Immunolocalization of the 38.3 kDa calponin-like protein in stratified muscles of the tail of Schistosoma japonicum cercariae

Calponins are proteins present in vertebrate smooth musculature where they occur in association with thin myofilaments. Calponins are not present in vertebrate or invertebrate striated muscles. The blood fluke Schistosoma japonicum expresses a 38.3-kDa protein that bears substantial homology with vertebrate calponin and occurs entirely within smooth musculature of adults. Calponin-like immunoreactivity has been demonstrated in smooth muscles of many invertebrate phyla. The Schistosoma japonicum calponin has been localised in smooth myofibrils of adults where it is associated with myofilaments and sarcoplasmic reticulum. In this study, the ultrastructural localisation of the protein in muscles of S. japonicum cercariae is described. The protein is present in smooth muscles of the forebody and the stratified muscle of the tail. Within the stratified layer, the protein occurs predominantly in transverse arrays of sarcoplasmic reticulum. The localisation data suggest that the calponin-like protein of S. japonicum is involved in contraction of the stratified tail muscle. Furthermore, the presence of a calponin system in the stratified muscle suggests that this muscle is simply a superior form of muscle, closely related to smooth muscles that use a caldesmin-calponin system in contraction. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

HPP1: A transmembrane protein-encoding gene commonly methylated in colorectal polyps and cancers

Adenomas are the precursors of most colorectal cancers. Hyperplastic polyps have been linked to the subset of colorectal cancers showing DNA microsatellite instability, but little is known of their underlying genetic etiology. Using a strategy that isolates differentially methylated sequences from hyperplastic polyps and normal mucosa, we identified a 370-bp sequence containing the 5' untranslated region and the first exon of a gene that we have called HPP1. Rapid amplification of cDNA ends was used to isolate HPP1 from normal mucose. Using reverse transcription-PCR, HPP1 was expressed in 28 of 30 (93%) normal colonic samples but in only seven of 30 (23%) colorectal cancers (P < 0.001). The 5' region of HPP1 included a CpG island containing 49 CpG sites, of which 96% were found to be methylated by bisulfite sequencing of DNA from colonic tumor samples. By COBRA analysis, methylation was detected in six of nine (66%) adenomas, 17 of 27 (63%) hyperplastic polyps, and 46 of 55 (84%) colorectal cancers. There was an inverse relationship between methylation level and mRNA expression in cancers (r = -0.67; P < 0.001), and 5-aza-2-deoxycytidine treatment restored HPP1 expression in two colorectal cancer cell lines. In situ hybridization of HPP1 indicated that expression occurs in epithelial and stromal elements in normal mucosa but is silenced in both cell types in early colonic neoplasia. HPP1 is predicted to encode a transmembrane protein containing follistatin and epidermal growth factor-like domains. Silencing of HPP1 by methylation may increase the probability of neoplastic transformation.

Compartmentalization of Ras proteins

The Ras GTPases operate as molecular switches that link extracellular stimuli with a diverse range of biological outcomes. Although many studies have concentrated on the protein-protein interactions within the complex signaling cascades regulated by Ras, it is becoming clear that the spatial orientation of different Ras isoforms within the plasma membrane is also critical for their function. H-Ras, N-Ras and K-Ras use different membrane anchors to attach to the plasma membrane. Recently it has been shown that these anchors also act as trafficking signals that direct palmitoylated H-Ras and N-Ras through the exocytic pathway to the cell surface but divert polybasic K-Ras around the Golgi to the plasma membrane via an as yet-unidentified-route. Once at the plasma membrane, H-Ras and :K-Ras operate in different microdomains. K-Ras is localized predominantly to the disordered plasma membrane, whereas H-Ras exists in a GTP-regulated equilibrium between disordered plasma membrane and cholesterol-rich lipid rafts. These observations provide a likely explanation for the increasing number of biological differences being identified between the otherwise highly homologous Ras isoforms and raise interesting questions about the role membrane microlocalization plays in determining the interactions of Ras with its effecters and exchange factors.

NMR structure and backbone dynamics of a concatemer of epidermal growth factor homology modules of the human low-density lipoprotein receptor

The ligand-binding region of the low-density lipoprotein (LDL) receptor is formed by seven N-terminal, imperfect, cysteine-rich (LB) modules. This segment is followed by an epidermal growth factor precursor homology domain with two N-terminal, tandem, EGF-like modules that are thought to participate in LDL binding and recycling of the endocytosed receptor to the cell surface. EGF-A and the concatemer, EGF-AB, of these modules were expressed in Escherichia coli. Correct protein folding of EGF-A and the concatemer EGF-AB was achieved in the presence or absence of calcium ions, in contrast to the LB modules, which require them for correct folding. Homonuclear and heteronuclear H-1-N-15 NMR spectroscopy at 17.6 T was used to determine the three-dimensional structure of the concatemer. Both modules are formed by two pairs of short, anti-parallel beta -strands. In the concatemer, these modules have a fixed relative orientation, stabilized by calcium ion-binding and hydrophobic interactions at the interface. N-15 longitudinal and transverse relaxation rates, and {H-1}-N-15 heteronuclear NOEs were used to derive a model-free description of the backbone dynamics of the molecule. The concatemer appears relatively rigid, particularly near the calcium ion-binding site at the module interface, with an average generalized order parameter of 0.85 +/- 0.11. Some mutations causing familial hypercholesterolemia may now be rationalized. Mutations of D41, D43 and E44 in the EGF-B calcium ion-binding region may affect the stability of the linker and thus the orientation of the tandem modules. The diminutive core also provides little structural stabilization, necessitating the presence of disulfide bonds. The structure and dynamics of EGF-AB contrast with the N-terminal LB modules, which require calcium ions both for folding to form the correct disulfide connectivities and for maintenance of the folded structure, and are connected by highly mobile linking peptides. (C) 2001 Academic Press.

Crystallization and preliminary X-ray diffraction studies of FHA domains of Dun1 and Rad53 protein kinases

Forkhead-associated (FHA) domains are modular protein–protein interaction domains of ~130 amino acids present in numerous signalling proteins. FHA-domain-dependent protein interactions are regulated by phosphorylation of target proteins and FHA domains may be multifunctional phosphopeptide-recognition modules. FHA domains of the budding yeast cell-cycle checkpoint protein kinases Dun1p and Rad53p have been crystallized. Crystals of the Dun1-FHA domain exhibit the symmetry of the space group P6122 or P6522, with unit-cell parameters a = b = 127.3, c = 386.3 Å; diffraction data have been collected to 3.1 Å resolution on a synchrotron source. Crystals of the N-terminal FHA domain (FHA1) of Rad53p diffract to 4.0 Å resolution on a laboratory X-ray source and have Laue-group symmetry 4/mmm, with unit-cell parameters a = b = 61.7, c = 104.3 Å.

Availability of folate bound to folate-binding protein: Environmental effects

The effect of FBP on folate bio-availability depends on its environment. The FBP of whole WPC enhances bioavailability of folates more than does purified FBP and its efficacy might be even greater when lipids are removed from the WPC. FBP polymerises and folate release from the polymer is found to be slower than that from the monomer. FBP has a role also as a folate receptor at cell surfaces and in this role folate binding increases polymerisation of FBP attached to lipid membranes.

A potential role for the analytical ultracentrifuge in the experimental measurement of protein valence

A method is described whereby sedimentation velocity is combined with equilibrium dialysis to determine the net charge (valence) of a protein by using chromate as an indicator ion for assessing the extent of the Donnan redistribution of small ions. The procedure has been used in experiments on bovine serum albumin under slightly alkaline conditions (pH 8.0, I 0.05) to illustrate its application to a system in which the indicator ion and protein both bear net negative charge and on lysozyme under slightly acidic conditions (pH 5.0, I 0.10) to illustrate the situation where chromate is a counterion. (C) 2001 Elsevier Science.

Analysis of thermodynamic non-ideality in terms of protein solvation

The effects of thermodynamic non-ideality on the forms of sedimentation equilibrium distributions for several isoelectric proteins have been analysed on the statistical-mechanical basis of excluded volume to obtain an estimate of the extent of protein solvation. Values of the effective solvation. parameter delta are reported for ellipsoidal as well as spherical models of the proteins, taken to be rigid, impenetrable macromolecular structures. The dependence of the effective solvated radius upon protein molecular mass exhibits reasonable agreement with the relationship calculated for a model in which the unsolvated protein molecule is surrounded by a 0.52-nm solvation shell. Although the observation that this shell thickness corresponds to a double layer of water molecules may be of questionable relevance to mechanistic interpretation of protein hydration, it augurs well for the assignment of magnitudes to the second virial coefficients of putative complexes in the quantitative characterization of protein-protein interactions under conditions where effects of thermodynamic non-ideality cannot justifiably be neglected. (C) 2001 Elsevier Science B.V. All rights reserved.

Dipeptidyl-peptidase II and cathepsin B activities in amelogenesis of the rat incisor

A body of published evidence suggests that a significant portion of enamel matrix protein synthesized by ameloblasts localises in the lysosomal-endosomal organelles of these enamel organ cells. Little is known regarding the lysosomal proteolytic activities during amelogenesis. The aims of this study were to detect and measure the activities of lysosomal peptidases cathepsin B (E.C. 3.4.22.1) and dipeptidyl-peptidase II (E.C. 3.4.14.2) in the enamel organ of the rat incisor and to ascertain whether rat enamel matrix proteins are degraded by these peptidases in vitro. Whole enamel organs were dissected from rat mandibular incisors. Enamel protein was also collected from the rat teeth. Analysis indicated that the rat incisor enamel organs contained specific activities of both dipeptidyl-peptidase II and cathepsin B at levels comparable with those of kidney which is rich in both these lysosomal peptidases. Gel electrophoresis and immunoblotting demonstrated that both cathepsin B and dipeptidyl-peptidase II were able to substantially degrade the rat enamel proteins in vitro. Based on these observations, we propose that lysosomal proteases have roles in amelogenesis in the intracellular degradation of amelogenins.

Growth hormone binding protein in normal and aneuploid pregnancy: a paradoxical decrease in trisomy 18

Objective To explore whether abnormalities in growth hormone binding protein (GHBP) may underlie the growth restriction associated with fetal aneuploidy. Design A retrospective casecontrol study. Setting Monash Medical Centre, Clayton, Victoria, Australia. Population Twenty-one trisomy 18, and 30 trisomy 21 pregnancies, and 170 chromosomally normal pregnancies at 15-18 weeks of gestation representing three to five controls per case matched for source, gestation and duration of storage. Methods GHBP was measured using a ligand immunofunctional assay. Results In the chromosomally normal pregnancies GHBP levels decreased slightly but significantly across the narrow gestational window studied. Compared with controls, levels of GHBP, expressed as median (95% CI) multiples of the median (MoM), in the trisomy 21 pregnancies were similar, 1.0 (0.92-1.39) MoM and 1.27 (1.04-1.50) MoM, respectively; P = 0.061 (Mann-Whitney CI test) but were significantly reduced in the trisomy 18 pregnancies, 0.68 (0.51-0.84) MoM; P = 0.0014 (Mann-Whitney U test). Conclusions These data suggest that decreased levels of maternal growth hormone binding protein, and by implication growth hormone receptor complement, may underlie the early severe growth restriction that is characteristic of trisomy 18.