Volumetry of Mesiotemporal Structures Reflects Serostatus in Patients with Limbic Encephalitis.

BACKGROUND AND PURPOSE: Limbic encephalitis is an autoimmune disease. A variety of autoantibodies have been associated with different subtypes of limbic encephalitis, whereas its MR imaging signature is uniformly characterized by mesiotemporal abnormalities across subtypes. Here, we hypothesized that patients with limbic encephalitis would show subtype-specific mesiotemporal structural correlates, which could be classified by supervised machine learning on an individual level. MATERIALS AND METHODS: T1WI MPRAGE scans from 46 patients with antibodies against glutamic acid decarboxylase and 34 patients with antibodies against the voltage-gated potassium channel complex (including 10 patients with leucine-rich glioma-inactivated 1 autoantibodies) and 48 healthy controls were retrospectively ascertained. Parcellation of the amygdala, hippocampus, and hippocampal subfields was performed using FreeSurfer. Volumes were extracted and compared between groups using unpaired, 2-tailed t tests. The volumes of hippocampal subfields were analyzed using a multivariate linear model and a binary decision tree classifier. RESULTS: Temporomesial volume alterations were most pronounced in an early stage and in the affected hemispheric side of patients. Statistical analysis revealed antibody-specific hippocampal fingerprints with a higher volume of CA1 in patients with glutamic acid decarboxylase-associated limbic encephalitis (P = .02), compared with controls, whereas CA1 did not differ from that in controls in patients with voltage-gated potassium channel complex autoantibodies. The classifier could successfully distinguish between patients with autoantibodies against leucine-rich glioma-inactivated 1 and glutamic acid decarboxylase with a specificity of 87% and a sensitivity of 80%. CONCLUSIONS: Our results suggest stage-, side- and antibody-specific structural correlates of limbic encephalitis; thus, they create a perspective toward an MR imaging-based diagnosis.

Isolation, quantitation, and characterization of a stable complex formed by Lp[a] binding to triglyceride-rich lipoproteins.

Lipoprotein [a] (Lp[a]) is a cholesterol-rich lipoprotein resembling LDL to which a large polymorphic glycoprotein, apolipoprotein [a] (apo[a]), is covalently coupled. Lp[a] usually exists as a free-standing particle in normolipidemic subjects; however, it can associate noncovalently with triglyceride-rich lipoproteins in hypertriglyceridemic (HTG) subjects. In this study, 10-78% of the Lp[a] present in five HTG subjects was found in the triglyceride-rich lipoprotein (TRL) fraction. The Lp[a]-TRL complex was resistant to dissociation by ultracentrifugation (UCF) alone, but was quantitatively dissociated by UCF in the presence of 100 mM proline. Of this dissociated Lp[a], 70-88% was in the form of a lipoprotein resembling conventional Lp[a]. Incubation of Lp[a]-depleted TRL with native Lp[a] resulted in a reconstituted Lp[a]-TRL complex that closely resembled the native isolates in all examined properties. Complex formation was inhibited by several compounds in the order proline > tranexamate > epsilon-aminocaproate >> arginine > lysine. Neither plasminogen nor LDL inhibited binding of Lp[a] to TRL. We observed the preferential binding of Lp[a] containing higher apparent molecular weight apo[a] polymorphs to TRL both in native and reconstituted Lp[a]-TRL complexes. A disproportionate amount of Lp[a] was bound to the larger TRL particles. Although most apo[a] bound to TRL was in the form of conventional Lp[a] particles, lipid-free recombinant apo[a] was observed to bind TRL. These results provide unequivocal evidence of the existence of an Lp[a]-TRL complex under pathophysiologic conditions. The metabolic fate of the Lp[a]-TRL complex, which is more abundant in hypertriglyceridemia, may be different from that of conventional Lp[a], and may contribute uniquely to the progression or severity of cardiovascular disease.

Evaluation of Lp[a] and other independent risk factors for CHD in Asian Indians and their USA counterparts.

Conventional risk factors for coronary heart disease (CHD) do not completely account for the observed increase in premature CHD in people from the Indian subcontinent or for Asian Indians who have immigrated to the USA. The objective of this study was to determine the effect of immigration to the USA on plasma levels of lipoprotein [a] (Lp[a]) and other independent risk factors for CHD in Asian Indians. Three subject groups were studied: group 1, 57 subjects living in India and diagnosed with CHD (CHD patients); group 2, 46 subjects living in India and showing no symptoms of CHD (control subjects); group 3, 206 Asian Indians living in the USA. Fasting blood samples were drawn to determine plasma levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein [LDL cholesterol (LDL-Chol)], high density lipoprotein [HDL cholesterol (HDL-Chol)], apolipoprotein B-100 (apoB-100), and Lp[a]. Apolipoprotein [a] (apo[a]) size polymorphism was determined by immunoblotting. Plasma TG, apoB-100, and Lp[a] concentrations were higher in CHD patients than in control and USA groups. CHD patients had higher levels of TC and LDL-Chol and lower HDL-Chol than control subjects. However, the USA population had higher levels of TC, LDL-Chol, and apoB-100 and lower HDL-Chol than control subjects. Plasma Lp[a] levels were inversely correlated with the relative molecular weight of the more abundant of each subject's two apo[a] isoforms (MAI), and CHD patients showed higher frequencies of lower relative molecular weights among MAI. Our observed changes in lipid profiles suggest that immigrating to the USA may place Asian Indians at increased risk for CHD. This study suggests that elevated plasma Lp[a] confers genetic predisposition to CHD in Asian Indians, and nutritional and environmental factors further increase the risk of CHD. This is the first report implicating MAI size as a predictor for development of premature CHD in Asian Indians. Including plasma Lp[a] concentration and apo[a] phenotype in screening procedures may permit early detection and preventive treatment of CHD in this population.

Cytosolic enzymes from rat tissues that activate the cooked meat mutagen metabolite N-Hydroxyamino-1-methyl-6- phenylimidazo[4,5-b]pyridine (N-OH-PhIP).

Heterocyclic amines are formed during the cooking of foods rich in protein and can be metabolically converted into cytotoxic and mutagenic compounds. These "cooked-food mutagens" constitute a potential health hazard because DNA damage arising from dietary exposure to heterocyclic amines can modify cell genomes and thereby affect future organ function. To determine enzymes responsible for heterocyclic amine processing in mammalian tissues, we performed studies to measure genotoxic activation of the N-hydroxy form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) --a common dietary mutagen. O-Acetyltransferase, sulfotransferase, kinase, and amino-acyl synthetase activities were assayed using substrate-specific reactions and cytosolic enzymes from newborn and adult rat heart, liver, spleen, kidney, brain, lung, and skeletal muscle. The resultant enzyme-specific DNA adduct formation was quantified via (32)P-postlabeling techniques. In biochemical assays with rat tissue cytosolic proteins, O-acetyltransferases were the enzymes most responsible for N-hydroxy-PhIP (N-OH-PhIP) activation. Compared to O-acetyltransferase activation, there was significantly less kinase activity and even lesser amounts of sulfotransferase activity. Proyl-tRNA synthetase activation of N-OH-PhIP was not detected. Comparing newborn rat tissues, the highest level of O-acetyltransferase mutagen activation was observed for neonatal heart tissue with activities ranked in the order of heart > kidney > lung > liver > skeletal muscle > brain > spleen. Enzymes from cultured neonatal myocytes displayed high O-acetyltransferase activities, similar to that observed for whole newborn heart. This tissue specificity suggests that neonatal cardiac myocytes might be at greater risk for damage from dietary heterocyclic amine mutagens than some other cell types. However, cytosolic enzymes from adult rat tissues exhibited a different O-acetyltransferase activation profile, such that liver > muscle > spleen > kidney > lung > brain > heart. These results demonstrated that enzymes involved in catalyzing PhIP-DNA adduct formation varied substantially in activity between tissues and in some tissues, changed significantly during development and aging. The results further suggest that O-acetyltransferases are the primary activators of N-OH-PhIP in rat tissues.

Bioactivation of the proximal food mutagen 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) to DNA-binding species by human mammary gland enzymes.

We have investigated phase II activation of the food-derived mutagen 2-hydroxyamino-1-methyl-6-phenyl[4,5-b]pyridine (N-OH-PhIP) by cytosolic acetyltransferase, sulfotransferase, and tRNA synthetase/kinase enzymes from human breast tissue. Cytosol from homogenates of mammary gland tissue obtained from breast-reduction surgery or mastectomy was incubated with and without enzyme-specific cofactors, and mutagen binding of calf thymus DNA was quantified by 32P-postlabeling. In addition, microsomal fractions of mammary epithelial cells from some individuals were examined for prostaglandin H synthetase activation of N-OH-PhIP. Our results show that all four enzymes can participate in activating N-OH-PhIP, thus inducing PhIP-DNA adduct formation in human mammary cells. However, not all individuals exhibited all these activities; instead each individual showed a combination of one or more activation pathways. The present findings demonstrate that the human mammary gland has the capacity to metabolically activate a dietary mutagen by several enzyme systems, including acetyltransferase, sulfotransferase, tRNA synthetase/kinase, and prostaglandin hydroperoxidase catalysis.

Introduction, distribution, and removal of 7-methylguanine in different liver chromatin fractions of young and old mice.

The distribution and elimination of 7-methylguanine (m7Gua) from different liver DNA chromatin fractions has been studied after treating young and old mice with N-methyl-N-nitrosourea (MNU). Guanine methylation kinetics was first studied in total liver DNA following intraperitoneal injections of 25 mg/kg and 50 mg/kg MNU does. MNU-induced DNA alkylation, as measured by m7Gua levels, was dose-dependent in liver tissues of young (9-11 month) and old mice (28-29 month). However, liver DNA in old mice incurred approximately 50% more damage than young mice for weight-normalized doses of MNU. The kinetics of adduct removal from total DNA was biphasic. A more rapid phase of m7Gua removal was observed during the first 24 h to 48 h following MNU administration; thereafter, the remaining m7Gua adducts were hydrolyzed much more slowly. Similar amounts of m7Gua were removed by 24 h at both the 25 mg/kg and 50 mg/kg MNU doses for a single age-group, but old liver tissue removed significantly more m7Gua than young liver tissue during this initial phase. Following a single injection of carcinogen (50 mg/kg), liver nuclei were isolated and chromatin was sheared by limited Micrococcal nuclease digestion. Chromatin was separated into nuclease-soluble, low-salt, high-salt and nuclear matrix fractions. All four fractions of young liver chromatin were methylated to the same degree. In contrast, there were differences in m7Gua levels between old liver chromatin fractions. DNA in the nuclease-sensitive fraction was most heavily alkylated, whereas nuclear matrix sequences were modified the least. Removal of m7Gua occurred at relatively uniform rates in all chromatin fractions regardless of age, indicating that m7Gua was not preferentially repaired in different nuclease-susceptible regions of chromatin. These results suggest that the N-methylpurine-DNA glycosylase responsible for eliminating m7Gua from the mammalian genome is not deficient in senescent liver tissue. However, there may be age-related changes in chromatin composition or structure that make some genomic sequences more accessible to alkylating agents in liver tissue of older animals.

Lipoprotein(a) in plasma, arterial wall, and thrombus from patients with aortic aneurysm.

The plasma concentration of lipoprotein(a) [Lp(a)] is highly correlated with the incidence of cardiovascular and peripheral vascular disease. A positive physiological role for Lp(a) has not yet been clearly identified, although elevated plasma levels in pregnant women, long-distance runners, subjects given growth hormone, patients after cardiovascular surgery, and patients with cancer, diabetes, or renal disease suggest its involvement in tissue synthesis and repair. The hypothesis that Lp(a) is involved in repair/reinforcement of the aorta was tested in 38 patients undergoing surgery for aortic aneurysm. In 29 patients 1 day before surgery, the mean plasma Lp(a) protein level was 10.7 mg/dl. At about 1, 2, and 8 weeks after surgery, the level was 14.1, 15.1, and 15.2 mg/dl, respectively. These levels are significantly higher than those of a comparable group of normal subjects (6.4 mg/dl; n = 274). Specimens of resected aortic aneurysm showed extensive medial degeneration, discontinuous elastic fibers, and deposition of mucopolysaccharides; these specimens were treated with a detergent-containing buffer to extract entrapped lipoproteins. The mean Lp(a) protein level in aortic wall extracts was 14.6 ng/mg tissue; these individual values were significantly associated with plasma Lp(a) levels before surgery (r2 = 0.31, p = 0.0003). The mean Lp(a) protein level in aortic thrombus extracts was substantially higher at 69.6 ng/mg tissue; these individual levels also were significantly associated with plasma Lp(a) concentrations before surgery (r2 = 0.68, p < 0.0001). The observations that: (i) plasma Lp(a) protein is about 1.7-fold higher in patients with aortic aneurysms than in normal subjects; and (ii) that Lp(a) protein in the aneurysmic thrombus is about 4.8-fold higher than in the aortic wall suggest that this lipoprotein plays a significant and direct role in thrombus formation and in reinforcement of the aneurysmic aortic wall.

Interaction of apolipoprotein[a] with apolipoproteinB-100 Cys3734 region in lipoprotein[a] is confirmed immunochemically.

Monospecific polyclonal antibodies (MPAbs) to apoB-100 regions Cys3734 and Cys4190 were isolated by affinity chromatography using the synthetic polypeptides, Q3730VPSSKLDFREIQIYKK3746 and G4182IYTREELSTMFIREVG4198, respectively, coupled to a hydrophilic resin. Molecular modeling and fluroescence labeling studies have suggested that Cys67 located in kringle type 9 (LPaK9, located between residues 3991 and 4068 of the apo[a] sequence inferred by cDNA) of the apo[a] molecule is disulfide linked to Cys3734 of apoB-100 in human lipoprotein[a] (Lp[a]). This possibility has been further explored with MPAbs. Four species of MPAbs directed to a Cys3734 region of apoB-100 (3730-3746) were isolated from goat anti-human LDL serum by a combination of synthetic peptide (Q3730VPSSKLDFREIQIYKK3746) affinity chromatography and preparative electrophoresis (electrochromatography). MPAbs to the Cys4190 region of apoB-100, a second or alternative disulfide link-site between apo[a] and apoB-100, were also isolated using a synthetic peptide (G4182IYTREELSTMFIREVG4198) affinity resin. Results of immunoassays showed that binding of these four MPAbs to Lp[a] was significantly lower than to LDL. In contrast, MPAbs to the apoB-100 region 4182-4198 which contains Cys4190, a second or alternative disulfide link-site between apo[a] and apoB-100, displayed a less significant difference in binding to Lp[a] and LDL. These results provide additional evidence that the residues 3730-3746 of apoB-100 interact significantly with apo-a- in Lp-a-, and that Cys3734 is a likely site for the disulfide bond connecting apo[a] and apoB-100.

Aging affects the levels of DNA damage in postmitotic cells.

Cells are continuously exposed to DNA damaging agents that may cause mutations or lead to cell death. To counter this constant, ubiquitous attack on the genetic material, cells possess highly diverse and efficient systems to repair a variety of DNA lesions. For cells that are nondividing and are expected to remain functionally viable for many years, it is important that damage not accumulate in those genes that are essential to maintaining differentiated gene expression. If damage were to accumulate slowly in working genes, then the outcomes might appear as biological changes typically associated with senescence. Estimates on the types of DNA damage believed to arise spontaneously suggest that methylation of N7-guanine is one of the more frequently occurring events, exceeded only by single-strand breaks and possibly depurination. Previous studies have shown that the steady-state levels of m7Gua increase during aging of postmitotic mammalian tissues. To test for the possibility that repair of m7Gua might decline in senescent animals, we induced methyl adducts in young and old mice with single doses of MNU, and determined the kinetics of adduct removal. Liver, kidney and brain all exhibited some active repair of m7Gua as characterized by the rapid removal of the adduct from DNA. However, a fraction of damage was refractory to repair and was lost from DNA much more slowly. This repair-resistant fraction of damage was greater in DNA from the old tissues, but the interpretation of the data is not straightforward, because different amounts of damage were induced in young and old tissues with the same weight-normalized dose of MNU. Although old cells had higher levels of persistent adducts, initial repair rates were similar between age-matched tissues. Furthermore, experiments indicated that mRNA levels for 3-methyladenine glycosylase repair enzyme did not change with age. Our working hypothesis is that repair enzymes are present and active in senescent postmitotic tissues, but changes have occurred in old chromatin that have affected the ability of repair enzymes to efficiently process these adducts.

Interaction of LDL and Lp[a] with human skin fibroblasts.

We have studied the interaction of LDL and Lp[a] with fibroblasts. Our studies suggest that Lp[a] does not effectively compete with LDL for binding to the LDL receptor, and does not efficiently suppress the activity of the intracellular enzyme HMG-CoA reductase. However, Lp[a-], formed by reduction of the disulfide bond between apo[a] and apoB, behaves much like homologous LDL, whether or not apo[a] is removed from the mixture, and in spite of the fact that one or more apoB disulfides may also have been cleaved. In our studies we also noted that Lp[a] often enhanced binding of 125I-LDL by fibroblasts. Further investigation has suggested that this interaction is time-dependent. Experiments in receptor-negative fibroblasts indicate that the enhancement is not related to the presence of the LDL receptor; however, it is inhibited by the removal of calcium from the medium. The presence of sialic acid at millimolar concentrations in the medium inhibits much of the Lp[a]-enhanced binding of 125I-LDL to the cells. These studies suggest that Lp[] may in some way enhance LDL binding to cells, perhaps via interaction with cell surface glycosaminoglycans or proteoglycans or with collagen.

Identification and quantification of apolipoproteins in addition to apo[a] and apo B-100 in human lipoprotein[a].

The protein moiety of Lp[a] is widely believed to consist of one molecule of apo B-100 and one molecule of apo[a] per particle, linked by at least one disulfide bond. In this study we have re-examined the composition of Lp[a] to determine if other less abundant apolipoproteins might be present. Analysis of Lp[a] by sodium dodecyl sulfate-polyacrylamide electrophoresis under reducing conditions showed bands corresponding to < 200 kD but > 50 kD, 40 kD, 26 kD, 23 kD and 9 kD when stained with silver. Western immunoblot analysis of three preparations of Lp[a] revealed the presence of apoE and apoD. Enzyme-linked immunoassays were used to quantify apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, apoE and apo B-100 in Lp[a] and autologous LDL isolated from three healthy males. There is a significant amount of apoA-I in the Lp[a], although the levels varied widely among the different samples. ApoE concentrations were consistent in the three Lp[a] samples and were between 22 and 26% of relative apo B-100 concentrations. Relatively minor amounts of apoA-II and no apoCs were detectable in the three Lp[a] preparations. In contrast, the autologous LDL preparations contained relatively higher amounts of apoA-I, apoA-II, apoE, apoC-I, apoC-II and apoC-III. The identity of the multiple bands corresponding to < 200 kD and > 54 kD and 9 kD is not established.

Electron cryomicroscopy and digital image processing of lipoprotein(a).

Electron cryomicroscopy was used to study the structure of human lipoprotein(a) (Lp(a)), a plasma lipoprotein implicated in cardiovascular disease. An individual Lp(a) particle consists of a neutral lipid core within a shell of phospholipid, cholesterol and glycoprotein. In principle, electron cryomicroscopy images of single particles should contain structural detail attributable to the density differences among these components and the surrounding buffer. We observed such structural detail in images of frozen, hydrated Lp(a) particles. Lp(a) particles appeared to be roughly spherical in shape with an average diameter of 210 A. As is generally true for unstained samples in vitreous ice, imaged with a low electron dose, these images have low contrast with low signal-to-noise ratios. To increase the signal-to-noise ratio, we averaged classes of similar particles. We began with a set of 5813 randomly oriented Lp(a) particles and generated classes using a linear multivariate statistical method, followed by hierarchical ascendant classification. Our initial classification, based on only the first eight eigenvectors, separated particles on the basis of gross size and shape. After a rough reference-free alignment step, a second classification used the finer details in the images. This approach yielded class averages with structural detail only faintly visible in the raw, single images.

Proposed mechanisms for binding of apo[a] kringle type 9 to apo B-100 in human lipoprotein[a].

The protein component of human lipoprotein[a] consists primarily of two apolipoproteins, apo[a] and apo B-100, linked through a cystine disulfide(s). In the amino acid sequence of apo bd, Cys4057 located within a plasminogen kringle 4-like repeat sequence (3991-4068) is believed to form a disulfide bond with a specific cysteine residue in apo B-100. Our fluorescence-labeling experiments and molecular modeling studies have provided evidence for possible interactions between this apo[a] kringle type and apo B-100. The fluorescent probe, fluorescein-5-maleimide, was used in parallel experiments to label free sulfhydryl moieties in lipoprotein[a] and low-density lipoprotein (LDL). In apo B-100 of LDL, Cys3734 was labeled with the probe, but this site was not labeled in autologous lipoprotein[a]. The result strongly implicates Cys3734 of apo B-100 as the residue forming the disulfide linkage with Cys4057 of apo[a]. To explore possible noncovalent interactions between apo B-100 and apo[a], the crystallographic coordinates for plasminogen kringle 4 were used to generate molecular models of the apo[a] kringle-repeat sequence (3991-4068, LPaK9), the only plasminogen kringle 4 type repeat in apo[a] having an extra cysteine residue not involved in an intramolecular disulfide bond. The Cys4057 residue (henceforth designated as Cys67 in the LPaK9 sequence) is believed to form an intermolecular disulfide bond with a cysteine of apo B-100. In computer graphics molecular models of LPaK9, Cys67 is located on the surface of the kringle near the lysine ligand binding site. Selected segments of the LDL apo B-100 sequence that contain free sulfhydryl cysteines were subjected to energy minimization and docking with the ligand binding site and adjacent regions of the LPaK9 model. In the docking experiments, apo B-100 segment 3732-3745 (PSCKLDFREIQIYK) displayed the best fit and the largest number of van der Waals contacts with models of LPaK9. Other apo B-100 peptides with sulfhydryl cysteine were found to be less compatible when minimized with this kringle. These results support and extend previously suggested mechanisms for a complex interaction between apo[a] and apo B-100 that involve more than a simple covalent disulfide bond.

Age-related studies on the removal of 7-methylguanine from DNA of mouse kidney tissue following N-methyl-N-nitrosourea treatment.

To investigate the effects of age on DNA repair of alkylation damage, C57BL/6NNia mice ranging from 9 months to 29 months of age were injected by the intraperitoneal route with single doses of N-methyl-N-nitrosourea (MNU). The rates of removal of 7-methylguanine (m7Gua) in nuclear DNA from kidney were determined at various intervals from 1 to 288 h after injection of either 25 mg or 50 mg MNU per kg body weight. Reversed phase HPLC with electrochemical detection was used to monitor adduct disappearance from DNA hydrolysates. The kinetics of m7Gua removal from DNA were at least biphasic. Evidence was obtained that there was a rapid removal of m7Gua occurring in the first 24 h after MNU administration, followed by a slow phase of removal with a t1/2 greater than 150 h. We assume that these two phases of m7Gua removal correspond to active repair of DNA by N-alkylglycosylases and to passive elimination via spontaneous hydrolysis, respectively. Young and old kidney tissues all exhibited significant repair of m7Gua (55-73% of the induced adducts were removed in the first 24 h), but a substantial fraction of m7Gua was removed slowly, indicating that there are methylated bases which were refractory to repair processes. At both doses of MNU studied, old tissues showed active repair of m7Gua that, within the limits of detection, had similar initial rates of removal as young tissues. However, old kidney did not remove this adduct with the same overall efficiency as young kidney. Therefore, the amount of m7Gua in the repair-resistant fraction was greater in the senescent tissues. The biochemical mechanisms responsible for the less efficient DNA repair in senescent kidney are not known, but we suggest that such differences are due in part to structural alterations in the chromatin.

Gene expression of an endogenous retrovirus-like element during murine development and aging.

We have measured intracisternal A-particle (IAP) RNA levels during development and aging in C57BL/6J mouse tissues to determine possible age-dependent changes in gene expression of these retrovirus-like sequences. Total RNA was isolated from tissues of embryonic and new born mice and mice ranging in age from 2 months to 32 months of age. RNA samples were either slot-blotted directly or fractionated on denaturing agarose gels and transferred to nylon membranes. Hybridization with cloned, 32P-labeled IAP sequences showed that both the mass amounts and the relative proportions of IAP transcripts varied between tissues and as a function of age. IAP gene products were higher in brain and kidney tissues than in liver and heart tissues. The relative proportion of transcripts increased in embryonic tissues until birth and following birth, was highest in neonatal or 2-month-old tissues. The adult levels of IAP-related RNAs did not change significantly from 6 to 24 months of age. However, 32-month-old tissues exhibited the lowest content of IAP transcripts, with the exception of heart tissue which did not change with age. A 5.4-kb RNA was the predominant IAP transcript in most samples, but each tissue had a characteristic size distribution of IAP-related transcripts. These results demonstrate that transcription of IAP genes continues throughout the life span of this mouse strain with tissue-specific and age-dependent regulation of expression.

Mouse satellite DNA is transcribed in senescent cardiac muscle.

Mouse satellite DNA consists of highly repetitive tandem sequences located in the centromeric heterochromatin. It is generally assumed that these simple sequences are not transcribed. We have analyzed total cellular RNA preparations from mouse liver, kidney, brain, and heart tissues at different ages for satellite transcripts. Using recombinant probes containing the major mouse satellite sequence, satellite transcripts were detected only in the heart RNA samples. These transcripts were not detected in the heart muscle of young adult animals (2 and 6 months), but then appeared at the age of 12 months and continued to increase over 2-fold up to the age of 32 months. The transcripts were resistant to DNase I and sensitive to RNases and alkaline treatment. Northern hybridization experiments showed a large and heterogeneous size range of satellite transcripts. Control studies using short-interspersed (B1 and B2) and long-interspersed (L1 and IAP) repetitive DNA sequence probes did not show a similar age-related pattern of transcription. These results indicate that satellite transcription does occur in mice but is highly tissue- and age-specific. The unique occurrence of satellite transcription only in adult and senescent heart tissue indicates age to be an important determinant of gene activity. An understanding of the regulatory mechanisms involved could lead to new insights in the biological role of satellite DNA, gene derepression of reiterated DNA sequences, and the aging processes of cardiac muscle.

Steady-state levels of 7-methylguanine increase in nuclear DNA of postmitotic mouse tissues during aging.

The major DNA product formed by methylating agents in vitro and in vivo is 7-methylguanine (m7Gua). In untreated rodent genomes, this damage is thought to arise as a consequence of endogenous processes. Using 2 independent HPLC systems and 2 methods of detection, we observed that low levels of m7Gua are present in nuclear DNA of normal 23-month-old postmitotic mouse tissues. We then asked whether the steady-state levels of indigenous m7Gua change as a function of age in these tissues. C57BL/6NNia male mice 11 months, 23 months, and 28 months of age were analyzed. The results showed that in nuclear DNA of brain, liver, and kidney tissues, the steady-state levels of m7Gua increased approximately 2-fold between the young and old age groups. The persistence of N-methyl-N-nitrosourea (MNU)-induced m7Gua in these tissues in treated animals was also studied. Following a 25 mg MNU/kg body weight dose, administered by the intraperitoneal route, m7Gua appeared to be at least partially persistent for a period of up to 20 days. The degree of persistence of m7Gua, however, appeared to be independent of tissue or age. Since m7Gua has intrinsic mutagenic potential and the content of m7Gua is generally a good indicator of overall alkylation damage to DNA, an age-related increase in the steady-state amounts of m7Gua may be relevant to basic mechanisms of aging and carcinogenesis.