Enhanced activity of serum and urinary hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats

Using streptozotocin-induced diabetic Wistar and GK rats as models of type 1 and type 2 diabetes, respectively, we investigated the changes in serum and urinary hyaluronidase activity with the pathological progress. The serum hyaluronidase levels of streptozotocin-induced rats started to increase on the third day after injection and thereafter maintained approximately threefold higher levels compared with control rats; those of GK rats were already higher ( approximately twofold) from the beginning of the experiment. The increases of serum hyaluronidase activity in both diabetic rats were similar to those of blood glucose level, indicating that diabetes mellitus was accompanied by enhanced activity of circulating hyaluronidase from the early phase of its development. In zymography, every serum from diabetic and control rats gave two hyaluronidase isomers, a major 73-kDa band (Hyal-1 type) and a minor 132-kDa band, suggesting that the increases in serum hyaluronidase activity were not due to the appearance of novel isomers. The hyaluronidase activity in 24-h urine of streptozotocin-induced rats was 3-, 7-, and 11-fold higher at the 8th, 15th, and 18th week than that of control rats, respectively, and the urinary hyaluronidase activity of GK rats was not significantly different from controls. There was a good correlation between the urinary hyaluronidase activity and the albumin excretion. Thus the increase in urinary hyaluronidase activity may reflect enhanced glomerular permeability in streptozotocin-induced diabetic rats and may be a useful marker for diabetic nephropathy. Relative resistance to SDS-denaturation in zymography of rat serum and urinary hyaluronidases compared with human serum hyaluronidase are also shown.     

Glycoprofiling with micro-arrays of glycoconjugates and lectins

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.     

N-glycan branching requirement in neuronal and postnatal viability

The structural variations among extracellular N-glycans reflect the activity of glycosyltransferases and glycosidases that operate in the Golgi apparatus. More than other types of vertebrate glycans, N-glycans are highly branched oligosaccharides with multiple antennae linked to an underlying mannose core structure. The branching patterns of N-glycans consist of three types, termed high-mannose, hybrid, and complex. Though most extracellular mammalian N-glycans are of the complex type, some cells variably express hybrid and high-mannose forms. Nevertheless, a requirement for hybrid and complex N-glycan branching exists in embryonic development and postnatal function among mice and humans inheriting defective Mgat1 or Mgat2 alleles. The resulting defects in formation N-glycan branching patterns cause multiple abnormalities, including neurologic defects, and have inferred the presence of distinct functions for hybrid and complex N-glycan branches among different cell lineages. We have further explored N-glycan structure-function relationships in vivo by using Cre-loxP conditional mutagenesis to abolish hybrid and complex N-glycan branching specifically among neuronal cells. Our findings show that hybrid N-glycan branching is an essential posttranslational modification among neurons. Loss of Mgat1 resulted in a unique pattern of neuronal glycoprotein deficiency concurrent with caspase 3 activation and apoptosis. Such animals exhibited severe locomotor deficits, tremors, paralysis, and early postnatal death. Unexpectedly, neuronal Mgat2 deletion resulting in the loss of complex but not hybrid N-glycan branching was well tolerated without phenotypic markers of neuronal or locomotor dysfunction. Structural features associated with hybrid N-glycan branching comprise a requisite posttranslational modification to neuronal glycoproteins that permits normal cellular function and viability.     

Agrin is required for posterior development and motor axon outgrowth and branching in embryonic zebrafish

Although recent studies have extended our understanding of agrin's function during development, its function in the central nervous system (CNS) is not clearly understood. To address this question, zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS and in nonneural structures such as somites and notochord. In agrin morphant embryos, acetylcholine receptor (AChR) cluster number and size on muscle fibers at the choice point were unaffected, whereas AChR clusters on muscle fibers in the dorsal and ventral regions of the myotome were reduced or absent. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon-Beard sensory neurons were also observed, which included truncation of axons and increased branching of motor axons. Moreover, agrin morphants exhibit significantly inhibited tail development in a dose-dependent manner, as well as defects in the formation of the midbrain-hindbrain boundary and reduced size of eyes and otic vesicles. Together these results show that agrin plays an important role in both peripheral and CNS development and also modulates posterior development in zebrafish.     

The shedding of syndecan-4 and syndecan-1 from HeLa cells and human primary macrophages is accelerated by SDF-1/CXCL12 and mediated by the matrix metalloproteinase-9

We recently demonstrated that stromal cell-derived factor-1(SDF-1/CXCL12) forms complexes with CXCR4, but also with syndecan-4expressed by human primary lymphocytes and macrophages, andHeLa cells. We also suggested that syndecan-4 behaves as a SDF-1-signalingmolecule. Here, we demonstrate that SDF-1 strongly acceleratesthe shedding of syndecan-4 ectodomains and to a lesser extentthat of syndecan-1 from HeLa cells. The fact that this accelerationwas not inhibited by the CXCR4 antagonist AMD3100, anti-CXCR4mAb 12G5, and CXCR4 gene silencing suggests its CXCR4-independence.Pre-treating the cells with heparitinases I, III, or with theprotein kinase C (PKC) inhibitor, bisindolylmaleimide, significantlyinhibited this accelerated shedding, which suggests the involvementof both cell-surface heparan sulfate and PKC transduction pathway.In contrast, Map Kinase or NF-B pathway inhibitors had no effect.Moreover, SDF-1 increases the matrix metalloproteinase-9 (MMP-9)mRNA level as well as MMP-9 activity in HeLa cells, and MMP-9silencing by RNA interference strongly decreases the syndecan-1and -4 ectodomain shedding accelerated by SDF-1. Finally, SDF-1also accelerates in a CXCR4-independent manner, the sheddingof syndecan-1 and -4 from human primary macrophages, which issignificantly inhibited by anti-MMP-9 antibodies. This stronglyindicates the role of MMP-9 in these events occurring in botha tumoral cell line and in human primary macrophages. BecauseMMP-9 plays a crucial role in extracellular matrix degradationduring cancer cell metastasis and invasion, and shed ectodomainsof syndecans may likely be involved in tumor cell proliferation,these data further indicate the multiplicity of the roles playedby SDF-1 on tumor cell biology.     

Why and how marine-invertebrate larvae metamorphose so fast

It is argued that larviparous development has evolved at least eight times among extant animals. A 'need for speed hypothesis' is proposed to explain profound convergence on a pattern of small larvae and rapid metamorphosis across six marine invertebrate clades. Shared selection pressures include limits to larval size, the plankton-to-benthos transition, extreme hazards on the benthos, and the profound helplessness of metamorphosing animals. The adaptive mechanisms include: (1) development of juvenile structures in larvae before they are metamorphically competent; (2) external cues trigger metamorphosis; and (3) rapid cell-to-cell conductance of the metamorphic signal to bring about rapid loss of larval structures and release of juvenile structures. Both pattern and mechanisms contrast in every regard with those of the other two major larviparous clades, Insecta and Amphibia.     

Interaction profile of galectin-5 with free saccharides and mammalian glycoproteins: probing its fine specificity and the effect of naturally clustered ligand presentation

Cell-surface glycans are functional docking sites for tissue lectins such as the members of the galectin family. This interaction triggers a wide variety of responses; hence, there is a keen interest in defining its structural features. Toward this aim, we have used enzyme-linked lectinosorbent (ELLSA) and inhibition assays with the prototype rat galectin-5 and panels of free saccharides and glycoconjugates. Among 45 natural glycans tested for lectin binding, galectin-5 reacted best with glycoproteins (gps) presenting a high density of Galbeta1-3/4GlcNAc (I/II) and multiantennary N-glycans with II termini. Their reactivities, on a nanogram basis, were up to 4.3 x 10(2), 3.2 x 10(2), 2.5 x 10(2), and 1.7 x 10(4) times higher than monomeric Galbeta1-3/4GlcNAc (I/II), triantennary-II (Tri-II), and Gal, respectively. Galectin-5 also bound well to several blood group type B (Galalpha1-3Gal)- and A (GalNAcalpha1-3Gal)-containing gps. It reacted weakly or not at all with tumor-associated Tn (GalNAcalpha1-Ser/Thr) and sialylated gps. Among the mono-, di-, and oligosaccharides and mammalian glycoconjugates tested, blood group B-active II (Galalpha1-3Gal beta1-4GlcNAc), B-active IIbeta1-3L (Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc), and Tri-II were the best. It is concluded that (1) Galbeta1-3/4GlcNAc and other Galbeta1-related oligosaccharides with alpha1-3 extensions are essential for binding, their polyvalent form in cellular glycoconjugates being a key recognition force for galectin-5; (2) the combining site of galectin-5 appears to be of a shallow-groove type sufficiently large to accommodate a substituted beta-galactoside, especially with alpha-anomeric extension at the non-reducing end (e.g., human blood group B-active II and B-active IIbeta1-3L); (3) the preference within beta-anomeric positioning is Galbeta1-4 > or = Galbeta1-3 > Galbeta1-6; and (4) hydrophobic interactions in the vicinity of the core galactose unit can enhance binding. These results are important for the systematic comparison of ligand selection in this family of adhesion/growth-regulatory effectors with potential for medical applications.     

Molecular genetics of crustacean feeding appendage development and diversification

Arthropods dominate our seas, land, and air and have done so for hundreds of millions of years. Among the arthropods, crustaceans present us with a rich history of morphological change, much of which is still represented among extant forms. Crustacea largely interact with their environment via their appendages; thus vast amounts of variation exist among the different appendages of a single individual and between appendages from different species. Comparative studies of crustacean appendage development present us with an important story regarding the evolution of morphology over both relatively short (a few million years) and relatively long (a few hundred million years) evolutionary time scales. Recent studies have used the genetic and molecular data from Drosophila development to try to understand the molecular basis for some of the variations seen in crustacean limbs. Here we review some of these data based on the expression patterns of the genes Ultrabithorax, abdominal - A, Sex combs reduced, and Distal-less.     

Affinity of galectin-8 and its carbohydrate recognition domains for ligands in solution and at the cell surface

Galectin-8 has two different carbohydrate recognition domains (CRDs), the N-terminal Gal-8N and the C-terminal Gal-8C linked by a peptide, and has various effects on cell adhesion and signaling. To understand the mechanism for these effects further, we compared the binding activities of galectin-8 in solution with its binding and activation of cells. We used glycan array analysis to broaden the specificity profile of the two galectin-8 CRDs, as well as intact galectin-8s (short and long linker), confirming the unique preference for sulfated and sialylated glycans of Gal-8N. Using a fluorescence anisotropy assay, we examined the solution affinities for a subset of these glycans, the highest being 50 nM for NeuAcalpha2,3Lac by Gal-8N. Thus, carbohydrate-protein interactions can be of high affinity without requiring multivalency. More importantly, using fluorescence polarization, we also gained information on how the affinity is built by multiple weak interactions between different fragments of the glycan and its carrier molecule and the galectin CRD subsites (A-E). In intact galectin-8 proteins, the two domains act independently of each other in solution, whereas at a surface they act together. Ligands with moderate or weak affinity for the isolated CRDs on the array are bound strongly by intact galectin-8s. Also galectin-8 binding and signaling at cell surfaces can be explained by combined binding of the two CRDs to low or medium affinity ligands, and their highest affinity ligands, such as sialylated galactosides, are not required.     

Rapid structural characterization of the arabinogalactan and lipoarabinomannan in live mycobacterial cells using 2D and 3D HR-MAS NMR: structural changes in the arabinan due to ethambutol treatment and gene mutation are observed

Mycobacteria possess a unique, highly evolved, carbohydrate- and lipid-rich cell wall that is believed to be important for their survival in hostile environments. Until now, our understanding of mycobacterial cell wall structure has been based upon destructive isolation and fragmentation of individual cell wall components. This study describes the observation of the major cell wall structures in live, intact mycobacteria using 2D and 3D high-resolution magic-angle spinning (HR-MAS) nuclear magnetic resonance (NMR). As little as 20 mg (wet weight) of [13C]-enriched cells were required to produce a whole-cell spectra in which discrete cross-peaks corresponding to specific cell wall components could be identified. The most abundant signals of the arabinogalactan (AG) and lipoarabinomannan (LAM) were assigned in the HR-MAS NMR spectra by comparing the 2D and 3D NMR whole-cell spectra with the spectra of purified cellular components. This study confirmed that the structures of the AG and LAM moieties in the cell wall of live mycobacteria are consistent with structural reports in the literature, which were obtained via degradative analysis. Most important, by using intact cells it was possible to directly demonstrate the effects of ethambutol on the mycobacterial cell wall polysaccharides, characterize the effects of embB gene knockout in the M. smegmatis DeltaembB mutant, and observe differences in the cell wall structures of two mycobacterial species (M. bovis BCG and M. smegmatis.) Herein, we show that HR-MAS NMR is a powerful, rapid, nondestructive technique to monitor changes in the complex, carbohydrate-rich cell wall of live mycobacterial cells.     

Functional design in the evolution of embryos and larvae

A function of development is to put the right kind of cells in the right place at the right time. Other functional analyses help define what is right. As examples, functional analyses offer explanations for the unicellular bottleneck in life histories that necessitates embryos, evolutionary divergences in embryonic cell cycles, conditions permissive of loss of larval structures and consequent change in embryonic development, and the decoupled development of larval bodies and juvenile rudiments. Functional analyses also reveal the specifications required of morphogenesis, hence defining developmental phenomena to be explained.     

Characterization and role of fucose mutarotase in mammalian cells

L-Fucose for mammalian glycosylation contains an anomeric carbon atom generating alpha- and beta-L-fucoses. Based on sequence comparison of mouse and human homologs with the prokaryotic fucose mutarotases (FucU) characterized previously, we investigated their function in mammalian cells. By nuclear magnetic resonance (NMR) measurement with saturation difference analysis, the purified mammalian mutarotases were demonstrated to be involved in an interconversion between the two anomeric forms with comparable efficiency as that of the Escherichia coli FucU. The mouse gene was widely expressed in various tissues and cell lines, including kidney, liver, and pancreas, although expression was marginal in muscle and testis. By generating stably expressed cell lines for mutarotase genes in HepG2, it was shown that fucose incorporations into cellular proteins were increased as demonstrated by an incorporation of radiolabeled fucose into the cells. Furthermore, intracellular levels of GDP-L-fucose, measured with high performance liquid chromatography (HPLC), were enhanced by an overproduction of cellular mutarotase, which was reversed by gene silencing of mutarotase based on RNA interference. The results suggest that the mammalian mutarotase is functional in facilitated incorporation of fucose through the salvage pathway.     

Expression and regulation of chemokine genes in the mouse uterus during pregnancy

Leukocytes accumulate in the pregnant mouse uterus following mating, during implantation and during placental development. Changes in leukocyte number are primarily due to recruitment from the blood, not local proliferation, but the underlying recruitment mechanisms are poorly understood. Mating-induced granulocyte and macrophage recruitment is due in part to pro-inflammatory and chemotactic factors present in seminal plasma. Accumulation of macrophages later in pregnancy appears to be caused in part by ovarian hormone-stimulated CSF-1 production and in part by other as yet unidentified uterine chemotactic factors. The current study was performed to assess chemokine production in the uterus during pregnancy. Northern blotting was used to demonstrate NSI/KC (KC), macrophage chemotactic protein-1 (MCP-1), macrophage inflammatory protein one alpha (MIP1alpha) and regulated inactivation, normal T expressed and secreted protein (RANTES) mRNA in the uterus. Oestrogen and progesterone induced intrauterine production of all four chemokines and may have done so through the autocrine/paracrine activities of IL-1. The data suggest that C-C chemokines play a role in accumulation of macrophages in the uterus during pregnancy.     

Reactive oxygen species and mitochondrial diseases

A variety of diseases have been associated with excessive reactive oxygen species (ROS), which are produced mostly in the mitochondria as byproducts of normal cell respiration. The interrelationship between ROS and mitochondria suggests shared pathogenic mechanisms in mitochondrial and ROS-related diseases. Defects in oxidative phosphorylation can increase ROS production, whereas ROS-mediated damage to biomolecules can have direct effects on the components of the electron transport system. Here, we review the molecular mechanisms of ROS production and damage, as well as the existing evidence of mitochondrial ROS involvement in human diseases.     

Differential structure and activity between human and mouse intelectin-1: human intelectin-1 is a disulfide-linked trimer, whereas mouse homologue is a monomer

Human intelectin-1 (hITLN-1) is a 120-kDa lectin recognizing galactofuranosyl residues found in cell walls of various microorganisms but not in mammalian tissues. Although mouse intelectin-1 (mITLN-1) has been identified previously, its biochemical properties and functional characteristics have not been studied. Therefore, we have compared structures and saccharide-binding specificities of hITLN-1 and mITLN-1 using recombinant proteins produced by mammalian cells. Recombinant hITLN-1 is a trimer, disulfide-linked through Cys-31 and Cys-48, and N-glycosylated at Asn-163. Despite 84.9% amino acid identity to hITLN-1, recombinant and intestinal mITLN-1 are unglycosylated 30-kDa monomers. Recombinant hITLN-1, as well as recombinant and intestinal mITLN-1 were purified by Ca(2+)-dependent adsorption to galactose-Sepharose. In competitive binding studies, hITLN-1 was eluted from galactose-Sepharose by 100 mM 2-deoxygalactose, a galactofuranosyl disaccharide, d-xylose, and both d- and l-ribose. In contrast, mITLN-1 was partially eluted by the galactofuranosyl disaccharide, and only minimally by the other saccharides indicating that the two intelectins have different saccharide-binding specificities. When the N- and C-terminal regions of hITLN-1 were replaced, respectively, with those of mITLN-1, galactose-Sepharose binding was associated with the C-terminal regions. Finally, hITLN-1 binding to galactose-Sepharose was not affected by the substitution of the Cys residues in the N-terminal region that are necessary for oligomer formation, nor was it affected by the removal of the N-linked oligosaccharide at Asn-163. Although both hITLN-1 and mITLN-1 recognize galactofuranosyl residues, our comparative studies, taken together, demonstrate that these intelectins have different quaternary structures and saccharide-binding specificities.     

Molecular cloning and characterization of a novel human beta1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain

Protein O-linked fucosylation is an unusual glycosylation associated with many important biological functions such as Notch signaling. Two fucosylation pathways synthesizing O-fucosylglycans have been reported on cystein-knotted proteins, that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin Type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC) analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous beta3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by beta-glucosidase from almond and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in Escherichia coli was first fucosylated by the recombinant protein O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for the recombinant beta3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.     

Galectin-3 and soluble fibrinogen act in concert to modulate neutrophil activation and survival: involvement of alternative MAPK pathways

Galectin-3 (Gal-3), a member of a family of highly conserved carbohydrate-binding proteins, has recently emerged as a novel cellular modulator at inflammatory foci. Here we investigated the effects of Gal-3 on central effector functions of human neutrophils, including phagocytosis, exocytosis of secretory granules, and survival. We examined the effects of Gal-3 alone or in combination with soluble fibrinogen (sFbg), an extracellular mediator that plays a key role during the early phase of the inflammatory response through binding to integrin receptors. In addition we evaluated the intracellular signals triggered by these mediators in human neutrophils. Human neutrophils incubated with recombinant Gal-3 alone increased their phagocytic activity and CD66 surface expression. In contrast to the known antiapoptotic effect of Gal-3 on many cellular types, Gal-3 enhanced PMN apoptotic rate. Preincubation with Gal-3 primed neutrophils to the effects of sFbg, resulting in a synergistic action on degranulation. On the other hand, Gal-3 and sFbg had opposite effects on PMN survival, and the simultaneous action of both agonists partially counteracted the proapoptotic effects of Gal-3. In addition, although sFbg induced its effects through the activation of the ERKs, Gal-3 led to p38 phosphorylation. Disruption of this signaling pathway abrogated Gal-3-mediated modulation of neutrophil degranulation, phagocytosis, and apoptosis. Together, our results support the notion that Gal-3 and sFbg are two physiological mediators present at inflammatory sites that activate different components of the MAPK pathway and could be acting in concert to modulate the functionality and life span of neutrophils.     

Reciprocal Negative Regulation of PDK1 and ASK1 Signaling by Direct Interaction and Phosphorylation

Cell survival and death-inducing signals are tightly associated with each other, and the decision as to whether a cell survives or dies is determined by controlling the relationship between these signals. However, the mechanism underlying the reciprocal regulation of such signals remains unclear. In this study, we reveal a functional association between PDK1 (3-phosphoinositide-dependent protein kinase 1), a critical mediator of cell survival, and ASK1 (apoptosis signal-regulating kinase 1), an apoptotic stress-activated MAPKKK. The physical association between PDK1 and ASK1 is mediated through the pleckstrin homology domain of PDK1 and the C-terminal regulatory domain of ASK1 and is decreased by ASK1-activating stimuli, such as H₂O₂, tumor necrosis factor α, thapsigargin, and ionomycin, as well as insulin, a PDK1 stimulator. Wild-type PDK1, but not kinase-dead PDK1, negatively regulates ASK1 activity by phosphorylating Ser⁹⁶⁷, a binding site for 14-3-3 protein, on ASK1. PDK1 functionally suppresses ASK1-mediated AP-1 transactivation and H₂O₂-mediated apoptosis in a kinase-dependent manner. On the other hand, ASK1 has been shown to inhibit PDK1 functions, including PDK1-mediated regulation of apoptosis and cell growth, by phosphorylating PDK1 at Ser³⁹⁴ and Ser³⁹⁸, indicating that these putative phosphorylation sites are involved in the negative regulation of PDK1 activity. These results provide evidence that PDK1 and ASK1 directly interact and phosphorylate each other and act as negative regulators of their respective kinases in resting cells.     

Identification of thermolabile Escherichia coli proteins: prevention and reversion of aggregation by DnaK and ClpB

We systematically analyzed the capability of the major cytosolic chaperones of Escherichia coli to cope with protein misfolding and aggregation during heat stress in vivo and in cell extracts. Under physiological heat stress conditions, only the DnaK system efficiently prevented the aggregation of thermolabile proteins, a surprisingly high number of 150-200 species, corresponding to 15-25% of detected proteins. Identification of thermolabile DnaK substrates by mass spectrometry revealed that they comprise 80% of the large (>/=90 kDa) but only 18% of the small (相似文献