A Chaperone in the HSP70 Family Controls Production of Extracellular Fibrils in Myxococcus xanthus

Three independent Tn5-lac insertions in the S1 locus of Myxococcus xanthus inactivate the sglK gene, which is nonessential for growth but required for social motility and multicellular development. The sequence of sglK reveals that it encodes a homologue of the chaperone HSP70 (DnaK). The sglK gene is cotranscribed with the upstream grpS gene, which encodes a GrpE homologue. Unlike sglK, grpS is not required for social motility or development. Wild-type M. xanthus is encased in extracellular polysaccharide filaments associated with the multimeric fibrillin protein. Mutations in sglK inhibit cell cohesion, the binding of Congo red, and the synthesis or secretion of fibrillin, indicating that sglK mutants do not make fibrils. The fibR gene, located immediately upstream of the grpS-sglK operon, encodes a product which is predicted to have a sequence similar to those of the repressors of alginate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida. Inactivation of fibR leads to the overproduction of fibrillin, suggesting that M. xanthus fibril production and Pseudomonas alginate production are regulated in analogous ways. M. xanthus and Pseudomonas exopolysaccharides may play similar roles in a mechanism of social motility conserved in these gram-negative bacteria.     

Reactivities of the cysteine residues of the reduced pancreatic trypsin inhibitor.

The six cysteine residues of the reduced pancreatic trypsin inhibitor have been found to be equally reactive toward iodoacetate under the conditions used for refolding of the protein. The rates of reaction of each residue were comparable to those observed with model thiol compounds. It is concluded that the reduced inhibitor has no stable conformational properties that affect the cysteine residues. The results corroborate the previous conclusion that all six cysteine residues participate in forming the first disulphide bond during refolding of the reduced inhibitor and confirm that disulphide bond formation is an accurate probe of the conformational transitions that occur during protein folding.     

Differentiation of rat lens epithelial cells in tissue culture. (I) Effects of cell density, medium and embryonic age of initial culture.

The growth and differentiation of rat lens epithelial cells in tissue culture were studied. Cells could be maintained for a number of generations in an undifferentiated state in suspension culture. When cultured as monolayers, they grew and differentiated in a series of six defined stages described here. These stages include morphological changes (elongation, followed by cell "spreading" or formation of cell aggregates), and biochemical changes (appearance of nu-crystallin protein as detected by immunofluorescence). The process of differentiation appeared to be accelerated in the vicinity of elongated cells, occurred more rapidly at high cell density, and required frequent changes of medium. This suggests that cell-cell communication, and not medium factors, may be essential for promoting differentiation. The final morphology of the differentiated cells differed, depending on the embryonic age of the rats used as a source of lens epithelial cells. This implies that the programme for differentiation changes as a function of the embryonic age of the lens.     

The single-disulphide intermediates in the refolding of reduced pancreatic trypsin inhibitor

The intermediate species with one disulphide bond in the renaturation of reduced pancreatic trypsin inhibitor have been trapped, isolated, and the Cys residues involved in the disulphide bonds determined. Approximately half the intermediate species had the disulphide bond between Cys-30 and 51, a disulphide bond also present in the native inhibitor. The next most predominant species, representing one-quarter of the total, had a disulphide bond between Cys-5 and 30, and two more minor species involving Cys-30 and 55 and Cys-5 and 51 were detected; these disulphide bonds are not present in the native inhibitor.The nature of the disulphide bonds present are concluded to reflect primarily the conformational forces acting at this stage of folding, which may be primarily interactions between segments with propensities for secondary structure, either helices or β-sheet. The general importance of such interactions in protein folding is discussed.     

Immunochemical analysis of the conformational properties of intermediates trapped in the folding and unfolding of bovine pancreatic trypsin inhibitor.

Immunochemical methods have been used to examine the conformational properties of the entire polypeptide chain in the various trapped intermediate states which are kinetically important in the unfolding and refolding of pancreatic trypsin inhibitor. The interactions of each of the trapped intermediates, having their disulphide bonds frozen, with antibodies specific for either the native, folded or the reduced, unfolded states of the entire protein have been used to determine the probabilities of the various segments of the polypeptide chain adopting either conformation recognized by the antibodies.The results are considered with regard to the kinetic roles of the various species and to their conformational properties during folding and unfolding inferred from the observed propensities of each of the six cysteine residues to participate in disulphide bond formation, interchange, or breakage. It is concluded that no segment of the polypeptide chain adopts a stable native-like conformation until the entire polypeptide chain is able to do so simultaneously. The best correlation of conformation with the kinetic role in refolding of the intermediates is observed not with their propensity to adopt native-like conformation, but with their stability to full unfolding as measured by their interaction with antibodies against the reduced protein.     

Static allometry of mammalian teeth and the correlation of tooth size and body size in contemporary mammals

Functional least squares regression was used to fit the allometric equation, y = bx^k, to data on head-body lengths and body weights for 123 species of mammals. These measurements were found to be highly correlated and to scale isometrically. The scaling of M ₁ area with head-body length was investigated for 288 species of terrestrial mammals. A method was described for estimating the body size of extinct mammals from M ₁ area and the tooth size-body size relationships discovered among contemporary forms.     

Conformational restrictions on the pathway of folding and unfolding of the pancreatic trypsin inhibitor

The kinetic roles of the partially folded, intermediate protein species with two disulphide bonds in folding and unfolding of the pancreatic trypsin inhibitor have been investigated further. Formation of a second disulphide bond between Cys5 and Cys55 during refolding of the reduced inhibitor, which would yield the species with the 30–51 and 5–55 disulphide bonds and, possibly, the native-like conformation of the protein, is not significant. Instead, three other second disulphide bonds (5–14, 5–38 and 14–38) are formed approximately 10⁵ times more readily, but each of these two-disulphide species then rearranges intramolecularly to the native-like, two-disulphide intermediate. Therefore, the reduced protein does not simply form sequentially the three disulphide bonds of the native state. Unfolding of the native state takes place by the reverse of this process.The kinetic importance for folding and unfolding of this transition between two-disulphide intermediates under the conditions used here was illustrated experimentally by a modified form of the inhibitor in which the thiols of Cys14 and Cys38 were blocked irreversibly. In the folded conformation, this modified protein is more stable to unfolding than normal, but after unfolding cannot readily regain the native-like conformation, because Cys14 or Cys38 are required to be involved in disulphide bonds during the interconversion of the two-disulphide intermediates.Some conception of the conformational transitions that take place at each stage of the folding transition may be inferred from the relative propensities of the six cysteine residues to make or rearrange disulphide bonds. It is concluded that the inhibitor probably does not refold by sequential adoption of the native conformation by the unfolded polypeptide chain. Instead, it appears that essentially all elements of the native conformation are attained simultaneously in the final stage of folding, within an unstable and flexible, yet relatively compact, form of the entire polypeptide chain produced by weak interactions between groups distant in the primary structure.     

N-(2-hydroxypropyl)methacrylamide copolymers of a glutathione (GSH)-activated glyoxalase i inhibitor and DNA alkylating agent: synthesis, reaction kinetics with GSH, and in vitro antitumor activities

The incorporation of anticancer prodrugs into polyacrylamide conjugates has been shown to improve tumor targeting via the so-called "enhanced permeability and retention" effect. This strategy has now been expanded to include two different classes of glutathione (GSH)-activated antitumor agents prepared by radical polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-methacryloyloxy-methyl-2-cyclohexenone (7) and/or with S-(N-4-chlorophenyl-N-hydroxycarbamoyl-thioethyl)methacrylamide (8), followed by treatment with 3-chloroperoxybenzoic acid, to give the HPMA copolymers of 7 and the 8-sulfoxide, respectively. In aqueous-buffered solution at pH 6.5, GSH reacts rapidly with poly-HPMA-8-sulfoxide (k approximately 2.3 mM(-1) min(-1)) to give S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione (1), a tight-binding transition state analogue inhibitor of the antitumor target enzyme glyoxalase I (K(i) = 46 nM), or with poly-HPMA-7 (k approximately 0.02 mM(-1) min(-1)) to give the electrophilic antitumor agent 3-glutathio-2-methylenecyclohexenone (4). Indeed, B16 melanotic melanoma in culture is inhibited by poly-HPMA-8-sulfoxide and by poly-HPMA-7 with IC(50) values of 168 +/- 8 and 284 +/- 5 microM, respectively. These values are significantly greater than those of the unpolymerized prodrugs suggesting that the cytotoxicity of the polymer prodrugs might be limited by slow cellular uptake via pinocytosis. This prodrug strategy should be applicable to a range of different GSH-based antitumor agents.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics