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PubMed1983

Skeletal framework of mammalian heart muscle. Arrangement of inter- and pericellular connective tissue structures.

We have studied the connective tissue of mammalian heart muscle in order to obtain an integrated description of extracellular structures and their dispositions relative to cardiac myocytes. Light microscopy and several types of electron microscopy have been employed in these investigations. The epimysium, the sheath of connective tissue that surrounds the muscle, contains relatively large fibers of collagen and elastin. In papillary muscles of rat, the large collagen fibers of the epimysium form a weave pattern at slack length (sarcomere lengths 1.8 to 2.0 micron) but are well aligned in states of stretch along the long axis of the muscle (sarcomere length 2.3 to 2.5 micron). We propose that the epimysial collagen network protects the sarcomeres from being stretched beyond lengths favorable to maximal force production. The endomysium is defined as the connective tissue that surrounds and interconnects myocytes; it consists of intercellular struts (bundles of collagen fibrils, often attached near Z-band level), a weave of bundles of collagen fibrils that envelopes myocytes, and a collagen fibril-microthread-granule lattice that bridges cells and fills the extracellular matrix. In contracted muscles festoons of sarcolemma are attached to Z-bands, thus forming regions for transmission of force across the sarcolemma. Perimysial bundles of collagen connect epimysium to endomysium and surround groups of myocytes. Collagen fibers often have a twisted configuration, probably for enhanced tensile strength. Superimposed on the large extracellular structures is the polyanion-rich lattice comprised of unit collagen fibrils, microthreads, and granules. Amorphous ground substance forms a matrix in which the fibrils of collagen fibers are embedded; it appears continuous with the cell coat in regions of fiber attachment. Elastic fibers interconnect cells and helically wind around myocytes. Circumferential forces from elastin stretched about shortened, thickened myocytes in systole should promote elongation in tandem with intramyocyte forces of elongation.

Animals↗
PubMed1994

Polarized distribution of metalloproteinases in the bovine interphotoreceptor matrix.

Previous studies from this laboratory had indicated that metalloproteolytic activity was present in the interphotoreceptor matrix. This report extends those observations by providing evidence for the presence of multiple forms of metalloproteinase and for their polarized distribution within the interphotoreceptor matrix as determined by zymogram analysis on substrate-loaded gels. Retinal pigment epithelium-associated interphotoreceptor matrix, both unfractionated as well as fractions separated by gel filtration, exhibited bands of proteolytic activity on gelatin-loaded gels at about 70-75 kDa and 90 kDa, possibly due to gelatinases A and B (MMP-2 and MMP-9, respectively). In contrast, no neutral proteolytic activity was seen in retina-associated interphotoreceptor matrix unless it was first fractionated by gel filtration, whereupon a diversity of forms was exhibited, including additional major bands of proteolytic activity at about 150 kDa and 100 kDa, on gelatin-loaded gels, and at about 180 kDa, on casein-loaded gels, along with many minor species. In all cases, all proteinase activity was inhibited by chelating agents. Since these enzymes may be involved in the turnover and remodeling of components present in the interphotoreceptor matrix, many of which are distributed in a compartmentalized fashion, this unequal distribution of metalloproteinases may be correlated with substrate specificity.

Animals↗
PubMed1998

Analysis of differential gene expression in rat tibia after an osteogenic stimulus in vivo: mechanical loading regulates osteopontin and myeloperoxidase.

The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague-Dawley rats, approximately 8 months old, received either a sham or bending load using a four-point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse-transcribed into cDNA, and used in DDRT-PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4-fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4- to 10-fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load-induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function.

Animals↗
PubMed1975

Ultrastructural identification of extracellular matrix and cell surface components during limb morphogenesis in man.

Development of the human hand plate (stages 16-17) has been analyzed with emphasis on differentiation of elements within the extracellular matrix and the composition of the mesenchymal cell surface. The epithelial-mesenchymal interface contains a basal lamina and a sublaminar matrix exhibiting: (a) collagen fibrils with characteristic 63-64 nm banding: (b) non-banded filaments, 10-15 nm in diameter; (c) ruthenium red-positive particles, 12-15 nm in diameter; and (d) attenuated threads, 3-5-5-0 nm in diameter which inter-connect particles, fibrils, filaments and the basal lamina. Processes of mesenchymal cells penetrate this matrix network. In addition to staining with ruthenium red, components of basal laminae bind to ferritin-conjugated Concanavalin A, greatest binding being localized on the mesenchymal surface of the lamina. Asymmetry of binding is removed by incubation of exposed laminae with trypsin (5 mug/ml). Regional differences in these staining and binding characteristics within the subepithelial matrix have not been observed in the hand plate. However, precartilaginous extracellular zones deep within the plate are notably unstructured in comparison to the sublaminar region. Ruthenium red-positive materials at mesenchymal cell surfaces display sensitivity to testicular hyaluronidase, Pronase and trypsin but resist removal with neuraminidase and EDTA. These features of the substrate in situ may be important in the regulation of mesenchymal cell behavior during limb morphogenesis in man.

Binding Sites↗
PubMed2005

The relationship between adsorption energies of methyl on metals and the metallic electronic properties: a first-principles DFT study.

A theoretical study of CH3 adsorbed on the (111) surface of some transition and noble metal surfaces M (M = Cu, Ni, Rh, Pt, Pd, Ag, Au) and on the Fe(100) is presented. We find that the hollow site is preferred more than the top one for Fe, Ni, Rh, and Cu, but it is the other way for Pt, Pd, Au, and Ag. In addition, a good linear relationship was observed between the chemisorption energy and d-band center for Group VIII metals or the square of the coupling matrix element for Group IB metals at the hollow site. Interestingly, with a detailed comparison of the adsorption energies at the top and hollow sites, we find that the adsorption energies among each group are very similar on the top site, which supports the theoretical model of Hammer and Norskov that the coupling between the HOMO of adsorbate and sp states of the metal is dominant and almost equal, and that the second coupling to the d-band contributes less but reflects the change of the adsorption energy. It confirms that the coupling to the d band comprises two opposite factors, that is, the d-band center was attractive and the square of the coupling matrix element was repulsive, such that the contributions from the two factors can counteract each other at the top site.

Journal Article↗
PubMed2001

Ultrastructure of collagen fibers and distribution of extracellular matrix in the temporomandibular disk of the human fetus and adult.

We quantitatively examined the distribution of these differences in extracellular matrices (collagen types I, III, and fibronectin) and elastic fibers under confocal laser scanning microscopy and electron scanning microscopy in terms of their contribution to the mechanics of the TMJ during development and in adults. Elastic fibers were found in the anterior and posterior bands in adults aged 40 years, and a few elastic fibers in the anterior band of the disk in adults aged 80 to 90 years. The extracellular matrix contents of the TMJ disk are shown in various detected levels in the anterior, intermediate, posterior bands of TMJ disk. During development, collagen fibers are arranged in a complex fashion from 28 weeks' gestation. These ultrastructures of the embryonic TMJ are resembled to that of adults aged the 40s, however the difference in extracellular matrix distribution found in embryonic stages and adults. They might reflect the differences in function between mastication and sucking or the changes in shape and form as results of functional disorders of the TMJ.

Adult↗
PubMed2006

Toll-like receptor 9 agonists promote cellular invasion by increasing matrix metalloproteinase activity.

Toll-like receptor 9 (TLR9) recognizes microbial DNA. We show here that TLR9 protein is expressed in human breast cancer cells and clinical breast cancer samples. Stimulation of TLR9-expressing breast cancer cells with the TLR9 agonistic CpG oligonucleotides (1-10 mumol/L) dramatically increased their in vitro invasion in both Matrigel assays and three-dimensional collagen cultures. Similar effects on invasion were seen in TLR9-expressing astrocytoma and glioblastoma cells and in the immortalized human breast epithelial cell line MCF-10A. This effect was not, however, dependent on the CpG content of the TLR9 ligands because the non-CpG oligonucleotides induced invasion of TLR9-expressing cells. CpG or non-CpG oligonucleotide-induced invasion in MDA-MB-231 cells was blunted by chloroquine and they did not induce invasion of TLR9(-) breast cancer cells. Treatment of MDA-MB-231 cells with CpG or non-CpG oligonucleotides induced the formation of approximately 50-kDa gelatinolytic band in zymograms. This band and the increased invasion were abolished by a matrix metalloproteinase (MMP) inhibitor GM6001 but not by a serine proteinase inhibitor aprotinin. Furthermore, CpG oligonucleotide treatment decreased tissue inhibitor of metalloproteinase-3 expression and increased levels of active MMP-13 in TLR9-expressing but not TLR9(-) breast cancer cells without affecting MMP-8. Neutralizing anti-MMP-13 antibodies inhibited the CpG oligonucleotide-induced invasion. These findings suggest that infections may promote cancer progression through a novel TLR9-mediated mechanism. They also propose a new molecular target for cancer therapy, because TLR9 has not been associated with cancer invasiveness previously.

Antibodies↗
PubMed1998

Insulin-like growth factor binding protein-5 expression by human trabecular meshwork.

PURPOSE: The authors found transcript expression for insulin-like growth factor binding protein-5 (IGFBP-5) while screening for uniquely expressed trabecular meshwork (TM) mRNAs. Because the insulin-like growth factor (IGF) autocrine-paracrine system may provide an important signaling mechanism between TM cells and the outflow pathway, the expression of IGFBP-5 and IGF-I receptor in the TM was characterized. METHODS: Poly(A+) RNA was isolated from cell cultures of human TM, ciliary body, retinal pigment epithelium, and skin fibroblasts and subjected to reverse transcription-polymerase chain reaction (RT-PCR) differential display analysis. A unique 980-bp band present in the TM was cloned and sequenced. Additional PCR and Northern analyses were used to define trabecular IGFBP-5 expression. Western immunoblots and confocal immunohistochemistry were used to evaluate the protein expression patterns of IGFBP-5 and the IGF-I receptor. IGF-I and IGF-II were added to trabecular cells in culture, and matrix metalloproteinase production was evaluated. RESULTS: A unique differential display band was identified in the TM. Sequencing of this band identified it as the 3'-untranslated region of IGFBP-5. RT-PCR, using a variety of specific primers for IGFBP-5, Northern analysis, Western immunoblots, and immunohistochemical analysis, confirmed that IGFBP-5 was expressed in the TM. However, IGFBP-5 was also present at low levels in the ciliary body and skin fibroblasts by Northern and Western analysis, in contrast with the differential display findings. In addition, the IGF-I receptor was expressed by the TM and showed cell-surface staining by immunohistochemistry. Trabecular IGFBP-5 was distributed throughout the meshwork in the extracellular matrix and the cells with more staining in the juxtacanalicular region than in the uveal meshwork. IGF-I, but not IGF-II, modestly increased trabecular stromelysin and gelatinase B but not collagenase, gelatinase A, or tissue inhibitor of metalloproteinases 1 or 2. CONCLUSIONS: IGFBP-5 and IGF-I receptor were expressed at significant levels by TM cells and may serve an important role in trabecular function.

Blotting, Northern↗
PubMed1982

Discrimination of several classes of nonhistone proteins in chromatin fractions from liver and thymus nuclei of rats.

Nonhistone proteins (NHPs) of salt-soluble chromatin (Chromatin S) and of the residual nuclei (Chromatin P) from rat liver and thymus were studied by SDS-polyacrylamide gel electrophoresis. The two chromatin fractions of the liver showed significant differences in their NHP patterns with most of the hnRNP and matrix proteins occurring in Chromatin P. In accordance with the low protein content of thymus nuclei, the corresponding thymus fractions exhibited electrophoretic patterns with a markedly lower amount of NHPs than in liver. Chromatin P from thymus, in contrast to the liver fraction, revealed only a very low content of hnRNP-specific proteins of molecular weight 30,000-40,000 (30 K to 40 K) (informosomal proteins) consistent with the significantly lower RNA content of thymus nuclei. In the region of the matrix proteins (60-75 K) Chromatin P showed only two bands of about 64 K and 73 K in thymus, whereas in liver five strong bands at 64 K, 66 K, 69 K, 73 K, and 75 K were found. RNase digestion was employed to discriminate hnRNP-specific protein from "real" chromosomal NHPs. At least about 65% and 25% of the NHPs from Chromatin P and S of liver, respectively, were found to be RNP-specific. The two chromatin fractions were further fractionated by sucrose gradient centrifugation and isopycnic banding in metrizamide. After centrifugation the main peaks, both of Chromatin S and P, contained only minor amounts of NHPs with a predominating protein of 38 K. By the centrifugation procedures described in this paper, a small subfraction of chromatin could be separated which was enriched in newly synthesized RNA, informosomal proteins, matrix- and other high molecular weight proteins. This subfraction might be related to transcriptionally active chromatin.

Animals↗
PubMed1992

Development of the rabbit craniomandibular joint in association with tooth eruption.

The adult rabbit craniomandibular joints (CMJs) are stress-bearing joints. The two CMJs and the teeth form an articular triad. In early fetal life the developing triad consists of the CMJ primordia, the tooth germs for the entire set of deciduous teeth, as well as the posterior extensions of the dental lamina, which will give rise to the permanent teeth with no deciduous predecessors. During postnatal life, before occlusion is established, there is a remodelling stage in which the CMJ builds up its matrix components such as collagenous and elastic fibres, proteoglycans and type II collagen. Remodelling gradually diminishes into the maintenance stage once occlusion is fully established and after eruption of the first and second molars. Chondrocytes first appear in the CMJ articular disc during the second week of postnatal development. These cells localize in the band areas of the disc and establish an extensive cartilaginous matrix 3-4 weeks postnatally. This study supports the concept that the full development of a fibrocartilaginous articular disc, rich in proteoglycans, occurs as adult occlusion is established.

Animals↗
PubMed1991

Is Plasmodium falciparum aldolase useful for rational drug design?

P. falciparum lacks a functional citric acid cycle. Unlike most tissues of the mammalian host, it is totally dependent on glycolysis for energy generation. A compound which selectively inhibits the parasite's ATP-generating machinery is therefore a potential antimalarial agent. Such a drug may interact in two ways: a) by inhibiting the activity of an enzyme or b) by disturbing the micro-organization of consecutive enzymes in a metabolic pathway. In mammalian tissues the glycolytic pathway involves the cytoskeleton as a matrix to keep phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase in an optimal sterical position for rapid substrate conversion. For instance, these three enzymes bind to the band 3 protein in erythrocytes or to actin in muscle cells. P. falciparum aldolase binds with very high affinity to the band 3 protein of human erythrocyte ghosts. However, the true in vivo site of association is believed to be actin II of P. falciparum. This actin has a sequence element which is almost identical to that of the band 3 aldolase binding site. We therefore suppose that plasmodia exploit a similar matrix organization. If true, the association of these enzymes with the cytoskeleton is a target for novel antimalarials. In contrast to all vertebrate aldolases, P. falciparum and P. berghei aldolases have two neighbouring lysine residues near the carboxy-terminus. We show here that mutagenesis of these basic residues has an effect on the catalytic constants Vmax and KM and moreover, the ability to bind to band 3 is reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Scaffold attachments within the human genome.

It is generally agreed that, above the level of the 30 nm fibre, eukaryotic chromatin is constrained into loops, but there is disagreement about the nature of the substructure that serves to anchor loops and the DNA sequences that act as the attachment sites. This problem may stem from the very different methods that all purport to separate loop and attached DNAs. We have tested ideas about how the genome is arranged into loops by analysing the average loop size over different cytologically resolvable regions of human chromosomes using fluorescence in situ hybridisation with loop and attached DNA fractions. Variations in average loop size, along and between chromosomes, measurable at this level of resolution were small but significant and were dependent on the extraction method. This emphasises the fundamental differences between the nuclear substructure probed by different protocols. DNA attached to the nuclear 'scaffold' or 'matrix' hybridises preferentially to gene-poor regions of the genome (G-bands). Conversely, fractions attached to the nuclear 'skeleton' hybridise preferentially to gene-rich R-bands and sites of high levels of transcription. The inactive X chromosome has a deficit of associations with the nuclear skeleton but not with the matrix or scaffold. A large excess of attached sequences is found at some sites of constitutive heterochromatin, but not at centromeres.

Cell Nucleus↗
PubMed1998

Janus kinases and focal adhesion kinases play in the 4.1 band: a superfamily of band 4.1 domains important for cell structure and signal transduction.

The band 4.1 domain was first identified in the red blood cell protein band 4.1, and subsequently in ezrin, radixin, and moesin (ERM proteins) and other proteins, including tumor suppressor merlin/schwannomin, talin, unconventional myosins VIIa and X, and protein tyrosine phosphatases. Recently, the presence of a structurally related domain has been demonstrated in the N-terminal region of two groups of tyrosine kinases: the focal adhesion kinases (FAK) and the Janus kinases (JAK). Additional proteins containing the 4.1/JEF (JAK, ERM, FAK) domain include plant kinesin-like calmodulin-binding proteins (KCBP) and a number of uncharacterized open reading frames identified by systematic DNA sequencing. Phylogenetic analysis of amino acid sequences suggests that band 4.1/JEF domains can be grouped in several families that have probably diverged early during evolution. Hydrophobic cluster analysis indicates that the band 4.1/JEF domains might consist of a duplicated module of approximately 140 residues and a central hinge region. A conserved property of the domain is its capacity to bind to the membrane-proximal region of the C-terminal cytoplasmic tail of proteins with a single transmembrane segment. Many proteins with band 4.1/JEF domains undergo regulated intra- or intermolecular homotypic interactions. Additional properties common to band 4.1/JEF domains of several proteins are binding of phosphoinositides and regulation by GTPases of the Rho family. Many proteins with band 4. 1/JEF domains are associated with the actin-based cytoskeleton and are enriched at points of contact with other cells or the extracellular matrix, from which they can exert control over cell growth. Thus, proteins with band 4.1/JEF domain are at the crossroads between cytoskeletal organization and signal transduction in multicellular organisms. Their importance is underlined by the variety of diseases that can result from their mutations.

Amino Acid Sequence↗
PubMed2004

[Onychomatricoma, a rare lesion of the nail].

Onychomatricoma is a rare fibroepithelial lesion of the nail matrix with peculiar clinical and histological features. Clinically, it is characterized by a longitudinal band of yellow thickening of the nail plate with transverse overcurvature and splinter hemorrhages. Nail avulsion exposes a villous tumor of the matrix with filamentous digitations extending into multiple holes of the nail plate. Histologically, a thick keratogenous zone forms a thickened nail plate. The lesion in its proximal portion is characterized by deep epithelial invaginations and by a stroma organized in two layers. The distal zone corresponds to multiple fibroepithelial projections extending into the nail plate. The diagnosis can be difficult in the presence of misleading clinical features or when the specimen is incomplete or examined with an improper orientation. Surgical resection is the recommended treatment.

Humans↗
PubMed1998

Pathologic fibrosis and connective tissue matrix in left ventricular hypertrophy due to chronic arterial hypertension in humans.

OBJECTIVE: To investigate pathologic fibrosis and connective tissue matrix in left ventricular hypertrophy due to chronic arterial hypertension in humans. DESIGN AND METHODS: Seventeen human hearts were studied. Group 1 consisted of control hearts (four hearts, weighing 280 +/- 40 g each), from subjects who had had no evidence of heart disease and for whom the diagnoses of death were noncardiac. Groups 2 (five hearts, weighing 440 +/- 50 g each), 3 (five hearts, weighing 560 +/- 50 g each), and 4 (three hearts, weighing 680 +/- 60 g each) consisted of hearts from subjects who had had a history of systemic hypertension. All hearts had no valvular deformities and no evidence of ischemic disease at the postmortem examination. A cell-maceration method was employed to evaluate the myocardial connective matrix after removal of the nonfibrous elements of myocardial tissue, leaving behind a noncollapsed matrix, thus allowing a better three-dimensional view. Myocardial tissue was also processed for conventional light microscopic and morphometric studies. RESULTS: The minor transverse diameter of myocytes from hearts in groups 1-4 hearts were 13.7 +/- 7.8, 23.7 +/- 3.4, 26.6 +/- 3.7, and 32.8 +/- 5.8 microm, respectively. The volume fraction of fibrosis of the controls was 6.5%, whereas the volume fractions in hypertensive hearts increased progressively according to heart weight: 15.4, 22.9, and 31.1% for hearts in groups 2, 3, and 4, respectively. The most striking feature was the diffuse marked increase in amount of pericellular collagen weave fibers (endomysial matrix), parallel to the increase of heart weight. The hypertrophied myocytes were encased in a dense weave of collagen fibrils continuous with those of adjacent myocytes. The muscle fibers in hypertrophied hearts were markedly larger than normal, although this was extremely variable from an area to another. Besides, a diffuse increase in the number of thick collagen fibers constituting broad bands and sheets of collagen surrounding disorganized muscle bundles (perimysial matrix) was observed. Scattered dense scar-like foci, apparently replacing areas of myocyte loss, could be seen, mainly on the periphery of muscle bundles. This latter finding was more commonly observed among hypertrophied hearts from group 3 and, mainly, among hypertrophied hearts of group 4. Importantly, a progressive disarray of the connective tissue skeleton of the myocardium could be seen in parallel to the progressive increase of cardiac hypertrophy. CONCLUSIONS: The progressive accumulation of interstitial collagen fibers in left ventricular hypertrophy, in parallel to an increase in heart weight, can be expected to contribute to a spectrum of ventricular dysfunction involving either the diastolic or systolic phase of the cardiac cycle, or both, that is associated with the greater than normal arrhythmogenic risk for a hypertensive heart. Moreover, the methodology used is useful for studying the spatial organization of the collagen fibrils of the myocardium under normal and pathologic conditions.

Adult↗
PubMed1998

A dehydrogenase-mediated recycling system of NADPH in plant peroxisomes.

The presence of the two NADP-dependent dehydrogenases of the pentose phosphate pathway has been investigated in plant peroxisomes from pea (Pisum sativum L.) leaves. Both enzymes, glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44), were present in the matrix of leaf peroxisomes, and their kinetic properties were studied. G6PDH and 6PGDH showed a typical Michaelis-Menten kinetic saturation curve, and had specific activities of 12.4 and 29.6 mU/mg protein, respectively. The Km values of G6PDH and 6PGDH for glucose 6-phosphate and for 6-phosphogluconate were 107.3 and 10.2 microM, respectively. Dithiothreitol did not inhibit G6PDH activity. By isoelectric focusing of peroxisomal matrices, the G6PDH activity was resolved into three isoforms with isoelectric points of 5.55, 5.30 and 4.85. The isoelectric point of peroxisomal 6PGDH was 5.10. Immunoblot analyses of peroxisomal matrix with an antibody against yeast G6PDH revealed a single cross-reactive band of 56 kDa. Post-embedment, EM immunogold labelling of G6PDH confirmed that this enzyme was localized in the peroxisomal matrices, the thylakoid membrane and matrix of chloroplasts, and the cytosol. The presence of the two oxidative enzymes of the pentose phosphate pathway in plant peroxisomes implies that these organelles have the capacity to reduce NADP+ to NADPH for its re-utilization in the peroxisomal metabolism. NADPH is particularly required for the ascorbate-glutathione cycle, which has been recently demonstrated in plant peroxisomes [Jiménez, Hernández, del Río and Sevilla (1997) Plant Physiol. 114, 275-284] and represents an important antioxidant protection system against H2O2 generated in peroxisomes.

Ascorbic Acid↗
PubMed1986

Protein-protein and protein-DNA interactions in calf thymus nuclear matrix using cross-linking by ultraviolet irradiation.

Nuclear matrices from calf thymus contained 30-50 protein species with one prominent band at 70 kilodaltons tentatively identified by its isoelectric point, apparent molecular weight, charge modification, and abundance as bovine lamin. The amount of DNA present in the matrix fraction was strongly dependent on the extent of digestion of the nuclei by micrococcal nuclease. The size of the DNA was higher than two kilobase pairs, although the chromatin DNA had been digested down to short oligonucleosomes. The lamin band was preferentially dissociated from isolated matrices during repeated treatment by 2 M NaCl or 5 M urea. Irradiation of calf thymus nuclear matrices at 313 nm induced protein-protein and protein-DNA cross-linking, as well as double-strand breaking of DNA, presumably at unprotected, protein-free regions. Lamin protein was more dramatically affected than other protein species by ultraviolet (UV) irradiation. In situ DNA hydrolysis, after the separation of the cross-linked matrix components on polyacrylamide-sodium dodecyl sulfate gels, followed by two-dimensional electrophoresis, showed lamin to be the major protein that was cross-linked to the DNA. Lamin molecules were also cross-linked by UV light to each other to form lamin homo-oligomers. A discrete size DNA fragment of approximately 450 base pairs is protected by lamin homo-oligomers from breakdown during UV irradiation. It is proposed that the direct contact between lamin and DNA found in this study is responsible for anchoring chromatin loops (domains) to a stable, immobile matrix structure.

Animals↗
PubMed1998

Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry.

A highly enriched spindle pole preparation was prepared from budding yeast and fractionated by SDS gel electrophoresis. Forty-five of the gel bands that appeared enriched in this fraction were analyzed by high-mass accuracy matrix-assisted laser desorption/ ionization (MALDI) peptide mass mapping combined with sequence database searching. This identified twelve of the known spindle pole components and an additional eleven gene products that had not previously been localized to the spindle pole. Immunoelectron microscopy localized eight of these components to different parts of the spindle. One of the gene products, Ndc80p, shows homology to human HEC protein (Chen, Y., D.J. Riley, P-L. Chen, and W-H. Lee. 1997. Mol. Cell Biol. 17:6049-6056) and temperature-sensitive mutants show defects in chromosome segregation. This is the first report of the identification of the components of a large cellular organelle by MALDI peptide mapping alone.

Amino Acid Sequence↗