Biomedical subjects

K Song

Publications and source records attributed to K Song.

At least 19 recordsLinked to original sources

PubMed1996

Elevated expression of Bcl-X and reduced Bak in primary colorectal adenocarcinomas.

Expression of several members of the BCL-2 family of genes was investigated by immunohistochemical methods in 30 primary colorectal adenocarcinomas and 24 adenomatous polyps. When compared to the intensity observed in adjacent normal mucosal epithelial cells, the intensity of Bcl-X immunostaining was elevated in 18 of 30 (60%) carcinomas (P = 0.0001) and 12 of 24 (50%) adenomatous polyps (P = 0.0001). Immunoblot analysis of five pairs of tumors and adjacent normal colonic tissue indicated marked elevations in the relative levels of the anti-apoptotic Bcl-XL, protein in all cases. In contrast to the increased Bcl-X expression, the intensity of Bcl-2 immunostaining was greater than that of normal colonic mucosa in only 3 of 30 (10%) carcinomas and, in fact, was lower than that of adjacent normal epithelia] cells in 25 (83%) cases (P = 0.0001). Furthermore, the percentage of Bcl-2 immunopositive cells was generally lower in carcinomas than in adenomas (mean +/- SE, 44 +/- 6% versus 73 +/- 5%, respectively; P = 0.001) and in moderately or poorly differentiated tumors than in well-differentiated tumors (39 +/- 6% versus 70 +/- 11%, respectively; P = 0.045). In addition, the proportion of tumors in which the Bcl-2 immunointensity was more than or equal to that of normal colonic mucosa was significantly lower in carcinomas than adenomas (5 of 30 versus 15 of 24, respectively; P < 0.001), suggesting that decreases in Bcl-2 expression represent a later event associated with the progression of colorectal cancers. When compared to that of normal adjacent colonic epithelium, the intensity of Mcl-1 immunostaining was reduced in 20 of 30 (67%) of carcinomas (P = 0.0001) compared to only 1 of 24 adenomas, suggesting that decreases in Mcl-1 expression represent a later event associated with progression from a benign to a malignant phenotype or with transition to a less-differentiated state, because most of the carcinomas evaluated here (25 of 30; 83%) were not well differentiated. The intensity of immunostaining for the pro-apoptotic protein Bak was reduced compared to that of normal mucosal epithelial cells in 27 of 30 (90%) carcinomas and 22 of 24 (92%) adenomas, suggesting that reductions in Bak expression occur early in colorectal tumor progression (P = 0.0001). In contrast, the intensity of immunostaining for the pro-apoptotic protein Bax was not significantly altered in carcinomas; compared to that of normal colonic mucosa, Bax immunointensity was reduced in only 7 of 30 (23%) carcinomas and 3 of 24 (13%) adenomas, and the percentage of Bax immunopositive cells was also not significantly different in any of the histological subgroups. Taken together, these results suggest that expression of Bcl-XL is increased in undifferentiated primary colorectal cancers, often with accompanying reciprocal decreases in the anti-apoptotic proteins Bcl-2 and Mcl-1 and the pro-apoptotic protein Bak, whereas Bax expression is relatively constant. Thus, a shift from expression of the anti-apoptotic proteins Bcl-2 and Mcl-1 to the Bcl-XL protein may occur during progression of colorectal tumors.

Adenocarcinoma↗

PubMed1996

Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle.

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolar membranes in vivo. Caveolin interacts directly with heterotrimeric G-proteins and can functionally regulate their activity. Recently, a second caveolin gene has been identified and termed caveolin-2. Here, we report the molecular cloning and expression of a third member of the caveolin gene gamily, caveolin-3. Caveolin-3 is most closely related to caveolin-1 based on protein sequence homology; caveolin-1 and caveolin-3 are approximately 65% identical and approximately 85% similar. A single stretch of eight amino acids (FED-VIAEP) is identical in caveolin-1, -2, and -3. This conserved region may represent a "caveolin signature sequence" that is characteristic of members of the caveolin gene family. Caveolin-3 mRNA is expressed predominantly in muscle tissue-types (skeletal muscle, diaphragm, and heart) and is selectively induced during the differentiation of skeletal C2C12 myoblasts in culture. In many respects, caveolin-3 is similar to caveolin-1: (i) caveolin-3 migrates in velocity gradients as a high molecular mass complex; (ii) caveolin-3 colocalizes with caveolin-1 by immunofluorescence microscopy and cell fractionation studies; and (iii) a caveolin-3-derived polypeptide functionally suppresses the basal GTPase activity of purified heterotrimeric G-proteins. Identification of a muscle-specific member of the caveolin gene family may have implications for understanding the role of caveolin in different muscle cell types (smooth, cardiac, and skeletal) as previous morphological studies have demonstrated that caveolae are abundant in these cells. Our results also suggest that other as yet unknown caveolin family members are likely to exist and may be expressed in a regulated or tissue-specific fashion.

Amino Acid Sequence↗

PubMed1996

Immunohistochemical analysis of bcl-2, bax, bcl-X, and mcl-1 expression in prostate cancers.

Proteins encoded by bcl-2 family genes are important regulators of programmed cell death and apoptosis. Alterations in the expression of these apoptosis-regulating genes can contribute to the origins of cancer, as well as adversely influence tumor responses to chemo- and radiotherapy. Using antibodies specific for the Bcl-2, Bax, Bcl-X, and Mcl-1 proteins in combination with immunohistochemical methods, we examined for the first time the expression of these bcl-2 family genes in 64 cases of adenocarcinoma of the prostate, including 10 Gleason grade 2 to 4 tumors, 21 grade 5 to 7 tumors, 17 grade 8 to 10 tumors, 8 lymph node metastases, and 8 bone metastases. In addition, 24 cases of prostatic intraepithelial neoplasia (PIN) or PIN coexisting with carcinoma were also evaluated. All immunostaining results were scored with regard to approximate percentage of positive tumor cells and relative immunostaining intensity. Expression of the anti-apoptotic protein Bcl-2 was present in 16 of 64 (25%) adenocarcinomas and tended to be more frequent in high grade tumors (Gleason grade 8 to 10; 41%) and nodal metastases (38%) than in lower grade (Gleason 2 to 7) primary tumors (16%; P < 0.05). Bcl-X was expressed in all 64 (100%) tumors evaluated. Bcl-X immunointensity was generally stronger in high grade primary tumors (grade 8 to 10) and metastases compared with PIN and low grade neoplasms (P < 0.0001). In addition, the proportion of specimens with > 50% Bcl-X-immunopositive tumor cells also was higher in advanced grade primary tumors (Gleason 8 to 10) and metastases than in PIN and low grade tumors (Gleason 2 to 7; P < 0.005). The anti-apoptotic protein Mcl-1 was expressed in 52 of 64 (81%) tumors, compared with only 9 of 24 (38%) cases of PIN (P < 0.001). In addition, the percentage of Mcl-1-positive cells was typically higher in Gleason grade 8 to 10 tumors and metastases than in PIN or lower grade tumors (P = 0.025). In contrast, the pro-apoptotic protein Bax was expressed in all prostate cancers evaluated, with high percentages of immunopositive cells and strong immunointensity typically occurring regardless of tumor grade. The findings suggest that expression of several anti-apoptotic members of the bcl-2 gene family, including bcl-2, bcl-X, and mcl-1 increases during progression of prostate cancers, a finding that may be relevant to the hormone-insensitive, metastatic phenotype of most advanced adenocarcinomas of the prostate.

Adenocarcinoma↗

PubMed1995

MSX1 inhibits myoD expression in fibroblast x 10T1/2 cell hybrids.

Transfer of human chromosome 11, which contains the myoD locus, from primary fibroblasts into 10T1/2 cells results in activation of myoD. In contrast, hybrids that retain human chromosome 11 and additional human chromosomes fail to activate myoD. We show that human chromosome 4 inhibits myoD activation. myoD enhancer/promoter reporter constructs show that repression is at the transcriptional level. Chromosome fragment-containing hybrids localize the repressing activity to the region of 4p that contains the homeobox gene MSX1. MSX1 is expressed in primary human fibroblasts and in 10T1/2 cells containing human chromosome 4, while parental 10T1/2 cells do not express Msx1. Forced expression of Msx1 represses myoD enhancer activity. Msx1 protein binds to the myoD enhancer and likely represses myoD transcription directly. Antisense MSX1 relieves repression mediated by chromosome 4. We conclude that MSX1 inhibits transcription of myoD and that myoD is a target for homeobox gene regulation.

Animals↗

PubMed1995

Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution.

Although the evolutionary success of polyploidy in higher plants has been widely recognized, there is virtually no information on how polyploid genomes have evolved after their formation. In this report, we used synthetic polyploids of Brassica as a model system to study genome evolution in the early generations after polyploidization. The initial polyploids we developed were completely homozygous, and thus, no nuclear genome changes were expected in self-fertilized progenies. However, extensive genome change was detected by 89 nuclear DNA clones used as probes. Most genome changes involved loss and/or gain of parental restriction fragments and appearance of novel fragments. Genome changes occurred in each generation from F2 to F5, and the frequency of change was associated with divergence of the diploid parental genomes. Genetic divergence among the derivatives of synthetic polyploids was evident from variation in genome composition and phenotypes. Directional genome changes, possibly influenced by cytoplasmic-nuclear interactions, were observed in one pair of reciprocal synthetics. Our results demonstrate that polyploid species can generate extensive genetic diversity in a short period of time. The occurrence and impact of this process in the evolution of natural polyploids is unknown, but it may have contributed to the success and diversification of many polyploid lineages in both plants and animals.

Biological Evolution↗

PubMed1995

Angiotensin II receptor subtypes in the human central nervous system.

The distribution of the AT1 and AT2 subtypes of angiotensin II receptor was mapped in the adult human central nervous system using quantitative in vitro autoradiography. Binding in all forebrain, midbrain, pontine, medullary and spinal cord sites where angiotensin II receptors have previously been described is of the AT1 subtype, as is binding in the small and large arteries in the adjacent meninges and in choroid plexus. By contrast, both AT1 and AT2 receptors occur in the molecular layer of the cerebellum. Angiotensin II AT1 receptors in the brain show a moderate degree of conservation across mammalian species studied so far, whereas expression of AT2 receptors is more variable, and is more restricted in the human CNS than in many other mammals. These differences between the subtype distributions in humans and other animals indicate the need for care when extrapolating the results of animal studies involving the brain angiotensin system.

Aged↗

PubMed1995

Identification and characterization of a human cDNA homologous to yeast SKI2.

A monoclonal antibody, 170A1, which recognizes a nucleolar peptide of molecular weight 90,000, was raised. The protein was conserved among various vertebrates. To characterize the antigen, we screened a human fetal liver expression library using the monoclonal antibody as a probe. Molecular analyses of immunopositive clones suggested the presence of a novel cDNA. It appeared to be a single-copy gene and encoded about 4- and 5-kb mRNAs. The gene appeared to be expressed in every cell tested so far. Its deduced amino acid sequence revealed an overall homology to recently described yeast SKI2. The SKI2 gene of Saccharomyces cerevisiae encoded a nucleolar protein that is involved in the antiviral system. We report here the partial human cDNA sequence and the localization of the corresponding gene on chromosome 6p21.

Amino Acid Sequence↗

PubMed1995

Mutational analyses of fs(1)Ya, an essential, developmentally regulated, nuclear envelope protein in Drosophila.

The fs(1)Ya protein (YA) is an essential, maternally encoded, nuclear lamina protein that is under both developmental and cell cycle control. A strong Ya mutation results in early arrest of embryos. To define the function of YA in the nuclear envelope during early embryonic development, we characterized the phenotypes of four Ya mutants alleles and determined their molecular lesions. Ya mutant embryos arrest with abnormal nuclear envelopes prior to the first mitotic division; a proportion of embryos from two leaky Ya mutants proceed beyond this but arrest after several abnormal divisions. Ya unfertilized eggs contain nuclei of different sizes and condensation states, apparently due to abnormal fusion of the meiotic products immediately after meiosis. Lamin is localized at the periphery of the uncondensed nuclei in these eggs. These results suggest that YA function is required during and after egg maturation to facilitate proper chromatin condensation, rather than to allow a lamin-containing nuclear envelope to form. Two leaky Ya alleles that partially complement have lesions at opposite ends of the YA protein, suggesting that the N- and C-termini are important for YA function and that YA might interact with itself either directly or indirectly.

Alleles↗

PubMed1994

The human stromelysin promoter contains a previously unreported 1.0-kb sequence.

Cloning and characterization of the promoter region controlling the gene encoding human stromelysin (Str) has been previously reported [Quinones et al., J. Biol. Chem. 264 (1989) 8339-8344]. We have characterized independently isolated genomic clones of the STR promoter, designated pSKStrB and 682, that are considerably different from the published sequence. Although the sequences up to an XbaI site at -480 of the 5' regions are identical, a novel 1.0-kb segment exists upstream from -480. This sequence is absent from the published clone, but its presence in the genomic DNA from twelve individuals has been confirmed by both PCR analysis and restriction mapping. Upstream of the novel 1-kb segment, the sequence of the published clone reappears, but in pSKStrB exists in inverse orientation.

Base Sequence↗

PubMed1994

AA1, a newly synthesized monovalent lipophilic cation, expresses potent in vivo antitumor activity.

Certain lipophilic cations have been reported to display anticarcinoma activities because of their selective uptake and retention by mitochondria of cancer cells. Thus, these agents may comprise a unique class of agents directed against carcinoma. After screening more than 1000 lipophilic cations, we found that the monovalent lipophilic cation, 2,6-bis(4-amino-phenyl)-4-[4-(dimethylamino)phenyl]thiopyrylium chloride (AA1), displayed remarkable anticarcinoma activity both in vitro and in vivo. Unlike most other lipophilic cations, AA1 is stable and displays minimal light sensitivity. In vitro testing showed that AA1 was 10 times more toxic to the carcinoma cell line CX-1 than to the normal epithelial cell line CV-1. In vivo animal experiments showed that AA1 significantly prolonged the survival of mice implanted with tumors. For C57BL x DBA/2 F1 mice implanted with the mouse bladder carcinoma cell line, MB49, the treated:control ratio was 344%. For Swiss nu/nu mice implanted i.p. with the human melanoma cell line, LOX, the treated:control ratio was 341%. The most significant observation was obtained with Swiss nu/nu mice that were implanted i.p. with the human ovarian cell line, OVCAR-III. The treated:control ratio in this situation was greater than 450%. In all these tumor models, AA1 produced minimal toxicities. AA1 exhibited little inhibition of electron transport in isolated rat liver mitochondria; however, it inhibited mitochondrial ATPase with 50% inhibitory concentration of 6 microM. Compared with previously reported anticarcinoma lipophilic cations such as rhodamine 123 and dequalinium chloride, AA1 appeared to display more effective in vivo anticarcinoma activity. Thus, AA1 could be considered for further clinical development as a candidate for anticarcinoma chemotherapy.

Adenocarcinoma↗

PubMed1994

The Drosophila fs(1)Ya protein, which is needed for the first mitotic division, is in the nuclear lamina and in the envelopes of cleavage nuclei, pronuclei, and nonmitotic nuclei.

The Drosophila melanogaster fs (1) Ya gene encodes a nuclear envelope protein needed to initiate embryonic cleavage divisions. In cleavage stage embryos the fs (1) Ya gene product is localized to the nuclear envelope in a cell cycle dependent manner. We show here that fs(1)Ya protein is also present in polar bodies of early cleavage stage embryos as well as in endoreplicating yolk nuclei. This observation suggests that the fs(1)Ya protein is necessary but not solely responsible for embryonic mitosis. Furthermore, cell cycle stage dependent nuclear localization is also seen for ectopically produced fs(1)Ya protein in tissue culture cells, neuroblasts, and male accessory glands, which are free of maternal products. We therefore conclude that cell cycle dependent nuclear localization does not require any exclusively maternal products. Finally, our immunoelectron microscopy shows that the fs(1)Ya protein distribution parallels that of lamin, indicating that it is a nuclear lamina protein.

Animals↗

PubMed1994

A method for examining expression of homologous genes in plant polyploids.

One of the essential issues regarding evolution of polyploid species is how duplicate genes are expressed. Most studies on gene expression in polyploids have been based on isozyme analyses; RNA analysis has not been widely used partially due to difficulties in distinguishing homologous transcripts which usually have the same length and similar or almost identical sequences. In this study, a method combining RT-PCR with RFLP was used to analyze transcripts of homologous genes in natural and synthetic Brassica amphidiploids. Sequences coding for several known genes were selected and used to synthesize gene-specific primers. Total RNAs were used as templates for RT-PCR to amplify homologous transcripts in three diploid parental species, three cultivated amphidiploid species and six synthetic amphidiploids. For each gene, initial PCR products amplified in all species had identical length; however, homologous transcripts in the diploid and amphidiploid species could be distinguished after digesting the PCR products with restriction enzymes. Preliminary results based on three genes indicated that both transcripts from the diploid parents were expressed in the synthetic and natural amphidiploids. This study represents the first application of RT-PCR and RFLP analysis to investigate expression of homologous genes in higher plants. The technique is a sensitive, simple and efficient method for distinguishing homologous transcripts in a mixed RNA population and can be applied to many types of studies on expression of homologous genes.

Acyl Carrier Protein↗

PubMed1994

Mandibular reconstruction using computer-generated three-dimensional solid models.

In mandibular reconstruction, it is necessary to know the exact, three-dimensional extent of the mandible and its defect; the bone graft must be the exact size and dimension of the defect, to assure a precise three-dimensional configuration of the mandible. Previously, the bone graft had to be reshaped during the operation by trial and error, often a time-consuming procedure. The operative procedure has been simulated in advance using three-dimensional, solid models, which has shortened the operating time required.

Bone Transplantation↗

PubMed1994

Localization and quantitation of active renin in monkey kidney by radioinhibitor binding and in vitro autoradiography.

We developed an in vitro autoradiographic method to localize and quantify active renin in primate tissues. Active renin in monkey kidney sections was labeled with the primate specific renin inhibitor, 3H-CGP29287, and quantitated with autoradiography and computerized densitometry. Microscopic emulsion autoradiography was carried out to clarify the detailed localization of the binding. Non-specific binding to aspartyl proteases other than renin was blocked using 1 mumol/L of N-acetyl-pepstatin. To assess the usefulness of this procedure, binding of 3H-CGP29287 was examined both by film and emulsion autoradiography in the kidneys of monkeys (Macaca fuscata) that were given chronically either an angiotensin converting enzyme inhibitor (trandolapril), an angiotensin II receptor antagonist (E4177), or vehicle. 3H-CGP29287 was found to bind very selectively to the juxtaglomerular apparatus (JGA) under control conditions. In monkeys treated with trandolapril or E4177, 3H-CGP29287 binding was increased in proportion to the increase in renal renin concentration determined enzymatically; in these kidneys, emulsion autoradiography revealed radioinhibitor binding extending far from the JGA. The potency of a series of unlabeled renin inhibitor in competing for 3H-CGP29287 binding in the autoradiographic system closely paralleled their potencies, as determined in inhibiting renin by an enzymatic assay. This technique permits specific labeling of the catalytic site of renin in the monkey kidney sections.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1994

Blockade by intravenous losartan of AT1 angiotensin II receptors in rat brain, kidney and adrenals demonstrated by in vitro autoradiography.

1. The in vivo inhibition of angiotensin II (AII) receptor binding in the rat brain, kidney and adrenal was investigated after intravenous administration of the AT1-selective AII receptor antagonist losartan. 2. Male Sprague-Dawley rats were administered intravenously either vehicle, or losartan at doses of 1, 3 or 10 mg/kg. Plasma samples were collected and tissues removed at 1, 2, 8 or 24 h after administration of the antagonist. The effects of losartan on AII receptor binding were assessed by quantitative in vitro autoradiography. 3. Losartan significantly increased plasma renin activity (PRA) by six-fold and nine-fold at doses of 1 and 10 mg/kg, respectively (P < 0.05). Plasma losartan concentrations rose from 0.83 micrograms/mL at 1 mg/kg to 46.5 micrograms/mL at 10 mg/kg 1 h after administration of the drug. Plasma renin activity returned to control, whilst losartan was undetectable 24 h after injection of the antagonist. 4. In the brain, losartan produced a dose-dependent inhibition of AII receptor binding to the brain structures which express exclusively, or predominantly, AT1 receptors both outside and within the blood brain barrier. By contrast, losartan did not affect binding to the nuclei which contain exclusively, or predominantly, AT2 receptors. 5. In the kidney, losartan blocked AII receptor binding to all anatomical sites in a dose-dependent manner. The inhibition peaked at 1 h and persisted beyond 24 h despite the fact that PRA had returned to control, and losartan was not detectable in the circulation. In the adrenal gland, where AT1 and AT2 receptors occur in both the cortex and medulla, losartan caused partial inhibition at both regions. 6. These results indicate that losartan, administered intravenously at these doses, and/or its active metabolites, partially penetrate the blood brain barrier to selectively inhibit central AT1 receptors, and exert selective and prolonged blockade at AT1 receptors in peripheral target tissues.

Adrenal Glands↗

PubMed1994

Quantitative localization of angiotensin II receptor subtypes in spontaneously hypertensive rats.

Angiotensin II (Ang II) receptors were labelled by in vitro autoradiography using 125I-[Sar1,Ile8]Ang II as a ligand in the kidney, adrenal gland, thoracic aorta, and hindbrain of adult spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Ang II receptors were differentiated into subtypes by susceptibility to subtype 1 (AT1) and subtype 2 (AT2) antagonists. In both rat strains, the adrenal cortex contained predominantly AT1 receptors, while AT2 receptors predominated in the adrenal medulla. The kidney contained exclusively AT1 receptors in glomeruli, proximal tubules, and the outer medulla. AT1 receptors were predominant in the thoracic aorta. The nucleus of the solitary tract (NTS), dorsal motor nucleus of the vagus (DM10), area postrema, and spinal trigeminal nucleus (Sp5) contained exclusively AT1 receptors, whereas the nucleus of the inferior olive contained AT2 receptors predominantly. Significant differences in receptor density were observed between SHR and WKY. The adrenal cortex, renal outer medulla, NTS, DM10, and Sp5 displayed higher AT1 receptor density in SHR than in WKY. These results indicate that expression of AT1 receptors is regulated differently in important targets of Ang II in SHR, and suggest that altered regulation of AT1 receptor expression may be relevant to the pathogenesis of hypertension in SHR.

Adrenal Glands↗

PubMed1993

Distribution of angiotensin II receptor subtypes in the rabbit brain.

We have determined the distribution of angiotensin II receptor subtypes in rabbit brain using in vitro autoradiography. AT1 receptors were found in very high concentrations in the forebrain circumventricular organs--the subfornical organ, organum vasculosum of the lamina terminalis, and the median eminence as observed in other mammals. However, there was very little labeling in the area postrema. In the paraventricular nucleus, median preoptic nucleus, supraoptic nucleus there were high levels of predominantly AT1 receptors. High densities of AT1 receptors were also found in the nucleus of the solitary tract and the rostral and caudal ventrolateral medulla. All of these regions have putative roles in the regulation of blood pressure and fluid and electrolyte balance. In the rabbit brain there is less AT2 receptor binding than the rat, with most AT2 binding found in the molecular layer of the cerebellum and in the septohypothalamic nucleus. In the subthalamic nucleus, the mediodorsal and ventroposterior nuclei of the thalamus, locus coeruleus and inferior olivary nuclei, areas containing mostly AT2 receptors in the rat, no binding was detected in the rabbit except in the locus coeruleus which contains moderate levels of AT1 receptors. Taken in conjunction with our previous results in the rat and human brains, these results reveal that AT1 receptors predominate in rostral forebrain, hypothalamus and autonomic control centers of the medulla oblongata in all three species. However, the distribution and density of AT2 bearing sites in regions such as the septum, thalamus subthalamic nuclei, locus coeruleus, cerebellum and inferior olivary nuclei show marked species differences.

Amygdala↗

PubMed1993

m-Calpain requires DNA for activity on nuclear proteins at low calcium concentrations.

m-Calpain (calpain II, m-CANP), which normally requires millimolar Ca2+ for activity in vitro, was capable of proteolyzing a number of matrix proteins in isolated rat liver nuclei at Ca2+ concentrations as low as 3 microM (Mellgren, R. L. (1991) J. Biol. Chem. 266, 13920-13924). Treatment of nuclei with deoxyribonuclease I eliminated the activity of m-calpain at low Ca2+ concentrations, while ribonuclease A and phospholipase C had no effect. Addition of DNA to DNase-treated nuclei restored m-calpain activity at low Ca2+. RNA had little if any effect. Eukaryotic and prokaryotic DNA were equally effective, and synthetic polydeoxyribonucleotides were also activators. m-Calpain did not bind to a DNA-cellulose column in the presence of 200 microM Ca2+, and m-calpain preincubated in the presence of DNA and 200 microM Ca2+ was not activated at low Ca2+ concentrations following removal of the DNA. DNA did not alter the Ca2+ requirement for m-calpain-catalyzed cleavage of casein. These results demonstrate that the Ca2+ requirement for proteolysis of nuclear matrix proteins by m-calpain can be dramatically decreased in the presence of DNA. Activation did not seem to be a result of DNA binding directly to calpain but appeared to require interaction of DNA, calpain, and calpain substrates in the nuclear matrix.

Animals↗