Characteristics of the cis- and trans-positions of chromatids of human acrocentric chromosomes in extreme old age

Cis- and trans-positions of chromatid associations of human acrocentric chromosomes were examined at extreme old age. Lymphocyte cultures were prepared by the usual method, from peripheral blood of 9 subjects aged 80-90 years (analysis of 179 metaphases) and 7 subjects aged 20-48 years (analysis of 124 metaphases). The functional difference between stalks of the sister chromatids was found. In the subjects at the age 80-90 years satellite stalks of chromatids-1 (in all DNA strands thymidine was substituted by 5-BrdU) of the D chromosome in cis-position are included into associations with lower frequency, as compared with the satellite stalks of chromatids-2 (thymidine+ is only substituted by 5-BrdU in a half DNA strands of the chromosome). This apparently reflects variability of regulation of functional activity of satellite stalks of sister chromatids.     

Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes.

The telomere hypothesis of cellular aging proposes that loss of telomeric DNA (TTAGGG) from human chromosomes may ultimately cause cell-cycle exit during replicative senescence. Since lymphocytes have a limited replicative capacity and since blood cells were previously shown to lose telomeric DNA during aging in vivo, we wished to determine: (a) whether accelerated telomere loss is associated with the premature immunosenescence of lymphocytes in individuals with Down syndrome (DS) and (b) whether telomeric DNA is also lost during aging of lymphocytes in vitro. To investigate the effects of aging and trisomy 21 on telomere loss in vivo, genomic DNA was isolated from peripheral blood lymphocytes of 140 individuals (age 0-107 years), including 21 DS patients (age 0-45 years). Digestion with restriction enzymes HinfI and RsaI generated terminal restriction fragments (TRFs), which were detected by Southern analysis using a telomere-specific probe (32P-(C3TA2)3). The rate of telomere loss was calculated from the decrease in mean TRF length, as a function of donor age. DS patients showed a significantly higher rate of telomere loss with donor age (133 +/- 15 bp/year) compared with age-matched controls (41 +/- 7.7 bp/year) (P < .0005), suggesting that accelerated telomere loss is a biomarker of premature immunosenescence of DS patients and that it may play a role in this process. Telomere loss during aging in vitro was calculated for lymphocytes from four normal individuals, grown in culture for 10-30 population doublings. The rate of telomere loss was approximately 120 bp/cell doubling, comparable to that seen in other somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation in pattern and frequency of acrocentric association in normal and trisomy-21 individuals

Summary Chromosomally normal and trisomy-21 individuals were studied for the ability of their nucleolus-organising chromosomes to form satellite associations in G-banded lymphocyte metaphases. Two types of parameter, absolute association frequency and relative association frequency, were used. There was no significant difference between females and males or between Caucasoids and Mongoloids for either type of association parameter in the controls, nor was there significant correlation between age (17–40 years) and either type of parameter in the controls.The pattern of two chromosome associations is accounted for by two related models in both normal and trisomic individuals. These models imply that there is an extensive polymorphism for associating ability and that this ability may be zero in individual chromosomes. Homologous do not associate preferentially with each other. The absolute frequency of acrocentric association is lower in trisomy 21 individuals than disomic controls, but the relative involvement of chromosome 21 (after correction for the trisomic state) is higher than in the controls.     

Meiosis in trisomic female mice with Robertsonian translocations. I. Prophase pairing

The prophase oocytes of two murine Robertsonian translocation (Rb) trisomies of chromosomes 16 and 19 were investigated using electron microscopy and a whole-cell micro-spreading technique after silver staining. About 20% of fetuses of each type were trisomic. They were obtained by mating animals heterozygous for two Rb's, monobrachially homologous for either chromosome 16 or 19, to an entirely acrocentric stock. Because of the almost inevitable prenatal mortality of the trisomic embryos, their fetal ovaries were "rescued" by an in vitro method for prophase studies. Analysis of the recovered oocytes showed frequent, close pairing associations of the three trisomic axes and evidence suggesting that the closely apposed axes coincided with the side-by-side formation of parallel, complete, true synaptonemal complexes; hence, the cytogenetic dogma that pairing is always two-by-two was contradicted. The presence of two parallel complexes has implications for crossing-over recombination. Triple associations of axes were found in almost half the trisomy 19 (Ts19) and in about 70% of the trisomy 16 (Ts16) prophases. The extent of triple associations varied and was greater in Ts16 than in Ts19 oocytes. Other relevant observations concerned the proportions of univalents and of univalence of the trisomic axes (21% in Ts16 and 46% in Ts19) and the distinctive, thickened appearance of all univalent axes. The pairing behaviour observed in balanced heterozygotes confirms what appears to be nonhomologous pairing and synaptic adjustment within the short-arm axes of the Rb trivalents.     

Double satellites do not increase the risk of chromosome 21 nondisjunction

The hypothesis concerning the increase of the risk of appearance of offsprings with trisomy 21 in parents having double satellite acrocentric chromosomes is checked. Chromosomes of 266 parents having offsprings with Down's disease were examined, including 73 donors of extrachromosome 21. The frequency of chromosomes with dNORs in the total and in the donor groups did not differ from that of the control group. From 42 donors of extrachromosome, who demonstrated a nondisjunction in the first meiotic division, double satellites were revealed only once. In addition, data on the frequency of Dp+ and Gp+ variants are presented and discussed.     

Segregation of acrocentric chromosome association in familial dicentric Robertsonian translocation t(14p;22p), aneuploidy (trisomy-21) and heteromorphism

The frequency and types of acrocentric chromosome association were quantitatively analysed in a Down syndrome child with unusual karyotype, 46, XX, -14, -22, t dic (14p;22p), +21, 21S+. Father and 4 sibs were heterozygous carriers for t dic (14p;22p). The variant 21S+ was inherited from the mother. The occurrence of translocation and trisomy in the same individual is extremely rare. Acrocentric chromosome association was analysed in this interesting family to understand the interrelationship of acrocentric chromosome association, Robertsonian translocation and heteromorphism, as possible predisposing factors for nondisjunction. Our findings suggest that acrocentric chromosome association is a heritable and nonrandom phenomenon. Heterozygous carriers for translocations and variants are likely to be at increased risk of nondisjunction. Long term family studies will enable to ascertain the causal-relationship of these factors more precisely.     

Effect of sex, age and cultivation time on number of satellites and acrocentric associations in man.

Lymphocyte cultures of 120 normal persons evenly distributed in relation to sex and age groups were studied. A total of 3900 cells were examined, 3600 in 3-day cultures and 300 in cultivations maintained for 2 days. In both age groups (10--13; 62--96 years) men showed higher numbers of satellites per cell but less cells with D/G associations. Older subjects presented a lower number of cells with D satellites and acrocentric associations; the pattern of these associations, however, did not seem to vary with age. The mean number of associations per cell decreased significantly in 3-day as opposed to 2-day cultures.     

Karyotypic and phenotypic changes during in vitro aging of human endothelial cells.

Karyotypic and phenotypic changes were found in human adult endothelial cells (EC) during aging in vitro. A trisomy of chromosome 11 was found in 11 out of 12 EC cultures examined, derived from 9 cell lines from 8 donors. The incidence of this trisomy in some cell lines increased over time from 0% to as much as 100% near the end of their in vitro life span. A number of oncogenes and other important genes are on chromosome 11. These genes might play a role in the changes observed. An increase in the percentage of polyploid cells was also found near the end of the in vitro life span in 6 lines. The cellular levels of two gene products characteristic of the EC, von Willebrand factor (vWF) or Factor VIII, and angiotensin converting enzyme (ACE) were also monitored. vWf was studied in 2 lines and was decreased in both with serial passage. ACE decreased in three out of the four lines examined. These chromosomal and phenotypic changes which occur with increasing age in vitro make the endothelial cell a suitable model to study in vitro culture-related changes, senescence, cardiovascular disease, and tumorigenesis.     

Chromosome banding and molecular cytogenetic study of two Mediterranean trachinoid fish species (Teleostei: Trachinidae, Uranoscopidae)

The chromosomes of Echiichthys vipera (Trachinidae) and Uranoscopus scaber (Uranoscopidae) were analyzed by means of various banding methods and fluorescence in situ hybridization (FISH) with telomeric and major rDNA probes, respectively. The karyotype of E. vipera was composed of 48 acrocentric chromosomes and NOR sites, as revealed by all detection methods, were situated pericentromerically on a single pair of middle-sized chromosomes. Blocks of constitutive heterochromatin were present in the pericentromeric regions of all pairs of chromosomes. The karyotype of U. scaber showed three karyomorphs: 2n = 30 (18 m + 12 a/st [m = metacentric, a = acrocentric and st = subtelocentric]), 2n = 28 (20 m + 8 a/st), 2n = 27 (21 m + 6 a/st). NORs, as revealed by FISH, were situated pericentromerically on a single pair of middle-sized chromosomes in spite of Ag-positive signals in the centromeres of all pairs of chromosomes. Robertsonian fusions were hypothesized for observed variation due to invariable number of chromosome arms FN = 48.     

Trisomy recurrence: a reconsideration based on North American data

Few reliable data exist concerning the recurrence risk for individual trisomies or the risk for recurrence of trisomy for a different chromosome. We collected records from two sources: (1) prenatal diagnoses performed at the Hopital Sainte-Justine in Montreal and (2) karyotype analyses performed at Genzyme. Using the standardized morbidity ratio (SMR), we compared the observed number of trisomies at prenatal diagnosis with the expected numbers, given maternal age-specific rates (by single year). SMRs were calculated both for recurrence of the same trisomy (homotrisomy) and of a different trisomy (heterotrisomy). After all cases with an index trisomy 21 were combined, the SMR for homotrisomy was 2.4 (90% CI 1.6-3.4; P=.0005). For women with both the index trisomy and subsequent prenatal diagnosis at age <30 years, the SMR was 8.0; it was 2.1 for women with both pregnancies at age >/=30 years. For the other index viable trisomies (13, 18, XXX, and XXY) combined, the SMR for homotrisomy was 2.5 (90% CI 0.7-8.0). For heterotrisomy, the SMR after an index trisomy 21 was 2.3 (90% CI 1.5-3.8, P=.0007); the SMR did not vary with maternal age at the first trisomy. When all cases with index viable trisomies were combined, the SMR for heterotrisomy was 1.6 (90% CI 1.1-2.4; P=.04). For prenatal diagnoses following a nonviable trisomy diagnosed in a spontaneous abortion (from Genzyme data only), the SMR for a viable trisomy was 1.8 (90% CI 1.1-3.0; P=.04). The significantly increased risk for heterotrisomy supports the hypothesis that some women have a risk for nondisjunction higher than do others of the same age.     

Chromosomal evolution in Cervidae

On the basis of chromosome data obtained on 30 species and 20 subspecies of Cervidae, a report is submitted on the karyosystematics of this family. The primitive karyotype of Cervidae may be inferred to be composed of 35 acrocentric pairs (2n = 70 FN = 70). During the phyletic evolution of this family different types of chromosome rearrangements were probably selected and the group may have differentiated karyologically into three branches: (1) the Cervinae that fixed a centric fusion resulting in a metacentric pair of autosomes (2n = 68, FN = 70), as shown by the basic karyotype of Cervus elaphus, and where Robertsonian fusions are the preeminent type of chromosome rearrangement; (2) the Odocoileinae, in which pericentric inversions and Robertsonian fusions were favored, yielding first a submetacentric X and then a submetacentric autosome pair. The most representative karyotype is 2n = 70, FN = 74--as in Odocoileus hemionus; and (3) the Muntiacinae, in which centric and tandem fusions were the most common chromosome rearrangements. While Muntiacus reevesi has a karyotype 2n = 46, FN = 46, the chromosome number drops down to 2n = 6 in the females of the M. muntjak vaginalis subspecies group and M. rooseveltorum. Therefore, while the karyotypes are conserved within the subfamilies Cervinae and Odocoileinae; the subfamily Muntiacinae appears to be the most chromosomally diversified group. The few karyological data on the Moschus berezovskii suggest that the Moschinae should be placed in a separate family, the Moschidae.     

Origin of acrocentric trisomies in spontaneous abortuses

Summary A total of 33 spontaneous abortuses with various acrocentric trisomies were studied for the origin of the extra chromosomes using Q- and R-band polymorphisms as markers. Eleven trisomic abortuses were informative: nine trisomic abortuses (one with trisomy 13, three with trisomy 21, and five with trisomy 22 including one with a 46,XX/47,XX,+22 mosaicism) originated at maternal first meiosis; a 21-trisomic abortus resulted from an error at maternal second meiosis (or first mitosis); and a 13-trisomic abortus was of maternal first or second meiotic origin. The abortus with mosaic trisomy 22 started as a 22-trisomic zygote resulting from an error at maternal first meiosis, followed by a mitotic (in vivo or in vitro) loss of the paternally derived chromosome 22.     

Quantitative studies on the arrangement of human metaphase chromosomes

Summary The association pattern was studied in 1182 mitoses of 21 patients with trisomy 13 and in a control group. In addition, 173 trisomic mitoses were compared with the same number of diploid mitoses in a case of mosaicism.The number of mitoses with associations was no higher in the trisomic cells than in cells with normal karyotypes. Some differences were observed in the frequency of associations per cell and of the types of associations in the patient group and in the trisomic cells of the mosaic case. The number of associations in which more than two acrocentric chromosomes were involved was unexpectedly low in the cells with a supernumerary chromosome 13.The result are interpreted as suggesting the existence of a compensatory mechanism activated by the additional acrocentric chromosome.Parts of this work are included in the doctoral (MD) thesis of DM     

Mosaicism for trisomy 21 and ring (21) in a male born to normal parents: A case report

We present a case of a ring (21) in a mentally challenged patient with mosaicism for trisomy 21 showing karyotype 47, XY,+21/47,XY,+21(r)/46,XY, born to normal parents. The parents and female sibling were phenotypically normal. This is a unique case report from Central India, on occurrence of trisomy 21 and r (21) in the same individual born to normal parents. Also being documented for the first time is the immuno-FISH analysis revealing differential expression of hTERT and a linked over expression of TRF2 in proband, probably corresponding to a high percentage of acrocentric associations.     

Antibodies and reactive T cells against the malaria heat-shock protein Pf72/Hsp70-1 and derived peptides in individuals continuously exposed to Plasmodium falciparum.

Pf72/Hsp70-1, a heat-shock protein of m.w. 72 kDa from Plasmodium falciparum is one of the Ag of interest to be included in a polyvalent vaccine against malaria. It is one of the major immunogens present in a fraction of purified blood stage parasites that elicited protection against experimental infection of Saimiri monkeys with blood stages of P. falciparum. It is present at all blood stages and one of its B cell epitopes is also detected on the surface of the infected hepatocyte. Moreover, Pf72 appears to be well conserved among different isolates of P. falciparum. We have examined the immune response against Pf72/Hsp70-1 in individuals from different age groups living in a holoendemic area (West Africa). The immune response against the native Ag (purified from schizonts and called Pf/Hsp70) was analyzed both at the humoral level by ELISA and at the cellular level by assessing in vitro proliferation and IFN-gamma production of PBMC. Of the individuals studied 52% had a statistically significant level of anti-Pf/Hsp70 antibodies as compared with unexposed individuals. These positive individuals showed a heterogeneous distribution because significant levels of antibodies were found in 70% of the adults but in only 26% of the children. The presence of Pf/Hsp70-specific reactive T cells in the blood was detected in 32% of the individuals. The total anti-Pf/Hsp70 antibody level (IgG+IgM) appeared strongly age related and correlated positively with parasite exposure, whereas the T cell response failed to correlate either with the antibody level or with age. Moreover, PBMC of donors responded to the Pf/Hsp70 in a dissociated way, namely, by either T cell proliferation or IFN-gamma production. Ten synthetic peptides based on sequences found in the C-terminal part of Pf72/Hsp70-1 were further tested as potential T cell epitopes. The proliferative response of PBMC from individuals continuously exposed to the parasite showed that three peptides more frequently trigger significant T cell proliferation (in 21% to 27% of the individuals) and three others less frequently (10%). None of these peptides allowed detection of reactive T cells in PBMC of Europeans with no previous exposure to malaria. Some of the stimulating peptides are highly similar to human heat-shock Hsc and Hsp70 with large stretches of identical amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of two means of identifying satellite associations in man]

Two methods of identifying satellite associations were compared in ten normal donors. Conventional Giemsa stain proved to identify by 31% less of 3-chromosomal associations, by 50% less of 4- and 5-, and by 66.6% less of 6-chromosomal associations than in staining with ammoniacal silver, whereas the number of 2-chromosomal associations was by 10% greater, this conditioning the prevalence of small associations. Silver staining used to expose intersatellite associations revealed acrocentric chromosomes mutually oriented, placed close to one another, but without any intersatellite connection. In Giemsa-stained preparations these are considered to be associating, which increases the number of multiple groups. Both errors of the conventional Giemsa method cannot be corrected. Identification of associations according to intersatellite connections is free of these errors after silver staining.     

Chromosome evolution of the blue sheep/bharal (Pseudois nayaur)

A male dwarf blue sheep was collected 60 km south of Batang east to the Jinsha Jiang river, and a male Subei blue sheep (Greater form) was collected from Gansu, China, representing two geographically separated blue sheep forms. Chromosome preparations were prepared from fibroblast cultures. The dwarf blue sheep has a 2n = 54 and a karyotype with three biarmed formations that resulted from acrocentric chromosome fusions (based on the 2n = 60 Capra autosomal equivalents) 14p/5q, 27p/1q, and 29p/2q from the largest to the smallest biarmed chromosome, respectively. The 14p/5q fusion is metacentric, whereas the 27p/1q and 29p/2q are submetacentric. The Subei blue sheep had a 2n = 56, with only the 27p/1q and 29p/2q biarmed chromosome fusions. The remainder of the chromosomes in both blue sheep are acrocentric; the X is the largest acrocentric chromosome and the Y is a minute biarmed chromosome. Our observation is one evidence showing that chromosome evolution within blue sheep has followed a series of centric fusions resulting in the reduction of chromosome number, which is typical of all extant genera within the tribe Caprini.     

Cytogenetic analyses of a Salvelinus hybrid reveal an evolutionary relationship between the parental species

Mitotic analyses of the brook trout (Salvelinus fontinalis) x arctic char (S. alpinus) hybrids (sparctic trout) revealed a mode of 2n = 82 with 18 metacentric and 64 acrocentric chromosomes. The brook trout had 2n = 84 with 16 metacentric chromosomes and the arctic char had 2n = 80 with 20 metacentric chromosomes; both species are derivatives of a single tetraploid event. Variable multivalent-like configurations that may be centromeric associations of bivalents were observed in C-banded pachytene figures of female sparctic trout. Metaphase I analyses of sparctic trout males indicated that two fusions of nonhomologous acrocentric chromosomes representing two duplicated chromosome sets must have occurred in the arctic char after its evolutionary divergence from the brook trout. A mode of seven tetravalent rods per cell suggests that preferential multivalent pairing occurs in the sparctic hybrid; metaphase I analyses of S. alpinus males revealed a mode of only five tetravalent rods per cell. The presence of multivalents implies that the arctic char, like the brook trout, is still undergoing diploidization. Cytochemical detection of the nucleolar organizer region (NOR) revealed intra- and interspecific as well as intraindividual variability in the numbers and types of chromosomes (metacentric or acrocentric) on which NORs appeared in arctic char and sparctic trout. Brook trout only had NORs on acrocentric chromosomes. This may indicate that different chromosomal fusions occurred in the evolution of brook trout from arctic char.     

The associations of acrocentric chromosomes in the parents of Down's syndrome children (a review of the literature)]

A point of view has been recently maintained that, as data are accumulated, the role of the association activity as a cause of acrocentric chromosome nondisjunction is not confirmed. Data are reviewed concerning all the studies available in the literature on the frequency and pattern of acrocentric associations in parents of the Down's syndrome patients. From these data it is evident that the nucleolar organizing activity is an important factor in etiology of trisomy 21. Reasons of some negative results occasionally reported are discussed.     

Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress

Impairment of mitochondria function and cellular antioxidant systems are linked to aging and neurodegenerative diseases. In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment that contributes to age-related visual dysfunction. Here, we examined changes in mitochondrial function in human RPE cells and sensitivity to oxidative stress with increased chronological age. Primary RPE cells from young (9–20)-, mid-age (48–60)-, and >60 (62–76)-year-old donors were grown to confluency and examined by electron microscopy and flow cytometry using several mitochondrial functional assessment tools. Susceptibility of RPE cells to H₂O₂ toxicity was determined by lactate dehydrogenase and cytochrome c release, as well as propidium iodide staining. Reactive oxygen species, cytoplasmic Ca²⁺ [Ca²⁺]_c, and mitochondrial Ca²⁺ [Ca²⁺]_m levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate, fluo-3/AM, and Rhod-2/AM, respectively, adenosine triphosphate (ATP) levels were measured by a luciferin/luciferase-based assay and mitochondrial membrane potential (ΔΨm) estimated using 5,5′,6,6′-tetrachloro 1,1′3,3′-tetraethylbenzimid azolocarbocyanine iodide. Expression of mitochondrial and antioxidant genes was determined by real-time polymerase chain reaction. RPE cells show greater sensitivity to oxidative stress, reduction in expression of mitochondrial heat shock protein 70, uncoupling protein 2, and superoxide dismutase 3, and greater expression of superoxide dismutase 2 levels with increased chronological age. Changes in mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity were more prominent in samples obtained from >60 years old compared to mid-age and younger donors. These mitochondria abnormalities correlated with lower ATP levels, reduced ΔΨm, decreased [Ca²⁺]_c, and increased sequestration of [Ca²⁺]_m in cells with advanced aging. Our study provides evidence for mitochondrial decay, bioenergetic deficiency, weakened antioxidant defenses, and increased sensitivity of RPE cells to oxidative stress with advanced aging. Our findings suggest that with increased severity of mitochondrial decay and oxidative stress, RPE function may be altered in some individuals in a way that makes the retina more susceptible to age-related injury.