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Age estimation in dental pulp DNA based on human telomere shortening.

Age estimation based on evidence found in teeth has received considerable attention within the field of forensic science. We determined the terminal restriction fragment (TRF) length, as telomere length, to estimate age. Using dental pulp DNA we found the average TRF length showed a tendency to shortening with aging. Our findings show that telomere shortening, based on dental pulp DNA is a new and useful approach to estimate age of the subject at the time of death.

Aging↗

Telomeric length and telomerase activity vary with age in peripheral blood cells obtained from normal individuals.

The telomerase activity and length of telomeres of peripheral blood mononuclear cells obtained from 124 healthy individuals aged 4-95 years was measured. Telomerase activity level was semiquantitatively assessed by a fluorescent-telomeric repeat amplification protocol (fluorescent-TRAP) using an internal telomerase assay standard, fluorescent primers and an automated laser fluorescent DNA sequencer. Telomeric length, measured by assay of terminal restriction fragments (TRFs), was determined in HinfI-digested DNA by Southern blot analysis using a (TTAGGG)4 probe. TRF length was determined in 80 individuals and age-related progressive reduction of size was observed. TRF length in peripheral blood mononuclear cells obtained from normal individuals (aged 4-39 years) decreased by approximately 84 bp per year, while in individuals aged > or = 40 years it decreased by 41 bp per year. In contrast, telomerase activity showed an apparent biphasic pattern with aging. Individuals aged 4-39 years showed a progressive decrease in telomerase activity, whereas 65% of those aged > or = 40 years showed relatively stable but very low telomerase activity, and the remaining individuals aged > or = 40 years had no detectable telomerase activity. These data obtained from normal individuals might in the future be of value to help risk stratify and manage the care of patients with leukemia.

Adolescent↗

Telomere shortening in the colonic mucosa of patients with ulcerative colitis.

Telomere length in human somatic cells gradually decreases with the number of cell divisions and is regarded as a marker of somatic cell turnover. Mucosal cells of the affected colon show rapid turnover in individuals with active ulcerative colitis (UC). Telomere length was determined by Southern blot analysis of terminal restriction fragments (TRFs) from the colonic mucosa of 17 patients with UC in remission, two of whom showed dysplasia, and 17 control subjects without colitis. For each individual, mean TRF length was compared between rectal mucosa and unaffected cecal mucosa. The mean TRF length of the rectal mucosa was significantly less than that of cecal mucosa in UC patients (7.87 +/- 0.36kb versus 8.77 +/- 0.21 kb; P = 0.0015, Wilcoxon signed rank test), whereas no significant difference was detected in the control subjects. The extent of telomere shortening was 10.6 +/- 3.35% in UC patients, compared with 0.8 +/- 0.64% in noncolitis controls (P = 0.0024, Mann-Whitney U-test). Four UC patients, two of whom had dysplasia, showed telomere shortening of more than 20% in the rectal mucosa. These observations suggest that telomere shortening in the colonic mucosa of individuals with UC may represent the history of mucosal inflammation during disease of long duration, and that it may contribute to aneuploidy in UC.

Adolescent↗

The Conners-28 teacher questionnaire in clinical and nonclinical samples of Greek children 6-12 years old.

It is accepted practice in child psychiatry to use more than one source of information in assessing behavioural problems in children and adolescents. Employing standardized tools for these assessments allows cross-cultural comparisons and better interchange of the findings. Achenbach's Child Behavior Checklist (CBCL) and Teacher's Report Form (TRF) are two widely accepted instruments that were standardized in Greece within the framework of the European Network for the Study of Hyperkinetic Disorder. We studied the Conners-28 teacher questionnaire in a Greek community sample of primary schoolchildren aged 6-12 years. The factor structure showed to be similar to that originally reported from the USA. Discrimination between the referred and nonreferred sample was high, especially for the Inattentive-passive scale. Conners-28 scores were highly correlated with the TRF (scored by the same informant), much less so with the CBCL (scored by a different informant). Our study demonstrates the usefulness and applicability of the Conners-28 item questionnaire.

Adolescent↗

Trace elements and some extracellular antioxidant proteins levels in serum of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic progressive autoimmune disorder with a wide spectrum of clinical and immunological abnormalities. In this study, we aimed to investigate the levels of serum zinc (Zn), copper (Cu), magnesium (Mg), manganese (Mn), iron (Fe), ceruloplasmin (Cp), transferrin (Trf), and albumin (Alb) in SLE and whether it is related to the severity of the clinical condition of this chronic disease. Cp and Cu levels were higher, while Trf, Alb, Zn, Mg, Mn, and Fe levels were lower in serum of patients with SLE (n = 27) compared with healthy controls (n = 20). The mechanisms by which these alterations occur in certain inflammatory conditions need to be elucidated. It is also obscure whether these alterations are a cause or a consequence of the inflammation. As a conclusion, alterations in the levels of the parameters in SLE may not be a reason for, but in fact a consequence of the disease itself.

Adolescent↗

Serum protein concentrations in Plasmodium falciparum malaria.

In patients with uncomplicated Plasmodium falciparum infection cytokine-mediated serum protein levels of C-reactive protein (CRP), coeruloplasmin (COE), beta 2-microglobulin (B2M), alpha 1-acid glycoprotein (AAG), alpha 1-antitrypsin (AAT), haptoglobin (HPT), prealbumin (PRE), retinol binding protein (RBP), albumin (ALB) and transferrin (TRF) were measured in an endemic area of the Amazonian rain forest. Semi-immune (SI) and nonimmune (NI) patients were investigated. In both patient groups the serum concentrations of CRP, COE and B2M were elevated on admission. In addition AAG and AAT concentrations were increased in NI patients compared to control subjects. Significantly lower serum concentrations of HPT, PRE, RBP, ALB and TRF were seen in both patient groups during the acute phase of the disease, and were more pronounced in NI patients. After a 28-day follow-up, AAT and B2M were normal in SI patients but HPT, AAT and B2M were still significantly altered in NI patients.

Acute-Phase Proteins↗

Europium chelate-loaded liposomes: a tool for the study of binding and integrity of liposomes.

Using the biotin-streptavidin interaction as a model, we investigated the suitability of lanthanide chelates as encapsulated liposomal labels in liposome-based binding assays. Large unilamellar phospholipid:cholesterol liposomes containing europium-DTPA chelate and biotinylated phosphatidylethanolamine were prepared by detergent dialysis. The resulting Eu-liposomes ([symbol: see text] 120 nm) bound specifically to streptavidin in microtiter wells as measured by time-resolved fluorometric assay (TRF). The intensity of fluorescence released from the bound liposomes was dependent on the concentration of biotin in the liposome membrane, the concentration of europium entrapped in the liposomes, the incubation time and the amount of liposomes used in the assay. The sensitivity of the TRF assay allowed the detection of binding of attomole quantities of liposomes. The streptavidin-immobilised liposomes subjected to porcine pancreatic phospholipase A2 (EC 3.1.1.4) and detergents displayed a dose-dependent release of the encapsulated europium. Lanthanide-chelate-liposomes should prove useful for studies addressing binding and stability of liposomes.

Avidin↗

Functional analysis of antigen-nonspecific T-cell suppression. I. Effect of mitogen-induced T suppressor cells on helper-T-cell clones.

The effect of mitogen-induced nonspecific suppressor T cells (Ts)2 on T-helper-cell activity was investigated using isolated clones of murine T-helper cells as targets. TNP-self-reactive Thy1+, Ly1+ T-cell clones were isolated after continuous culture of T cells derived from picryl chloride-sensitized mice and were characterized by their ability to proliferate in an antigen-specific and MHC-restricted manner. In addition, selected T-cell clones were found to produce both interleukin-2 (Il-2) and T-cell replacing factor (TRF), lymphokines characteristic of helper T cells. Concanavalin A (Con A)-induced Ts cells inhibited the antigen-specific proliferation of these helper-T cell clones in a noncytotoxic manner even in the presence of exogenous Il-2. This implied that failure to proliferate was not merely due to an inability of these clones to produce Il-2. The kinetics of suppression also suggested that early T-cell activation signals were not affected. Furthermore, coculture experiments indicated that while proliferation could be severely inhibited, the actual secretion of lymphokines such as Il-2 and TRF by the T-helper clones was not. Our data suggest that nonspecific Ts modulation of proliferation versus helper factor production are under separate control in cloned T-cell populations, with lymphokine secretion remaining intact in the presence of Con A-induced Ts cells.

Animals↗

Iron, transferrin, and haptoglobin levels after a single bout of exercise in men.

Twelve, healthy male university student volunteers, between the ages of 20 and 23, were studied. All subjects were considered normal after cardiopulmonary and electrocardiographic examination. The maximal aerobic capacity (Vo2 max) of each subject was determined. The exercise programmes were performed on a mechanically braked Monark cycle ergometer. The subjects were required to perform the three tests, one per week. Each subject had a catheter inserted in an antecubital vein and blood samples were drawn at rest and at the end of exercise. Before and immediately after each exercise session total proteins (TP), hematocrit (Hct), hemoglobin (Hb), and other hematological parameters were measured. Serum iron (Fe), transferrin (TRF), and haptoglobin (HPT) were also determined. Immediately after the end of the exercise (TPT, RST, and IET), TP, Hb, Hct, and RBC increased significantly. TRF and HPT concentrations remained unchanged and iron decreased significantly after maximum sustained test (RST).

Adult↗

Restricted feeding: a nonphotic zeitgeber in the rabbit.

The free-running circadian rhythms of five behavioral functions of the rabbit were masked by unsignalled restricted food access (RF). The rhythms were reorganized immediately, a large part of events being assembled around the end of food availability. In addition to masking a slower process of entrainment was running: a component of anticipatory activity (AA) was established 1-3 h before food access. AA consolidated in continuation of the camouflaged free-running rhythm. The time of AA establishment correlated significantly with the phase angle difference (PAD) between free-running rhythm and RF: it decreased with decreasing positive PAD. With the consolidation of AA, the rhythms had attained a stable phase relation to RF. At the termination of RF, the circadian rhythms started to free-run again, the phase being dependent from that of the preceding RF schedule. The period length of the RF zeitgeber (TRF) had some impact on tau of the circadian rhythm. This aftereffect was most evident after termination of TRF less than 24 h: the free-running rhythm continued for up to 49 days with a tau less than 24 h and turned longer thereafter. The results demonstrate the entrainment of circadian rhythms of the rabbit by RF in addition to masking.

Animals↗

Establishment of a bromelain-treated isologous red blood cell reactive B cell clone by somatic hybridization.

Splenic B cells of BALB/c mice were fused with 2.52M, a mutant of a B cell line, in the presence of polyethylene glycol and dimethyl sulfoxide. AT73.14 a subclone of a resulting hybridoma, expresses B cell surface antigens on the cell membrane, namely IAd, IgM, B220, and receptors for C3 fragment of complement (C3R), the Fc portion of IgG (Fc gamma R), and interleukin 2 (IL-2R). It also possesses a receptor molecule for mouse red blood cells treated with bromelain (Br-MRBC) on its surface, by rosette-forming assay with Br-MRBC. In contrast, parental 2.52M does not express IAd on the cell membrane and does not bind to Br-MRBC on the same conditions. Thus, it is likely that AT73.14 may be an autoreactive B cell clone specific for Br-MRBC. Interestingly, AT73.14 could generate a significant number of IgM-secreting cells when treated with Br-MRBC; this was followed by a marked decrease in the expression of B cell surface markers on the cell membrane. In addition, this differentiative response of the cells greatly augmented in the presence of B151-TRF, a B cell differentiation factor, although B151-TRF alone showed only a marginal effect on the generation of IgM-secreting cells. The result suggests that this kind of an autoreactive B cell clone may provide a good model for the study on the mechanism of autoimmune responses.

Animals↗

IFN-gamma is not an essential mediator of murine antibody responses in vitro.

We have used purified murine gamma-interferon (Mu IFN-gamma) and anti-Mu IFN-gamma monoclonal antibody to study the participation of gamma-interferon (IFN-gamma) in in vitro plaque-forming cell (PFC) responses to antigen. We have studied several supernatants with T-cell replacing factor (TRF) activity as well as helper T-cell dependent anti-sheep red blood cells (SRBC) PFC responses. Our findings demonstrate that neither TRF- nor T-cell mediated responses are critically dependent upon IFN-gamma.

Animals↗

DNA synthesis in cultured neonatal rat islets--a comparison of two methods.

To assess the effect of glucose, foetal calf serum (FCS) and iron-saturated transferrin (Fe-TRF) on DNA synthesis of fibroblast-free neonatal rat islets cultured in Ham's F-12 medium, we measured [3H]thymidine uptake in cultures and used immunohistochemistry to quantitate nuclear 5-bromo-2'-deoxyuridine staining. Addition of glucose to a concentration of 26.1 mmol/l resulted in a significant increase from baseline in DNA synthesis in islet cells as measured by both methods (22,591 counts/min per microgram DNA +/- 3628 S.E.M., vs. 9631 counts/min per microgram DNA +/- 1912 S.E.M., P < 0.002 and 19.19% positive beta-cells +/- 2.72 S.E.M., vs. 11.98 positive beta-cells +/- 0.26 S.E.M., P = 0.05). At a glucose concentration of 16.1 mmol/l we could demonstrate an increase compared with baseline only in [3H]thymidine uptake (15,700 counts/min per microgram DNA +/- 3323 S.E.M., P < 0.05). Supplementation with 10% or 15% FCS also increased [3H]thymidine uptake compared with baseline (to 15,809 counts/min per micrograms DNA +/- 136 +/- S.E.M., P < 0.05 and 23,746 counts/min per microgram DNA +/- 3114 S.E.M., P < 0.01, respectively) but did not significantly effect the BrDU labelling index. Addition of Fe-TRF to islet cultures significantly increased [3H]thymidine uptake at a concentration of 45 micrograms/ml compared with baseline (23,149 counts/min per microgram DNA +/- 6387 S.E.M., P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Transferrin, haematological indices and liver glycogen in field mice collected from a coal mining area and a control site in Poland.

A polymorphism for serum transferrin in field mice from polluted and control sites was compared with body-morphological and haematological parameters, and with liver glycogen concentration. The distribution of genotypes in the polluted area was not panmictic, due to higher frequencies of the rare allele Trf(c) occurring mainly in males. In the polluted area, transferrin-c was accompanied by slowly migrating, Fe-binding protein fractions. Since the Trf(c)/Trf(c) homozygote possessed higher red blood cell parameters, it is suggested that these extra proteins have an Fe-transport function. The morpho-physiology of heterozygotes from the polluted area did not differ significantly from those from the control site. The animals from the polluted area possessed a high concentration of liver glycogen, indicative of poor environmental conditions. Also, a generally lower level of red blood cell parameters suggests that non-oxygenic processes dominate over oxygenic ones in the polluted area.

Journal Article↗

Changes in right ventricular dimensions and performance after passive cardiac containment.

BACKGROUND: Previous studies have shown that the cardiac support device (CSD) improves left ventricular structure and function in patients with heart failure by preventing further cardiac enlargement. The aim of this study was to identify effects on the right ventricle (RV). METHODS: Ten male patients with idiopathic dilated cardiomyopathy underwent electron-beam computed tomographic (CT) examination within 1 month before, and 6 to 9 months after CSD implantation. The RV end-diastolic and end-systolic volumes (EDV, ESV) and diameters (EDD, ESD), stroke volume (SV), ejection fraction (EF), total and forward RV output (RVO, fRVO), and tricuspid regurgitation fraction (TRF) were calculated. RESULTS: The EDV measurements decreased from 182.1 +/- 49.6 to 137.5 +/- 37.0 mL, ESV from 114.8 +/- 47.0 to 68.3 +/- 23.8 mL, EDD from 48.2 +/- 6.6 to 41.6 +/- 7.1 mm, and ESD from 39.6 +/- 6.9 to 32.7 +/- 6.5 mm (p < 0.05 for each). Ejection fraction increased from 38.5 +/- 8.9 to 52.0% +/- 7.7% and fRVO from 4.0 +/- 0.8 to 4.6 +/- 1.1 L/min (each with p < 0.05). TRF decreased from 18.2 +/- 14.1 to 10.4% +/- 13.5%, whereas SV and RVO remained nearly unchanged. Postoperatively, RV volumes, EF, and fRVO were not different from 15 age- and gender-matched normal control patients. CONCLUSIONS: Implantation of a CSD leads to a decrease in RV size and improved RV performance. These data together with the results of previous studies demonstrating improved left ventricular structure and function confirm the biventricular nature of recovery with the CSD.

Cardiomyopathy, Dilated↗

Improvement the accuracy and reproducibility of telomere length measurement utilizing qPCR.

Telomere length serves as a well-established molecular biomarker for evaluating aging and age-associated diseases. Among various methods, quantitative PCR for telomere length detection is convenient, rapid, cost-effective, and capable of high-throughput analysis in large epidemiological cohorts. However, numerous studies have indicated that issues related to differences in DNA quality caused by DNA extraction process significantly affect the accuracy and reproducibility of qPCR-based telomere length quantification. Initially, we established a model of DNA integrity variation, by utilizing nucleic acid endonucleases of serial activity units to cleave genomic DNA, generating DNA with varying degrees of degradation. The integrity of DNA templates decreases, the reduction of long fragments and the increase of short fragments in the mixed telomere products are the causes of the disruption in Ct values. Moreover, compared with longer reference gene amplicons, the short-segment internal gene reference can reduce the impact of genomic integrity on its amplification. Subsequently, we utilized additional gel excision purification to reduce degradation products. It was found that gel excision processing provides the best stability for telomere length detection with the lowest coefficient of variation. Additionally, the introduce of another calibrator sample, which is used for to adjust the T/S value of the test sample, narrows the deviation between qPCR-derived telomere length and gold-standard Terminal Restriction Fragment (TRF) measurements. Collectively, these results reveal that gel excision purification supports stable telomere detection. Calculating the correction coefficient incorporating the short internal reference and calibrator minimizes measurement deviations relative to sample TRF values.

Telomere↗

The emerging role of poly(ADP-ribose) polymerase-1 in longevity.

In the present paper, the involvement of the family of poly(ADP-ribose) polymerases (PARPs), and especially of PARP-1, in mammalian longevity is reviewed. PARPs catalyse poly(ADP-ribosyl)ation, a covalent post-translational protein modification in eukaryotic cells. PARP-1 and PARP-2 are activated by DNA strand breaks, play a role in DNA base-excision repair (BER) and are survival factors for cells exposed to low doses of ionising radiation or alkylating agents. PARP-1 is the main catalyst of poly(ADP-ribosyl)ation in living cells under conditions of DNA breakage, accounting for about 90% of cellular poly(ADP-ribose). DNA-damage-induced poly(ADP-ribosyl)ation also functions as a negative regulator of DNA damage-induced genomic instability. Cellular poly(ADP-ribosyl)ation capacity in permeabilised mononuclear blood cells (MNC) is positively correlated with life span of mammalian species. Furthermore PARP-1 physically interacts with WRN, the protein deficient in Werner syndrome, a human progeroid disorder, and PARP-1 and WRN functionally cooperate in preventing carcinogenesis in vivo. Some of the other members of the PARP family have also been revealed as important regulators of cellular functions relating to ageing/longevity. In particular, tankyrase-1, tankyrase-2, PARP-2 as well as PARP-1 have been found in association with telomeric DNA and are able to poly(ADP-ribosyl)ate the telomere-binding proteins TRF-1 and TRF-2, thus blocking their DNA-binding activity and controlling telomere extension by telomerase.

Animals↗