Cellular immunity to nuclear antigens in systemic lupus erythematosus.

Cellular immune repsonses were determined by skin testing and mitogen- and antigen-induced blastic transformation of peripheral blood lymphocyte cultures in 24 patients with systemic lupus erythematosus (SLE) and 24 normal subjects. The incidence of positive skin tests with Candida albicans, PPD (tuberculin-purified protein derivative) intermediate strength, Trichophyton and histoplasmin was not significantly different in the two groups nor was lymphocyte stimulation by the mitogen phytohemagglutinin-M (PHA-M), implying that cellular immunity is normal in SLE. However, the SLE patients had a significantly increased incidence of positive skin tests and stimulated lymphocyte cultures to a number of nuclear antigens compared with normal subjects. No correlation could be made between the test results and the activity of the SLE at the time of study except for a significant association between lymphocyte culture stimulation by rabbit thymus native DNA and active SLE nephritis. Patients with a membranous antinuclear factor (ANF) pattern had positive skin tests with rabbit thymus native DNA and usually had active disease.

A study of an endogenous nucleolytic reaction and of the action micrococcal nuclease and DNAase I on a salt-soluble, compact form of chromatin.

The endogenous nucleolytic reaction occurring in rabbit thymus nuclear lysates has been studied at extended incubation times (up to 4 h). Production of nucleosomal polymers containing multiples of 205 base pairs of DNA was observed. The stability of the bands and the low release (1%) of acid-soluble nucleotides indicated there was only a small fraction of sensitive DNA between the subunits. The salt-soluble chromatin formed in the endogenous reaction at short incubation times (14--24 min) and purified over Sephadex G-200 has been treated with micrococcal nuclease and DNAase I. With micrococcal nuclease, nucleosomal polymers containing multiples of 201 base pairs of DNA were formed. Extensive digestion reaveled a core subunit containing 145 base pairs of DNA. With DNAase I only random degradation was observed and nucleosomal complexes were not produced.

Nuclear ribonucleoproteins as inhibitors of mammalian RNA polymerase.

A ribonucleoprotein complex isolated from rabbit thymus nuclear lysates was found to be an inhibitor of DNA-dependent RNA polymerase II. The inhibition appeared to be of a competitive type and was completely reversed by high concentration of DNA. Highest inhibition was observed when enzyme and complex were preincubated before addition of DNA while there was little inhibition after enzyme had started synthesis on the DNA template. The RNA isolated from the complex was equally inhibitory and was a more effective inhibitor than either tRNA or rRNA.

The nanomelic mutation in the aggrecan gene is expressed in chick chondrocytes and neurons.

We have established the presence of at least two large chondroitin sulfate proteoglycans in the developing chick brain, one that reacts exclusively with HNK-1, a carbohydrate epitope found on several neural specific molecules, and one that reacts with S103L, a defined peptide epitope in the CS-2 domain of the cartilage-specific chondroitin sulfate proteoglycan (CSPG), aggrecan. In order to determine the relationships between the two distinct S103L-reactive CSPGs from cartilage (chondrocytes) and brain (neurons), as well as among the three large CSPGs expressed in brain, S103L, HNK-1 and versican, we studied the expression of these multiple proteoglycan species in the brain of nanomelic chicks. We have previously shown that homozygous embryos expressing the nanomelic phenotype exhibit a single point mutation in the aggrecan gene. In the present study, the S103L CSPG is not accumulated or synthesized by embryonic chick CNS tissue or E8CH neuronal cultures derived from nanomelic chick embryo cerebral hemispheres. In contrast, expression of both versican and the HNK-1 CSPG was normal in the mutant embryo CNS. Pulse chase experiments demonstrated the presence of the 380 kDa precursor in normal neurons and the 300 kDa truncated precursor in nanomelic neurons. Northern blot analysis revealed normal-sized mRNA but reduced levels of expression of the S103L CSPG message in nanomelic neurons, while expression of the versican message was comparable in normal and nanomelic neurons. Most conclusively, the point mutation previously identified in nanomelic cartilage mRNA was also identified in nanomelic brain mRNA. Together these results provide evidence that a single aggrecan gene is expressed in both cartilage and CNS tissue leading to the production of identical core proteins which then undergo differential and tissue-specific post-translation processing, resulting in the characteristic tissue-specific proteoglycans. Furthermore, versican and the HNK-1 CSPG, although structurally and chemically similar to the S103L CSPG, are the products of separate genes.

Increased extracellular magnesium modulates proliferation in fetal neural cells in culture.

Retrospective studies have shown that antenatal magnesium may decrease the risk of cerebral injury in preterm infants, leading to several ongoing trials of tocolytic magnesium as a neuroprotective agent. However, other studies have indicated that antenatal magnesium actually increases neonatal mortality, leaving it unclear if magnesium is protective or dangerous to preterm infants. This controversy may be secondary to our limited understanding about the mechanisms of magnesium's action on the fetal brain. We therefore investigated the effect of increasing extracellular magnesium on cultures of neurons from embryonic day 6 telencephalon. Conversion of MTT (3-(4,5-dimethyl, thiazol-2-yl)-2,5-diphenyltetrazolium bromide) by intact mitochondria was taken as a measure of cell viability. Nuclear incorporation of BrdU (5-bromo-2'-deoxyuridine) was taken as a measure of cell proliferation. Exposure of cultures for 24 h to a 4-fold increase in magnesium (3.3 mM) increased both overall cell viability (P<0.002) and proliferation (P<0.02) by approximately 50%. Proliferating cells showed characteristics of glial cell precursors but magnesium had no effect on mature astrocyte proliferation. Increased Akt activation was observed following magnesium treatment, comparable to that observed with the growth factor insulin, suggesting one mechanism for proliferation. However, when apoptosis was induced in these cultures with the phosphatidylinositol-3-kinase inhibitor wortmannin, magnesium significantly enhanced cell death. Thus under normal conditions in the fetus, magnesium may be a positive factor but during stress it may exacerbate cell injury. This is the first time increased extracellular magnesium has been shown to increase cell proliferation in neural cells in culture or suggested to induce Akt activation.

On the binding of histone H1 in chromatin.

Nucleosomal subunits isolated from rabbit thymus nuclei in 0.04 M K2SO4-0.02 M Tris, pH 7.4 were devoid of histone H1, while whole chromatin prepared in the same buffer contained the full complement of histone H1. The question is asked why histone H1 dissociates from the subunits but not from the high molecular weight material. We propose that, at physiological salt concentrations, histone H1 is not bound to linker DNA as depicted in the current models; rather, alternate attachment sites, present only in the polymer, are involved.

The effect of low ionic strength on the circular dichroic spectrum of chromatin and nucleosomal subunits.

Circular dichroism has been used to measure the conformation changes in the DNA of chromatin and chromatin subunits as a function of ionic strength. Transfer of chromatin from 0.15 M to 0.25 mM salt led to an enhancement of the circular dichroic bands at 275 and 285 nm. Removal of histone H1 did not appreciably affect the circular dichroic spectrum when measured in 0.15 M salt, but in 0.25 mM salt H1 depletion led to a marked increase in the ellipticity. Conformation changes due to low ionic strength were also observed with a 145- and a 172-bp chromatin subunit. A linear combination of the ellipticities of the DNA of the two domains in chromatin, namely core and linker, was successful for measurements at 0.15 M salt, but large unexplained discrepancies appeared with the data from measurements in 0.25 mM salt.

RNA elongation by RNA polymerase II is not inhibited by N-ethylmaleimide or iodoacetamide.

The elongation of RNA by RNA polymerase II is not inhibited by N-ethylmaleimide or iodoacetamide. Three systems were studied: a soluble chromatin, purified RNA polymerase II with DNA, and a HeLa cell extract with an adenovirus 2 promoter sequence.

An improved method of sequential alcian blue and ammoniacal silver staining of chondroitin sulfate proteoglycan in polyacrylamide gels.

Partially deglycosylated chondroitin sulfate proteoglycan (CSPG) or peptide fragments obtained from CSPG are not readily detectable in gels by staining with Alcian blue 8GX or ammoniacal silver using the technique of Oakley et al. (B. Oakley, D. Kirsh, and N. Morris (1980) Anal. Biochem. 105, 361). Sequencial staining with both reagents allows visualization of intact CSPG or peptides derived from proteoglycans in polyacrylamide gels at protein concentrations as low as 2 ng/mm2, or glucuronic acid and galactosamine concentrations of 1 ng/mm2 or less. This method is significantly more sensitive and has broader applicability than that described by H. Min and M. Cowman (1986) Anal. Biochem. 155, 275) for staining glycosaminoglycan fragments in polyacrylamide gels.

Two immunologically and developmentally distinct chondroitin sulfate proteolglycans in embryonic chick brain.

Two different chondroitin sulfate proteoglycans (CSPG) in embryonic chick brain were distinguished by immunoreactivity either with S103L, a rat monoclonal antibody which reacts specifically with an 11-amino-acid region in the chondroitin sulfate domain of the core protein of chick cartilage CSPG (Krueger, R. C., Jr., Fields, T. A., Mensch, J. R., and Schwartz, N. B. (1990) J. Biol. Chem. 265, 12088-12097), or with HNK-1, a mouse monoclonal antibody which reacts with a 3-sulfoglucuronic acid residue on neural glycolipids and glycoproteins (Chou, D. K. H., Ilyas, A., Evans, J. E. Costello, C., Quarles, R. H., and Jungawala, F. B. (1986) J. Biol. Chem. 261, 11717-11725) but not with both antibodies. This specific immunoreactivity was used to separate the two CSPGs for further characterization. The S103L reactive brain proteoglycan had a core protein of similar size to cartilage CSPG (370 kDa) but exhibited a smaller hydrodynamic size (K(av) of 0.308). It was substituted predominantly with chondroitin sulfate chains and virtually no keratan sulfate chains. The HNK-1 reactive CSPG had a smaller core protein (340 kDa), an even smaller hydrodynamic size (K(av) of 0.564), and was substituted with both chondroitin sulfate and keratan sulfate chains. Glycosidase digestion patterns with endo-beta-galactosidase, N-glycosidase F, neuraminidase, and O-glycosidase, and reactivity with an antibody to the hyaluronate binding region also showed significant differences between the two brain CSPGs. Expression of the S103L reactive brain CSPG was developmentally regulated from embryonic day 7 through 19 with a peak in core protein on day 13, and in mRNA expression at day 10. In contrast the HNK-1 reactive brain CSPG was constitutively present from day 7 through hatching. These data suggest that these two distinct core proteins are immunologically and biochemically unique translation products of two different CSPG genes.

Completion of the mouse aggrecan gene structure and identification of the defect in the cmd-Bc mouse as a near complete deletion of the murine aggrecan gene.

Mouse cartilage matrix deficiency (cmd), an autosomal recessive phenotype caused by absence of aggrecan, maps to Chromosome (Chr) 7 and is caused by a 7-bp deletion in exon 5 generating a premature stop codon (Watanabe et al. 1994). Another spontaneous mutation with the same locus and phenotype, cmd-Bc, has now been defined as the complete loss of exons 2 to 18, resulting in a significantly shortened mRNA (1.2 kb). The upstream breakpoint is in intron 1, 18. 8 kb 3' of exon 1; the downstream breakpoint lies 10.5 kb past the final aggrecan exon 18. The deletion is flanked by sequences homologous to topoisomerase I and II cleavage sites and a 7-bp direct repeat, suggesting the defect resulted from a nonhomologous recombination event. Additionally, the size of the first intron and the intron-exon structure between exons 12 and 14 were determined, establishing the length of the murine aggrecan gene as 68.6 kb. This report completes the structural analysis of the murine aggrecan gene, defines a second null mutation, and reinforces the importance of aggrecan in development.

Deglycosylation of chondroitin sulfate proteoglycan and derived peptides.

In order to define the domain structure of proteoglycans as well as identify primary amino acid sequences specific for attachment of the various carbohydrate substituents, reliable techniques for deglycosylating proteoglycans are required. In this study, deglycosylation of cartilage chondroitin sulfate proteoglycan (CSPG) with minimal core protein cleavage was accomplished by digestion with chondroitinase ABC and keratanase, followed by treatment with anhydrous HF in pyridine. Nearly complete deglycosylation of secreted proteoglycan was verified within 45 min of HF treatment by loss of incorporated [3H]glucosamine label from the proteoglycan as a function of time of treatment, as well as by direct analysis of carbohydrate content and xylosyltransferase acceptor activity of unlabeled core protein preparations. The deglycosylated CSPG preparations were homogeneous and of high molecular weight (approximately 370,000). Comparison of the intact deglycosylated core protein preparations with newly synthesized unprocessed precursors (apparent Mr approximately 360,000) suggested that extensive proteolytic cleavage of the core protein did not occur during normal intracellular processing. Furthermore, peptide patterns generated after clostripain digestion of core protein precursor and of deglycosylated secreted proteoglycan were comparable. With the use of the clostripain digestion procedure, peptides were produced from unlabeled proteoglycan, and two predominant peptides from the most highly glycosylated regions (the chondroitin sulfate rich regions of the proteoglycan) were isolated, characterized, and deglycosylated. These peptides were found to follow similar kinetics of deglycosylation and to acquire xylose acceptor activity comparable to the intact core protein.

Hyperoxia alone causes changes in lung proteoglycans and hyaluronan in neonatal rat pups.

Specific changes in composition and content of lung extracellular matrix (ECM) proteoglycans (PGs) and hyaluronan (HA) have been observed during the acute response to damage in several forms of injury including infant respiratory distress syndrome (IRDS). These ECM components are thought to modulate the healing response. Hyperoxia, a contributing factor to IRDS, is known to damage both adult and developing lung, however, the extent and pattern of impairment depends on lung maturity. We hypothesized that exposing neonatal rats to hyperoxia alone might result in changes in lung HA, as well as in age-specific changes in lung PGs, similar to those shown to occur in IRDS. In control rats, lung HA decreased over the first 10 days of life, whereas rats exposed to hyperoxia exhibited a time-dependent, time-limited increase in both lung HA and lung wet weight. Histochemistry showed the HA in hyperoxia-exposed lungs to be accumulated in perivascular cuffs of medium sized arteries, and in the alveolar walls. Rats were then exposed to normoxia or hyperoxia for 7 days beginning at either 3 days of life (neonatal) or 21 days (adolescent), and lung tissue was cultured in the presence of [35S]-sulfate to label newly synthesized PGs. Proteoglycans were extracted, and analyzed by isopycnic CsCl gradient centrifugation, sequential enzymatic deglycosylation, size chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). When controlled for total protein extracted, 63% more label was incorporated into large molecular weight material in the tissue exposed to hyperoxia, with a 95% increase in incorporation in the most dense fraction, D1. [35S]-Sulfate incorporation into chondroitin and dermatan sulfate in hyperoxic tissue specifically increased 116% (242% in the D1 fraction), while incorporation into heparan sulfate remained essentially unchanged. There was a nearly fivefold increase in [35S]-sulfate incorporation into chondroitin sulfate chains in the D1 fraction. When the D1 fractions of extracts of treated and control rat lungs were compared on SDS-PAGE, a large chondroitin sulfate proteoglycan (CSPG; core protein of 195 kDa) was upregulated in the D1 fraction from hyperoxic tissue of neonatal rats, but was not detected in the lungs of adolescent animals exposed to hyperoxia. This CSPG and four additional large CSPGs were noted to be upregulated on western blotting by a polyclonal antibody directed against the G1 domain of the aggrecan protein core. We conclude that hyperoxia alone causes an increase in lung HA and lung water, and speculate that this contributes significantly to the clinical syndrome of IRDS. In addition, several large CSPGs are upregulated by hyperoxic exposure in a developmentally specific manner. We speculate that this increase in CSPGs may interfere with the normal developmental sequence of events, contributing to hypoalveolarization.

Chick cartilage chondroitin sulfate proteoglycan core protein. I. Generation and characterization of peptides and specificity for glycosaminoglycan attachment.

Peptides were derived from the large chondroitin sulfate proteoglycan from chick cartilage by clostripain digestion. Using differential chondroitinase ABC and keratanase treatment and direct carbohydrate analysis, three major peptides of 86, 75, and 27 kDa were shown to bear only chondroitin sulfate chains. Another major peptide of 65 kDa was shown to contain both chondroitin sulfate and keratan sulfate chains, allowing it to be separated from the peptides derived from the chondroitin sulfate domain by DEAE-cellulose chromatography. An additional new peptide (100 kDa) containing keratan sulfate chains was found only in clostripain digests of proteoglycan-hyaluronate-link protein aggregates. Unlike any of the other peptides derived from clostripain digestion of proteoglycan monomer or aggregate, this peptide had the properties of a functional hyaluronate binding region. All of these peptides were purified to apparent homogeneity by preparative electroelution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and deglycosylated with anhydrous hydrogen fluoride. Automated Edman degradation of the two largest chondroitin sulfate peptides revealed that they had unique N termini and several unrecognized residues, which were all subsequently revealed to be modified serine residues following deglycosylation. The keratan sulfate-bearing peptide also had a unique N terminus, which contained a single unrecognized residue, even after HF deglycosylation. Finally, the N terminus of the hyaluronate binding region was blocked. These studies allow estimates of core peptide masses in the absence of carbohydrate as well as provide primary amino acid sequence for O-xylosylated serine residues in the multiply substituted proteoglycans.

Chick cartilage chondroitin sulfate proteoglycan core protein. II. Nucleotide sequence of cDNA clone and localization of the S103L epitope.

A lambda gt11 expression library containing cDNA from total chick embryo was screened with S103L, a rat monoclonal antibody which reacts specifically with the core protein of the chick cartilage chondroitin sulfate proteoglycan. One clone was identified which produced a 220-kDa beta-galactosidase/S103L-binding fusion protein. Sequencing the entire 1.5-kilobase cDNA insert showed that it contained a single open reading frame, which encoded a portion of the proteoglycan core protein from the chondroitin sulfate domain. This was confirmed by comparison with amino acid sequence data from peptide CS-B, which was derived from the chondroitin sulfate domain (Krueger, R.C., Jr., Fields, T. A., Hildreth, J., IV, and Schwartz, N.B. (1990) J. Biol. Chem. 265, 12075-12087). Furthermore, the 3' end of the insert overlapped with 23 bases at the 5' end of the published sequence for the C-terminal globular domain (Sai, S., Tanaka, T., Kosher, R. A., and Tanzer, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5081-5085), which oriented this clone, as well as the CS peptide, along the protein core. The cDNA insert hybridized with a 9-kilobase mRNA from sternal chondrocytes as well as a similar sized message in brain but did not hybridize to any message from rat chondrosarcoma or from undifferentiated limb bud mesenchyme. In further studies, the fusion protein as well as a cyanogen bromide fragment (70 kDa) derived from it were isolated and shown to react with S103L, indicating that cleavage at methionine residues does not disrupt the antibody recognition site. Purification and N-terminal sequencing of the antigenic CNBr fragment derived from the fusion protein revealed that its N terminus is preceded by a methionine in the fusion protein and overlaps with the N terminus of peptide CS-B. As peptide CS-B is not recognized by S103L and the C terminus of peptide CS-B lies beyond the proteoglycan portion of the antigenic CNBr fragment, the S103L epitope is either contained within the 11 amino acids preceding the N terminus of peptide CS-B or it spans the clostripain cleavage site at the origin of the N terminus of peptide CS-B.

Brain aggrecan.

During development, the extracellular matrix (ECM) is a complex dynamic structure whose components and organization help to establish the requisite position and state of differentiation. Until recently, the large chondroitin sulfate proteoglycan, aggrecan, has been localized predominantly to skeletal tissue and considered a hallmark of cartilage differentiation. We have identified the presence of aggrecan in two other highly differentiated systems, brain and notochord, with clearly distinct expression patterns. In chick cartilage, aggrecan starts to be expressed at embryonic day 5 in limb rudiments, continues through the entire period of chondrocyte development, and remains a biochemical marker of the cartilage phenotype thereafter. In brain, aggrecan has a very low level of expression beginning at day 7, increases up to day 13, markedly decreases after day 16, and is not expressed posthatching. This pattern coincides with migration and establishment of neuronal nuclei in the chick telencephalon and has been proposed to be a component of the migration arrest mechanism. In very primitive embryos, aggrecan is detected as early as stage 16 in the notochord, long before chondrogenesis occurs, is then expressed up to day 5 and decreases thereafter. The expression of aggrecan occurs during the time of active neural crest migration and through the onset of sclerotomal differentiation, and correlates with the notochords' ability to inhibit neural crest cell migration. Animal models defective in aggrecan biosynthesis have been invaluable in delineating these functions. In addition we have characterized these proteoglycans by chemical, biosynthetic, and molecular analyses. Although significant post-translation differences distinguish the cell-specific aggrecan species, their core proteins are the products of a single gene. Our findings of the expression of the same gene (aggrecan) in multiple ontogenously unrelated differentiating tissue systems and at different times over the developmental life of an organism provide an elegant model system to study the regulation and interplay in expression of that gene, as well as the effect of alterations in that single gene simultaneously in several developing programs.