Characterization of a receptor for heat shock protein 70 on macrophages and monocytes

Heat shock proteins (Hsps) and molecular chaperones isolated from tumors or virally infected cells elicit an efficient CD8+ T cell response against bound antigenic peptides. This immune response is mediated by presentation of the peptides on MHC class I complexes of antigen-presenting cells (APCs), but the cellular mechanism of this presentation process is not yet understood. Here we provide evidence for the existence of a proteinaceous receptor on the surface of APCs that is specific for mammalian Hsp70. Using a flow cytometry-based assay, saturable binding of Hsp70 to the cell surface of macrophages and peripheral blood monocytes, but not of lymphocytes, can be demonstrated. The affinity of the receptor is in the sub-micromolar range (Kd < 100 nM). Only mammalian Hsc70/Hsp70, but not bacterial Hsp70, is bound with high affinity. Subsequent to binding, Hsp70 is taken up by endocytosis, resulting in an intracellular localization. Our results suggest that receptor-mediated endocytosis forms the basis for the demonstrated efficacy of Hsp70-peptide complexes as anti-tumor vaccines.     

Contribution of de novo protein synthesis to the hypertrophic effect of IGF-1 but not of thyroid hormones in adult ventricular cardiomyocytes

Background: Enhanced expression of IGF-1 occurs in left ventricular hypertrophy (LVH) associated with systemic hypertension. Cardiac dysfunction accompanied by LVH is also observed in hyperthyroidism. Objective: to assess the relative contributions of de novo protein synthesis and attenuated protein degradation to increased protein mass associated with cardiomyocyte hypertrophy elicited by IGF-1 and thyroid hormones (tri-iodo thyronine T₃, and l-thyroxine T₄), respectively. Methods: total mass of protein, and both the incorporation, and removal of previously incorporated l-U-¹⁴C-phenylalanine, indices of protein synthesis and degradation, respectively, were assessed in quiescent adult rat ventricular cardiomyocytes maintained in short-term culture, and corrected for DNA content, as a index of cell number. Results: IGF-1 (1 pM-100 nM) increased cell protein significantly, maximally at 1 nM and by 38% above basal value after 24 h. T₃ (10 pM-2 M) and T₄ (10 pM-2 M) increased cell protein significantly maximally at 1 M and by 33.2 and 30.5%, respectively, above basal value. IGF-1 ( 10 pM), T₃ (10 pM-2 M) and T₄ (10 pM-2 M) did not increase incorporation of l-U-₁₄C-phenylalanine above basal values. IGF-1 (100 pM-100 nM) increased incorporation of radiolabel significantly maximally at 100 nM and by 56%. T₄ (100 pM) and IGF-1 (10 pM), concentrations that did not stimulate de novo protein synthesis, attenuated the degradation of radiolabelled protein by 13.6 and 11.8%, respectively, compared to control values after 48 h. Conclusion: These data indicate that the acute hypertrophic response to (i) thyroid hormones cannot be attributed to initiation of de novo protein synthesis; (ii) IGF-1 comprises two components; the response elicited by IGF-1 (< 10 pM) is independent of, while the response elicited by IGF-1 (> 100 pM) is due to de novo protein synthesis.     

Altered transarcolemmal Ca transport modifies the myofibrillar ultrastructure and protein metabolism in cultured adult ventricular cardiomyocytes

The present study was designed to investigate how prolonged (24-72 h) exposure to modifiers of Ca transarcolemmal transport affects the myofibrillar structure, protein turnover and content of myofibrillar proteins in adult guinea pig cardiomyocytes maintained beating synchronously in long-term cultures. First we established the functional responses (the contractile activity and [Ca]_i transients) of the cultured myocytes to acute exposures to several drugs used in this study. The ultrastructural characteristics of these cultures under the various treatments were determined using immunohistochemistry and confocal scanning laser microscopy, and their biochemical properties were evaluated using analysis of total cellular protein content, myofibrillar protein content and SDS-PAGE electrophoretic examination. We compared the effects of 24, 36 and 72 h-long exposures to the various specific Ca-flux modifiers. Increased Ca influx via Ca_L-channel agonist (Bay K 8644) or via the reversed- mode of the Na/Ca exchanger (veratrine) did not alter the myofibrillar structure or the specific protein profiles or proteosynthesis. However, when cytosolic Ca was increased by three different types of inhibitors of Ca extrusion from the cells via Na/Ca exchange, (Na-free solution, 5 mM NiCl₂ and 10^-6 M ouabain), very significant changes in all investigated parameters occurred almost immediately. Twenty-four h-long exposure to Na-free did not affect significantly the total cellular protein (TCP), but the protein synthesis was decreased by 87% and the total myofibrillar protein (TMP) content was decreased by 38%. The myofibrils were heavily fragmented. Similarly, 24 h-long exposure to 5 mM NiCl₂ did not affect the TCP, but it reduced protein synthesis by about 90% and decreased the total myofibrillar protein content by 30%. These effects were even more pronounced at 72 h of exposure and they were accompanied with a complete disassembly of myofilaments. Exposure to 10^-6 M ouabain over 72 h resulted in > 80% inhibition of protein synthesis, a 45% decrease in TCP content and a 53% in TMP content. In contrast, 10^-7 M ouabain did not produce any such changes. The changes produced by the Na/Ca-exchange inhibitors were accompanied by only minor changes in DNA content, indicating that the myocytes remained viable. Moreover, these effects were not due to the associated contractile arrest, since exposure to Ca_L-channel antagonists (5-20 M nifedipine or 10 M verapamil) produced only very minor changes in the myofibrillar structure and in protein profiles.Our data demonstrate that short-term (up to 72 h) increased Ca influx or contractile arrest of well-interconnected, spontaneously beating adult cardiomyocytes does not affect their ultrastructural characteristics or their myofibrillar protein turnover greatly, while any situations leading to Ca accumulation (via inhibition of Na/Ca exchange) affect cardiomyocyte function and ultrastructure almost immediately. These data are in sharp contrast to those previously reported from immature, neonatal myocytes.     

Stigmatella aurantiaca Sg a15 carries genes encoding type I and type II 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases: involvement of a type II synthase in aurachin biosynthesis

3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases catalyse the first step of the shikimate pathway. Two unrelated DAHP synthase types have been described in plants and bacteria. Two type II (aroA(A2) and aroA(A5)) and one type I DAHP synthase gene (aroA001) were identified from the myxobacterium Stigmatella aurantiaca Sg a15. Inactivation of aroA(A5) leads to a mutant that is impaired in the biosynthesis of aurachins, which are electron transport inhibitors and contain an anthranilate moiety. Feeding of anthranilic acid to the mutant culture restores production of aurachins. Inactivation of aroA(A2) and aroA001 does not impair production of aurachins or other known secondary metabolites of S. aurantiaca Sg a15.     

Multi-allelic origin of congenital disorder of glycosylation (CDG)-Ic

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndrome, represent a family of genetic diseases with variable clinical presentations. Common to all types of CDG characterized to date is a defective Asn-linked glycosylation caused by enzymatic defects of N-glycan synthesis. Previously, we have identified a mutation in the ALG6 alpha1,3 glucosyltransferase gene as the cause of CDG-Ic in four related patients. Here, we present the identification of seven additional cases of CDG-Ic among a group of 35 untyped CDG patients. Analysis of lipid-linked oligosaccharides in fibroblasts confirmed the accumulation of dolichyl pyrophosphate-Man9GlcNAc2 in the CDG-Ic patients. The genomic organization of the human ALG6 gene was determined, revealing 14 exons spread over 55 kb. By polymerase chain reaction amplification and sequencing of ALG6 exons, three mutations, in addition to the previously described A333 V substitution, were detected in CDG-Ic patients. The detrimental effect of these mutations on ALG6 activity was confirmed by complementation of alg6 yeast mutants. Haplotype analysis of CDG-Ic patients revealed a founder effect for the ALG6 allele bearing the A333 V mutation. Although more than 80% of CDG are type Ia, CDG-Ic may be the second most common form of the disease.     

Molecular changes in the d-bifunctional protein cDNA sequence in australasian patients belonging to the bifunctional protein complementation group

The cDNA sequence for the human d-bifunctional protein (D-BP: 17β-hydroxysteroid dehydrogenase IV) was investigated in patients with peroxisomal disorders belonging to the BP complementation group (CG). In three cases, analysis of polymerase chain reaction products generated from the patients' cDNA indicated the presence of a deletion within the region corresponding to nucleotides 209–537 of the normal cDNA sequence. Subsequent sequencing revealed that, in two of the patients, 47 base pairs were missing, with the deletion corresponding to nucleotides 302/3–349/50 of the normal sequence. In the third patient, a smaller deletion of 22 bp (nucleotides 280/1–302/3) was characterized. Only the mutant sequence was detected in each of these cases, consistent with parental consanguinity. Both deletions cause a frameshift, and would lead to premature termination of the BP. Available family members were also investigated, and the findings conformed with expectations for an autosomal recessive disorder. In addition to the deletions, a number of other base changes have been identified in this series of patients. In particular, one patient, whose parents were also consanguineous, was homozygous for a base change, which results in a nonconservative substitution of serine 177 with a phenylalanine residue. The functional significance of this amino acid substitution, as well as the other identified changes, is still to be determined. Nevertheless, our data provide strong support for the hypothesis that defects in the gene for the D-BP are responsible for the β-oxidation defect in patients belonging to the BP CG.     

Localization and topology of ratp28, a member of a novel family of putative steroid-binding proteins

We have cloned ratp28, a membrane protein from rat liver homologous to the previously described hpr6.6, a putative steroid-binding protein in humans. Ratp28 has a type II topology as determined by protease digestion experiments on intact and detergent-solubilized membranes. Subcellular fractionation by sucrose density centrifugation revealed a distribution for ratp28 identical to Bip as a marker for membranes of the endoplasmic reticulum. In these experiments no association was found with markers for Golgi or plasma membranes, indicating that ratp28 is localized to the endoplasmic reticulum.     

Oligomerization of peptides analogous to the cytoplasmic domains of coatomer receptors revealed by mass spectrometry

Members of the p24 family of type I transmembrane proteins are involved in budding of coat protein type I (COPI)-coated vesicles. They serve as coat protein receptors, binding via their cytoplasmic domains to coatomer, a stable cytosolic protein complex that represents the major coat component of these vesicles. Experimental evidence suggest that p23, a member of the p24 family, binds to coatomer in an oligomeric state and that this binding triggers polymerization of the coat protein. Toward an understanding of this process at the molecular level, formation of noncovalent complexes and their relative stabilities were analyzed by Fourier transform ion cyclotron resonance mass spectrometry using nanoelectrospray ionization. Specificity and stability of oligomers formed were established to depend on characteristic peptide sequence motifs and were confirmed by mass spectrometric competition experiments with control peptides. Mutations in the peptide sequence caused decreased interaction and destabilization of the noncovalent complexes. The formation and relative stabilities of dimeric and tetrameric complexes were assessed to be formed by cytoplasmic tails of coatomer receptors. The direct molecular identification provided by mass spectrometry correlates well with biochemical results. Thus, electrospray ionization mass spectrometry proves to be a powerful tool to investigate physiologically relevant peptide complexes.     

Mechanism of substrate hydrolysis by a thermophilic endoglucanase from Thermotoga maritima†

A cellulase from the thermophile, Thermotoga maritima, hydrolyzed oligosaccharide substrates by an exoglucanase mode of action but acted as an endoglucanase to rapidly reduce the viscosity of the soluble polysaccharides carboxymethylcellulose and barley -glucan. The V _max for hydrolysis of the substrate, p-nitrophenyl -d-cellobioside, was 42 mol min^–1 (mg protein)^–1, while that for barley -glucan was 637. The enzyme had little activity on crystalline cellulose.     

Native American Postcolonial Psychology

Native American Postcolonial Psychology. Eduardo Duran and Bonnie Duran. Albany: State University of New York Press, 1995. 227 pp.