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H Bloemendal

Publications and source records attributed to H Bloemendal.

At least 37 records · Page 2Linked to original sources
PubMed1998

Localization of the stress proteins alpha B-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork.

PURPOSE: To examine the differential staining of two potential stress-response markers, alpha B-crystallin and the trabecular meshwork inducible glucocorticoid response protein (TIGR), in meshwork from normal and glaucomatous human eyes. METHODS: Trabecular meshwork from 35 eyes from 23 donors with either primary open-angle glaucoma, pseudoexfoliative glaucoma, or low-tension glaucoma, and from age-matched normal eyes, was examined. Sagittal and tangential frozen sections were stained with polyclonal antibodies to alpha B-crystallin or TIGR and then by a fluorescent secondary antibody. RESULTS: In normal eyes, labeling for alpha B-crystallin occurred in the subendothelial region of Schlemm's canal and outer corneoscleral regions, whereas TIGR labeling was found in the inner uveal meshwork region and the anterior portion of the meshwork. In contrast, in many glaucomatous eyes, labeling for alpha B-crystallin and TIGR occurred in more regions of the meshwork and appeared more intense than in normal eyes, regardless of the type or clinical severity of glaucoma. CONCLUSIONS: The differences in localization of alpha B-crystallin and TIGR may relate to functional specialization within meshwork tissues. The increase in the staining for these proteins in glaucomatous eyes could involve environmental and genetic factors in the disease processes.

Aged↗
PubMed1997

Detailed analysis of cell cycle kinetics upon proteasome inhibition.

We have studied specific effects of proteasome inhibition on cell cycle progression. To this end, the protease inhibitors MG115, calpain inhibitor I, and calpain inhibitor II, which display differential inhibitory effects on proteasomes, were used. Cell kinetic studies using bromodeoxyuridine pulse labeling revealed a complete block of G1/S and metaphase transitions and a delayed progression through S phase in cell cultures treated with 54 microM of MG115. Calpain inhibitor I in similar concentrations displayed a fivefold lower effect on cell cycle kinetics. Calpain inhibitor II and MG2M, which is a structural analogue of MG115, had no effect on the cell cycle. The inhibitory effect of MG115 treatment was reversible, because the cell cycle was immediately resumed when the MG115-containing culture medium was replaced by fresh culture medium. Because ubiquitinated proteins accumulated after MG115 treatment, it was confirmed that ubiquitin-dependent protein degradation, and thus proteasomal activity were blocked. By comparison of biochemical and in vitro proteasome inhibition experiments, it was hypothesized that chymotrypsin-like activity of proteasomes may play an important role in cell cycle kinetics.

Animals↗
PubMed1997

The human alpha-type proteasomal subunit HsC8 forms a double ringlike structure, but does not assemble into proteasome-like particles with the beta-type subunits HsDelta or HsBPROS26.

The eukaryotic proteasome is a barrel-shaped protease complex made up of four seven-membered rings of which the outer and inner rings may contain up to seven different alpha- and beta-type subunits, respectively. The assembly of the eukaryotic proteasome is not well understood. We cloned the cDNA for HsC8, which is one of the seven known human alpha-type subunits, and produced the protein in Escherichia coli. Recombinant HsC8 protein forms a complex of about 540 kDa consisting of double ringlike structures, each ring containing seven subunits. Such a structure has not earlier been reported for any eukaryotic proteasome subunit, but is similar to the complex formed by the recombinant alpha-subunit of the archaebacterium Thermoplasma acidophilum (Zwickl, P., Kleinz, J., and Baumeister, W. (1994) Nat. Struct. Biol. 1, 765-770). The ability of HsC8 to form alpha-rings suggests that these complexes may play an important role in the initiation of proteasome assembly in eukaryotes. To test this, we used two human beta-type subunits, HsBPROS26 and HsDelta. Both these beta-type subunits, either in the proprotein or in the mature form, exist in monomers up to tetramers. In contrast to the alpha- and beta-subunit of T. acidophilum, coexpression of the human beta-type subunits with HsC8 does not result in the formation of proteasome-like particles, which would be in agreement with the notion that proteasome assembly in eukaryotes is much more complex than in archaebacteria.

Cysteine Endopeptidases↗
PubMed1997

Biochemical differences between three subcell-lines derived from SV40-transformed hamster lens cells.

Clones derived from SV40-transformed hamster lens cells have at least three different stable morphologies. Biochemical differences between the three cell types that become detectable after transfection of the alpha A-crystallin gene do exist at the level of alpha B-crystallin and small heat shock protein (HSP27) expression. Furthermore one cell type is capable of alternative splicing of the hamster alpha A-crystallin gene, whereas another one cannot express alpha AIns-crystallin.

Animals↗
PubMed1997

Transgenic mice carrying chimeric or mutated type III intermediate filament (IF) genes.

Mice carrying chimeric, truncated or mutated genes encoding intermediate filament (IF) proteins type III do not show any detectable severe pathology. However, upon (over)expression of the transgene in the eye lens all animals develop lens opacification (cataract). At the cellular level the loss of visual acuity is preceded by interference with the terminal differentiation of lens fibre cells, plasma membrane damage, distorted assembly of the IF cytoskeleton and perturbation of the cytoskeleton-membrane complex. The degree of expression is paralleled by the extent of the damages.

Animals↗
PubMed1997

Towards a human crystallin map. Two-dimensional gel electrophoresis and computer analysis of water-soluble crystallins from normal and cataractous human lenses.

This paper describes a first approach to establish a master data base of human lens crystallins obtained by computer analysis of standardized two-dimensional lenticular protein patterns. To facilitate the eventual identification of the spots, the major crystallins have been separated into alpha-, beta H-, beta L- and gamma-crystallin fractions by gel filtration. The authors encourage colleague investigators to collaborate in a common effort in order to arrive eventually at a two-dimensional gel data base of all lenticular proteins.

Aging↗
PubMed1996

Immobilization of the C-terminal extension of bovine alphaA-crystallin reduces chaperone-like activity.

alpha-Crystallins occur as multimeric complexes, which are able to suppress precipitation of unfolding proteins. Although the mechanism of this chaperone-like activity is unknown, the affinity of alpha-crystallin for aggregation-prone proteins is probably based on hydrophobic interactions. alpha-Crystallins expose a considerable hydrophobic surface to solution, but nevertheless they are very stable and highly soluble. An explanation for this paradox may be that alpha-crystallin subunits have a polar and unstructured C-terminal extension that functions as a sort of solubilizer. In this paper we have described five alphaA-crystallins in which charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspartate does not substantially influence chaperone-like activity. In contrast, introduction of a hydrophobic tryptophan greatly diminishes functional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements show that the inserted tryptophan is located in a polar environment. However, NMR spectroscopy clearly demonstrates that the presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furthermore, the introduction of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing the polarity of the C-terminal extension of alphaA-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural and functional integrity.

Animals↗
PubMed1996

Human multidrug resistance 3-P-glycoprotein expression in transgenic mice induces lens membrane alterations leading to cataract.

We have generated mice transgenic for a human multidrug resistance (MDR)3 mini-gene driven by a hamster vimentin promoter. The MDR3 gene encodes a P-Glycoprotein that resembles the mouse multidrug resistance 2 P-Glycoprotein shown to be involved in the translocation of the phospholipid phosphatidylcholine through the hepatocyte canalicular membrane (Smit et al., 1993. Cell. 75:451-462). The vimentin promoter drives expression of the MDR3 transgene in mesenchymal tissues and in the eye lens. We show here that the presence of human multidrug resistance 3 P-Glycoprotein in the lens results in a severe lenticular pathology. Lens structural abnormalities initiate at a late embryonic stage and increase during postnatal lens development. Differentiation of the primary fibers is affected, and the terminal differentiation of the lens epithelium into secondary fibers is also perturbed. The ultrastructural alterations, particularly of the lens plasma membranes, resemble those identified in congenital mouse osmotic cataract.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1996

Transgenic mice: models for the study of cataractogenesis. A minireview.

Genetic manipulation followed by (over)expression of the transgene product in lens tissue results in: (I) inhibition of denucleation of lens fibers; (II) interference with lens cell differentiation; (III) cataract formation. The events reviewed are preceded by membrane damage, perturbed assembly of the IF cytoskeleton and distortion of the cytoskeleton-membrane complex.

Animals↗
PubMed1996

alpha-Crystallin: molecular chaperone and heat shock protein.

The relationship of alpha-crystallin with the family of small heat shock proteins has led to the discovery that the basic subunit alpha B-crystallin can, like other heat shock proteins, protect cells against heat stress. Here we show that the acidic subunit alpha A-crystallin, which in contrast to alpha B-crystallin is expressed mainly in the eye lens, shares this property. Furthermore we have investigated the in vitro molecular chaperone-like behavior of the natural mutant alpha A ins-crystallin that has a large insert peptide and occurs in rodents. We have found the chaperone-like activity of the mutant to be diminished compared to that of the wild type alpha A-crystallin.

3T3 Cells↗
PubMed1996

alpha B-crystallin in the primate ciliary muscle and trabecular meshwork.

The presence of alpha B-crystallin, a protein with heat-shock protein-like properties, has been demonstrated in ciliary muscle and trabecular meshwork derived from human and monkey eyes using immunohistochemical and polymerase chain reaction methods. Both frozen sections and cultured cells have been analyzed. In the ciliary muscle, alpha B-crystallin staining is localized in the region of the dense bands, in the cytoplasm of the muscle cells and in the Schwann cells of the nerves supplying the muscle. In the trabecular meshwork, two cell types could be distinguished on the basis of alpha B-crystallin occurrence. Whereas the trabecular cells covering the lamellae were virtually devoid of the protein, the subendothelial or cribriform cells contained relatively large amounts in parallel with a higher alpha B-crystallin mRNA level.

Adult↗
PubMed1996

Muscle-specific expression of a dominant negative desmin mutant in transgenic mice.

To extend our knowledge of the functions of desmin and vimentin intermediate filaments in the developing organism, a construct encoding a truncated desmin subunit driven by the desmin promoter (pDDV), was introduced into the murine germ line. The resulting mutant desmin subunit was assembly-incompetent and capable of disrupting both preexisting desmin and vimentin filaments in a dominant negative fashion in transfected C2C12 muscle cells and in transgenic mouse muscle tissue. Expression of the pDDV was tissue-specific in transgenic mice. High level expression of pDDV occurred in a small percentage of desmin-containing muscle cells. Immunohistochemical staining of muscle tissue showed a diffuse desmin pattern instead of the dots and clumps into which mutant desmin typically accumulates in undifferentiated C2C12 muscle cells in tissue culture. Disruption of the endogenous desmin filaments in Sartorius muscle results in ultrastructural abnormalities.

Animals↗
PubMed1995

The mutation Asp69-->Ser affects the chaperone-like activity of alpha A-crystallin.

alpha-Crystallins are members of the family of small heat-shock proteins. The conformation and mode of action of these 'junior chaperones' are unknown. To investigate the structure and chaperone-like activity, four mutants of bovine alpha A-crystallin were generated by site-directed mutagenesis. In comparison with wild-type alpha A-crystallin, the D69S mutant, in which a highly conserved charged residue has been replaced, forms larger multimers and displays a threefold reduced heat-protection capacity. The conformation and thermal stability of this mutant are not noticeably affected. Three other mutations, replacing hydrophobic by uncharged hydrophilic residues, were aimed at disturbing hydrophobic intersubunit interactions. None of these mutations resulted in major structural perturbations and only minor differences in heat-protective capacity were observed. Although it is assumed that small heat-shock proteins interact with denaturing proteins via their hydrophobic surfaces, this study clearly shows that charged residues in alpha-crystallin can also influence the efficiency of substrate binding.

Amino Acid Sequence↗
PubMed1995

Reduced chaperone-like activity of alpha A(ins)-crystallin, an alternative splicing product containing a large insert peptide.

alpha-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues. Like other members of the small heat shock protein family, alpha-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major alpha A- and alpha B-subunits, alpha-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, alpha A(ins)-crystallin. This polypeptide is identical to normal alpha A-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat alpha A- and alpha A(ins)-crystallin in Escherichia coli. The multimeric particles formed by alpha A(ins) are larger and more disperse than those of alpha A, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with alpha A, the alpha A(ins)-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of beta low-crystallin. Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.

Alternative Splicing↗
PubMed1995

Negative regulation of a special, double AP-1 consensus element in the vimentin promoter: interference by the retinoic acid receptor.

The growth-regulated vimentin gene contains a functional double AP-1 binding site formed by two nearly perfect inverted repeats. We present evidence for down-regulation of vimentin expression by the retinoic acid receptor (RAR) in two mesodermally derived cell types. By mutation analysis we prove that the double consensus element is responsible for this negative regulation. From in vitro protein-DNA interaction studies we conclude that AP-1 binding is inhibited at RAR amounts required for occupation of the cognate RAR binding site in nuclear extracts from 3T3 cells and differentiated embryonal carcinoma cells. Furthermore, we show that, unlike in other cases, trans-activation of the vimentin AP-1 enhancer element can occur in undifferentiated embryonal carcinoma cells, despite the low amount of Jun and Fos proteins present in these cells. Here, however, down-regulation by retinoic acid cannot be detected.

3T3 Cells↗
PubMed1995

Disruption of vimentin intermediate filaments in transgenic mice by expression of a dominant negative mutant desmin subunit.

To investigate putative functions of vimentin intermediate filaments in the context of intact tissues and the developing organism, a construct (pVDV), driven by the vimentin promoter and encoding a truncated desmin subunit, was introduced into the murine germ line. The mutant desmin was assembly-incompetent and capable of disrupting preexisting vimentin filaments in a dominant negative fashion, both in transgenic mouse tissues and in fibroblast cultures derived from these mice. Mutant desmin expression strongly enhanced vimentin turnover. In tissues of some transgenic mouse lines, high level expression of pVDV occurred in 10 to 40% of vimentin-containing cells and, surprisingly, in 1 to 10% of the skeletal and tongue muscle cells. Immunohistochemical staining of muscle tissue showed a diffuse staining pattern instead of the punctated aggregates into which mutant desmin typically accumulates in other cell types. The overexpression of pVDV and the concomitant disruption of the endogenous vimentin filament network and enhanced vimentin turnover in a significant percentage of cells did not cause detectable developmental abnormalities.

Animals↗
PubMed1994

Structure and modifications of the junior chaperone alpha-crystallin. From lens transparency to molecular pathology.

alpha-Crystallin is a high-molecular-mass protein that for many decades was thought to be one of the rare real organ-specific proteins. This protein exists as an aggregate of about 800 kDa, but its composition is simple. Only two closely related subunits termed alpha A- and alpha B-crystallin, with molecular masses of approximately 20 kDa, form the building blocks of the aggregate. The idea of organ-specificity had to be abandoned when it was discovered that alpha-crystallin occurs in a great variety of nonlenticular tissues, notably heart, kidney, striated muscle and several tumors. Moreover alpha B-crystallin is a major component of ubiquinated inclusion bodies in human degenerative diseases. An earlier excitement arose when it was found that alpha B-crystallin, due to its very similar structural and functional properties, belongs to the heat-shock protein family. Eventually the chaperone nature of alpha-crystallin could be demonstrated unequivocally. All these unexpected findings make alpha-crystallin a subject of great interest far beyond the lens research field. A survey of structural data about alpha-crystallin is presented here. Since alpha-crystallin has resisted crystallization, only theoretical models of its three-dimensional structure are available. Due to its long life in the eye lens, alpha-crystallin is one of the best studied proteins with respect to post-translational modifications, including age-induced alterations. Because of its similarities with the small heat-shock proteins, the findings about alpha-crystallin are illuminative for the latter proteins as well. This review deals with: structural aspects, post-translational modifications (including deamidation, racemization, phosphorylation, acetylation, glycation, age-dependent truncation), the occurrence outside of the eye lens, the heat-shock relation and the chaperone activity of alpha-crystallin.

Acetylation↗