, giving high ice-binding affinity and specificity. This binding site, substantiated by the structures and properties of several ice-binding site mutants, suggests that the AFP occupies a niche in the ice surface in which it covers the basal plane while binding to the prism face.     

Sequence-based recognition of protein folds using the threading method and frameworks of globular proteins

In studies on mammalian parental investment, time spent suckling is often used as a predictor of the milk transferred from mother to infant. It is assumed that the rate of milk transfer is positively correlated with the time spent suckling. However, this assumption has not been tested and empirical studies show conflicting results. Nevertheless, in species in which suckling can readily be observed, time spent suckling is still used to measure milk transfer, although an increasing number of workers recognize that the measure is potentially inaccurate. A meta-analysis on studies that have correlated measures of time spent suckling with milk intake estimates based on weight gain revealed a weak positive relationship and significant heterogeneity between studies. Isotope-labelling techniques for the measurement of milk transfer independent of behaviour have been in use since the 1970s, particularly in studies of species in which suckling is difficult to observe. Only one study has attempted to correlate behavioural measures with independent isotope measures, and it found no relationship between the two measures. I suggest that researchers have avoided such a test as it is unlikely that a strong relationship will be found between milk transfer and suckling behaviour, and I discuss the various factors that confound the relationship and contribute to high heterogeneity between studies. Consequently, the assumption that milk transfer can be measured by time spent suckling has inadequate empirical foundation, and needs to be tested using isotope-labelling methods. Copyright 1998 The Association for the Study of Animal Behaviour     

Adsorption of globular proteins on locally planar surfaces: models for the effect of excluded surface area and aggregation of adsorbed protein on adsorption equilibria

Equilibrium statistical-thermodynamic models are presented for the surface adsorption of proteins modeled as regular convex hard particles. The adsorbed phase is treated as a two-dimensional fluid, and the chemical potential of adsorbed protein is obtained from scaled particle theory. Adsorption isotherms are calculated for nonassociating and self-associating adsorbing proteins. Area exclusion broadens adsorption isotherms relative to the Langmuir isotherm (negative cooperativity), whereas self-association steepens them (positive cooperativity). The calculated isotherm for adsorption of hard spheres using scaled particle theory for hard discs agrees well with that calculated from the hard disc virial expansion. As the cross section of the adsorbing protein in the plane of the surface becomes less discoidal, the apparent negative cooperativity manifested in the isotherm becomes more pronounced. The model is extended to the case of simultaneous adsorption of a tracer protein at low saturation and a competitor protein with a different size and/or shape at arbitrary fractional saturation. Area exclusion by competitor for tracer (and vice versa) is shown to substantially enhance the displacement of tracer by competitor and to qualitatively invalidate the standard interpretation of ligand competition experiments, according to which the fractional displacement of tracer by competitor is equal to the fractional saturation by competitor.     

Surface area of globular proteins

A simplified practical histological classification of four stages of bladder epithelial tumours is proposed in accordance with the terminology adopted by WHO. The structural characters of neoplastic elements are considered with respect to their relations with the basal membrane delimiting the epithelia. This criterion gives valid information regarding real malignancy of the tumour and may be applied to biopsies obtained endoscopically. This is very important not only with regard to prognosis but also to therapeutic indications.     

Association-induced folding of globular proteins

It has generally been assumed that the aggregation of partially folded intermediates during protein refolding results in the termination of further protein folding. We show here, however, that under some conditions the association of partially folded intermediates can induce additional structure leading to soluble aggregates with many native-like properties. The amount of secondary structure in a monomeric, partially folded intermediate of staphylococcal nuclease was found to double on formation of soluble aggregates at high protein or salt concentrations. In addition, more globularity, as determined from Kratky plots of small-angle x-ray scattering data, was also noted in the associated states.     

Populations of hydrophobic amino acids within protein globular domains: identification of conserved "topohydrophobic" positions

Developmental anomalies resulting from prenatal toxicity can be manifested in terms of both malformations among surviving offspring and prenatal death. Although these two endpoints have traditionally been analyzed separately in the assessment of risk, multivariate methods of risk characterization have recently been proposed. We examined this and other issues in developmental toxicity risk assessment by evaluating the accuracy and precision of estimates of the effective dose (ED05) and the benchmark dose (BMD05) using computer simulation. Our results indicated that different variance structures (Dirichlet-trinomial and generalized linear model) used to characterize overdispersion yielded comparable results when fitting joint dose response models based on generalized estimating equations. (The choice of variance structure in separate modeling was also not critical.) However, using the Rao-Scott transformation to eliminate overdispersion tended to produce estimates of the ED05 with reduced bias and mean squared error. Because joint modeling ensures that the ED05 for overall toxicity (based on both malformations and prenatal death) is always less than the ED05 for either malformations or prenatal death, joint modeling is preferred to separate modeling for risk assessment purposes.     

Hydrational and intrinsic compressibilities of globular proteins

To evaluate further bacterial DNA immunization as a model to study antigen drive in the anti-DNA response, the specificity of induced monoclonal anti-DNA antibodies was characterized. A panel of IgM and IgG monoclonal anti-DNA antibodies was produced from spleen cells of BALB/c mice immunized with single-stranded DNA from E. coli complexed to methylated bovine serum albumin in complete Freund's adjuvant. The binding of these antibodies to DNA and non-DNA antigens was tested by ELISA to assess their range of polyspecificity. These monoclonal antibodies were found to bind to nucleic acid as well as non-nucleic acid antigens, such as beta-galactosidase, cardiolipin, Ro, La and Sm. These studies demonstrate that anti-DNA antibodies from normal mice, although induced by bacterial DNA, may display a broad range of antigen recognition and thus resemble lupus anti-DNA antibodies, many of which are polyspecific, in their pattern of cross-reactivity.     

Equivalent Roughness of Gravel-Bed Rivers

The relation between the equivalent roughness and different grain size percentiles of the sediment in gravel-bed rivers was determined under the hypothesis that the vertical distribution of the flow velocity follows a logarithmic law. A set of 954 data points was selected from rivers with gravel size sediment or larger, with a nonsinuous alignment and free of vegetation or obstacles. According to the results, the ks roughness is equivalent to approximately 2.4D90, 2.8D84, and 6.1D50. No correlation was detected between the sediment sorting and the sediment mobility index on one hand and, on the other, the coefficient of proportionality of each grain size percentile.     

A case of pulmonary arteriovenous fistula beneath the surface of the lung]

Mediastinal Hodgkin's disease has rarely been reported in the literature in Japan; however, it is not uncommon in Europe and North America. A 32-year-old woman with isolated mediastinal Hodgkin's disease mimicking thymoma is herein described. A preoperative diagnosis of thymoma led to a combined resection of the mediastinal tumor together with the entire thymus, left innominate vein, and left phrenic nerve. The resected tumor was histologically diagnosed to be Hodgkin's disease of the nodular sclerosis type. Adjuvant 40 Gy irradiation of the mediastinum and neck was added postoperatively. The patient is doing well at present with no signs of recurrence 8 months after the operation.     

Endoplasmic reticulum protein Hsp47 binds specifically to the N-terminal globular domain of the amino-propeptide of the procollagen I alpha 1 (I)-chain

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibroblasts, has gelatin-binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47-procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with 35[S]-methionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Sepharose 4B beads. The gelatin-bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro alpha 1(I)-N-propeptide at 25 micrograms/ml completely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86-90 of the pro alpha 1(I)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro alpha 1(I)-N-propeptide domain were confirmed by examination of polysome-associated pro alpha chains. The nascent pro alpha 1(I)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro alpha 1 (I)-N-propeptide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion protein constructs of residues 23-108 (NP1), 23-151 (NP2), and 23-178 (NP3) within the pro alpha 1 (I)- N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotranslation systems in which the complete pro alpha 1(I)- and pro alpha 2(I)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro alpha 2(I)-chains and hsp47, whereas there was strong interaction between pro alpha 1(I)-chains and nascent hsp47. SP1.D8 could not precipitate pro alpha 1(I)-chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a "chaperone" role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the pro alpha 1 (I) chain, and the pro alpha 1(I) amino-propeptide region in particular.     

Modeling of Microindentation with Consideration of the Surface Roughness

A finite element model of a microindentation is developed where the scanned surface topography data which represent the actual surface-roughness values are used in the construction of the finite element model of the workpiece. Indentation simulation with friction effect solely due to the surface deformation produces results which match the experimental ones in terms of residual indent size and pile-up heights. A roughness-dependent friction model is developed, which is comparable with the models proposed by Johnson (1985) and Alcála (2004).     

Evidence for the requirement of protein synthesis and protein kinase activity in the temperature regulated stability of a Tetrahymena surface protein mRNA

In Tetrahymena thermophila, the expression of the temperature-specific surface protein SerH3 is controlled primarily by a temperature-dependent change in the stability of its mRNA. The change in SerH3 mRNA stability occurs very rapidly after a shift in incubation temperature. This change in temperature could affect SerH3 mRNA stability directly by producing structural changes in the mRNA or regulatory factors acting on SerH3 mRNA. Alternatively, the temperature change could act indirectly through a signal transduction pathway leading to de novo synthesis of new regulatory factors or modifications of existing regulatory factors. To address these issues, we monitored the effect of temperature on an in vitro SerH3 mRNA decay assay and the in vivo effects of a variety of inhibitors against protein synthesis and protein kinases on SerH3 mRNA stability. The results of Northern analysis of SerH3 mRNAs in an in vitro mRNA decay assay indicate that temperature alone can not change the half-life of this mRNA. Furthermore, slot blot analysis of cytoplasmic RNAs show that protein synthesis and the action of protein kinases are not required for SerH3 mRNA turnover in cells grown at 30 degrees C. In contrast, our results indicate that the rapid decay of the SerH3 mRNA in cells grown at 30 degrees C and shifted to 40 degrees C requires a one time serine/threonine phosphorylation event which occurs at the temperature shift. In addition, the data show that a regulatory protein involved in rapid SerH3 mRNA decay must be newly and continuously synthesized following the temperature shift from 30 to 40 degrees C. These data show the complexity of temperature regulated mRNA decay and indicate that phosphorylation and protein synthesis are major factors in this process.